Sarah Hurtado-Bagès

TESI DOCTORAL UPF / 2019

Thesis Director: Marcus Buschbeck , Metabolism and Cell Fate group Josep Carreras Leukaemia Research Institute

The role of macroH2A1 variant in muscle metabolism and development

Je te la dédie mémé.

"nothing is lost, nothing is created, everything is transformed"

Antoine Lavoisier

Abstract

Introduction: Multipotent stem cells give rise to multiple differentiated cells sharing the same genetic information and yet performing distinct functions. This is achieved by epigenetic modifications, allowing rapid regulation of gene expression at the chromatin level, without altering the DNA sequence. In the eukaryotic nucleus, DNA, wrapped around a core of histone proteins, forms the basic unit of chromatin, the . Depending on the cellular context, replication-coupled can be exchanged by histone variants at the nucleosome level. In mammals, for instance, H2A can be replaced by several histone variants including three macroH2A proteins. H2AFY and H2AFY2 genes for macroH2A1 and macroH2A2 proteins, respectively. Alternative splicing of the macroH2A1 transcript further gives rise to macroH2A1.1 and macroH2A1.2 isoforms.

Aim: The aim of my study was to determine differences in the function of both isoforms during myogenic differentiation. As a side-objective I have further addressed the question about the evolutionary origin of these isoforms.

Results: We discovered that the expression of macroH2A1 splice isoforms switch during myogenic differentiation. From predominant expression of macroH2A1.2 in proliferating myoblasts to high expression of macroH2A1.1 in differentiated myotubes. This switch has two major consequences. First, both isoforms differentially regulate a number of genes and the dynamics of myotube formation through cell fusion. Second, macroH2A1.1 impacts on cellular metabolism by binding and inhibiting the major nicotinamide adenine dinucleotide-consuming in the nucleus, the cellular stress sensor PARP1. Finally, we provide evidence that the PARP1 inhibitory capacity of macroH2A is an ancestral function of the protein ranging back to the origins of multicellular life.

Conclusion: In conclusion, at distinct time points both macroH2A1 isoforms are essential for the proper myogenic differentiation and metabolic homeostasis.

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Resumen

Introducción: Las células madre multipotentes dan lugar a múltiples células diferenciadas que comparten la misma información genética y que, sin embargo, realizan funciones distintas. Esto se logra mediante la modificación epigenética, que permite una rápida regulación de la expresión génica a nivel de cromatina, sin alterar la secuencia de ADN. En el núcleo eucariótico, el ADN se encuentra envuelto alrededor de un núcleo de proteínas histonas formando la unidad básica de la cromatina: el nucleosoma. Dependiendo del contexto celular, las histonas acopladas a la replicación pueden intercambiarse por variantes de histonas a nivel de nucleosoma. En los mamíferos, por ejemplo, la histona H2A puede ser reemplazada por distintas variantes de histonas que incluyen tres proteínas macroH2A. Los genes H2AFY y H2AFY2 codifican las proteínas macroH2A1 y macroH2A2. El empalme alternativo de la transcripción de macroH2A1 da lugar a las isoformas macroH2A1.1 y macroH2A1.2.

Objectivos: El objetivo de mi estudio fue determinar las diferencias en la función de ambas isoformas de macroH2A1 durante la diferenciación miogénica. Como objetivo secundario, he abordado la pregunta sobre el origen evolutivo de estas isoformas.

Resultados: Descubrimos que la expresión de las isoformas de empalme macroH2A1 cambia durante la diferenciación miogénica: desde la expresión predominante de macroH2A1.2 en mioblastos en proliferación hasta la expresión alta de macroH2A1.1 en miotubos diferenciados. Este cambio tiene dos consecuencias principales. Primero, ambas isoformas regulan diferencialmente una serie de genes y la dinámica de la formación de miotubos a través de la fusión celular. En segundo lugar, la macroH2A1.1 tiene un impacto en el metabolismo celular al unirse e inhibir el sensor de estrés celular PARP1, la enzima que consume nicotinamida adenina dinucleótido en el núcleo. Finalmente, proporcionamos evidencia de que la capacidad inhibitoria de PARP1 de macroH2A es una función ancestral de la proteína que se remonta a los orígenes de la vida multicelular. Conclusión: En conclusión, en distintos puntos temporales, ambas

10 isoformas de macroH2A1 son esenciales para la correcta diferenciación miogénica y la homeostasis metabólica.

Acknowledgments

I need to say that it was the hardest part of the thesis I had to write, between exhaustion, sadness and happiness. My tears came several times for you guys. I know you are going to say that my acknowledgment is too long but how to say it... :)

Marcus Buschbeck's group Marcus, in five years we passed by many challenges. Nowadays, you often say that I changed a lot since the first time we met. Indeed, I changed a lot. You did too. I particularly enjoyed our last meetings when we understand and support each other. During these five years, I would like to thank you for all your support. You were here when I was going through the most stressful moments of my PhD's life. You were here when I needed your feedback for oral presentation, PhD report and you are still here today for the thesis writing and presentation. You will be here tomorrow, either for our next paper, for my switch to scientific communication and dissemination career, and for any collaboration we will establish in the near future. I learnt a lot beside you. Sometimes you failed, sometimes I failed, sometimes we both failed. But we also won battles several times, and we will win more in the future. I wish you, and all the members of your group, all the best for the next years to come. Melanija, you have been an inspiration for me. I started with your work, I finish with your work. I am really proud to present in this thesis our work. We went through complicated moments, but I will never forget the positive impact you had on my career. Julien, aïe Julien! les mots me manquent pour te remercier. Tu m'en as fait voir de toutes les couleurs :) Mais aujourd'hui tu as une place particulière dans ma vie et tu le sais. Ton soutien moral, ton feedback scientifique et surtout ton amitié sont inestimables. Je suis tellement heureuse pour toi que tu aies trouvé le calme et créé ta famille. On ne pouvait te souhaiter mieux. David, ay David! jajaja. Una otra persona en mi vida que ha tenido un impacto increíble. From day one, you were the best and we both know that ;) he tenido mucha suerte tenerte a mi lado durante todos estos años y no solamente en el laboratorio. Tengo mucho respeto para tu trabajo y también tu personalidad en oro. ¡Mi integración en este país no hubiera sido tan facíl sin ti! Gracias para todos estos momentos graciosos. I wish you all the best my friend, you deserve it! our story just started and does not finish here! Iva, ay Iva! few 12

years ago this bomb entered in the lab. You killed my neurones with so much scientific enthusiasm. We could talk for hours about science, ideas, experiments. I am crazy and I knew it. But, I didn't know I could be a scientist. With the PhD, and by exchanging all these ideas with you, this make me realized that nothing could stop us. You are a smart girl, and I am still surprized sometimes that people like you doubt about it... our scientific syndrome I guess. You are a warrior! lucky and proud to work with you! Marguerite- Marie, t'es dans la section "amies" en dehors du laboratoire ;) Vanesa, ay Vanesa! tantas cosas que han pasado durante estos años... Muchísimas gracias para todo tu apoyo. Gracias para todos estos momentos duros, graciosos, divertido. Tu risa a mis tonterías es el mejor regalo que me puedes dar. "Estas locas","Estas fatal ehhh","Donde esta el gatito?", "Te quiero", "hummm que rico este abrazo", "miaou". Roberto, ay Roberto! has ganado el premio del mejor humor negro del campus jajaja. Ro' gracias para tu apoyo y tu feedback sobre mi trabajo. Gracias para estas conversiones de cultura musical y de política interna XD. Las noches de GOT han sido geniales...como la pasta. Merci chef! Anna, ay Anna! mi paulaaaa. Mira donde has llegado... tan contenta para ti. Has ido una de las primeras que me han hecho sentir como a casa en Barcelona. Eres un sol y lo sabes. Me ha encantada trabajar contigo en el laboratorio y disfrutar de la vida afuera tambien. Neus, ay Neus! No nos hemos podido ver tanto una vez que te has ido. Pero cada momento contigo ha sido un placer inmenso. ¡Tu apoyo me ha dado mucha fuerza! Gracias guapi. Raquel, ay mi Raquelita! contigo...una otra bomba ha llegado en el laboratorio. Me siento honrada encontrarte. Eres un sol también y nuestra amistad tiene mucho valor a mis ojos. Espero haber podido ayudarte cuando lo necesitabas. ¡Veo mucho potencial en tí, cogelo y have fun it! Michael, ay Michael! Big thanks for all your time dedicated to correct my English... lot of work was needed there XD. I also see a lot of potential in you, believe in yourself. More importantly, have fun! Yes, a PhD can be fun if you see it not just as one the hardest, more challenging but also as one of the biggest inspiring experience in your life. Keep going bro, I'm proud! Jeannine, ay Jeannine! I hope that you will enjoy you return in the lab. I wish you the best with your family. Johanna, ay Johanna! my master's student XD. Thank you for your smile,

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enthusiasm and for the work you achieved in little time. Wish you the best for your next steps.

I would like to thanks all our collaborators involved in the projects. Particularly: Andreas Ladurner for its support since day one; Raffaele Teperino and Oscar Yanes for all their incredible work they performed for us; Ciro Rivera, Manjinder Cheema, Michelle Leger, Juan Ausio, Jose M. Eirin-Lopez, Iñaki Ruiz-Trill for their enthusiasm essential for the development of our evolution project. Thank you, Sonia Forcales and Pura Muñoz Cánovez, for your help and supervision.

Muchas Gracias Mar, para tu felicidad, para tu ayuda y para tus besos, un beso a Zoe. Yaiza... merci pour tout, merci pour ton soutient, ton amitié, je t'embrasse fort et merde pour la suite! Erika, muchas gracias para tu apoyo también y tu sonrisa, me ha encantado nuestras conversaciones. Thanks to all members of Fumi's lab. Your support, laughs, smiles, jokes and joy were a real inspiration. Thank you Fumi, Miyako and Emili!

Muchas Gracias a toda la gente que gestiona el mantenimiento como los recepcionistas y las señoras de limpieza (Irina, Awilda, Maribel, Emi, Oliver, Pala, Suguey). Nos olvidamos muchas veces de ellos, pero son los que te hacen reír, especialmente cuando estas abajo.

Gracias inmenso para las Saranas. Saranas = Sarah + Ana y más con un gran S :). He integrado ese grupo ese día cuando me has invitado a vivir contigo. Desde ese día, mi tiempo en Barcelona se ha mejorado cada día a vuestro lado. Ana, te quiero, muchas gracias para todo, todo y todo. Vivir contigo ha sido una de las mejores experiencias de mi vida. Hemos pasado por tantas cosas... sobre todo momentos maravillosos. Guapa te mereces lo mejor. Marta, ay Marta :) eres una persona en oro, unas de las grandes. Tu amistad tiene mucho valor para mí. Tu apoyo ha sido tremendo. Gracias para hacerme vivir esos momentos inolvidables y hacer parte de vida. ¡Te quiero mucho! Mucha gracias a tu prima Marta también, escuchar sus canciones durante la 14

tesis ha sido un suporte infalible. Anna, una relación que se ha reforzada poco a poco. Haces parte de la gente con quien quiero vivir más, más momentos graciosos y de locura. Tus masajes son geniales ya, así que guardo tu contacto jajaja. Melisa, ay mi amor, una otra mujer que nos da mucha inspiración. Te mereces el mejor también, y la ciencia tiene mucha suerte tener gente dedicada como tú. Alfonsina oyy la loca mona :) tu energía es una fuente de felicidad. Me encanta tu personalidad y me encanta siempre estar contigo. Good luck for the PhD guapa! Laura, una otra loca jijij. ¡Me ha encantado vivir todos estos momentos de locura y de felicidad contigo, gracias! Suerte con la tesis y los próximos pasos. Inés, una personalidad fuerte, es lo mínimo que podemos decir sobre ti. Gracias para las risas que me has dado con tus comentarios fuertes. Miriam, se siente siempre una paz alrededor de ti, gracias para tu calma y tus sonrisas. Raquel, ay Raquel! más de un año ya :) has entrado en mi vida como una bomba también. Tu energía, tu creatividad, tu arte, tus canciones, tus fotos, tu amor para la ciencia etc... me da mucha inspiración. Tendrás un papel en mi futuro, eso espero ¡Te quiero! Bea, ay Bea! un rayo de sol, una alegría imparable y una sonrisa increíble. Me encanta tu manera de ver la vida, nuestras conversiones son preciosas.

Blancita, casi 3 años viviendo contigo, hemos vivido muchas juntas. ¡Me reí tanto contigo! hoy estamos pasando a una nueva etapa de nuestra vida. I wish you all the best jefa! Un abrazo enorme. Miriam, pfff una persona preciosa, muchas gracias para tu ayuda para el piso y gracias para ser quien eres.

Thank you very much Luidmila for all these years of friend relationship. I am so glad that you find love with this great guy, Javi. You are both awesome. I enjoy every moment with you!

Let's go to the French part of my life. Sonia, Hélène, Sophie, Amandine, Elise, Laura, Cécile, Laure et Mathilde merci! Je vous ai déjà toutes remerciées dans mon mémoire de master mais encore une fois merci. Malgrè la distance vous êtes toujours là, sans faille. Le fait que certaines d'entre vous seront présentes à la thèse 15

me rempliera de bonheur. En vivant si loin de vous, j'en oublie mon Français, mais je n'oublie pas mon amour pour vous. Et puis... je reviens bientôt ;) Je vais enfin pouvoir me connecter à nouveau avec la vie de chacunes d'entre vous :) Je vous embrasse toutes très fort! Marguerite-Marie tant d'années ont passé et tu es toujours là. Merci pour ton soutien sans faille. Je n'ai pas toujours pu t'aider comme je l'aurai voulu. Je sais que tu passes par des moments difficiles. Je sais aussi que tu as la force en toi qui te permettra de te booster dans la bonne direction. Ne doute plus de tes capacités. Analyses tes compétences, utilises-les et combles les vides. Je crois en toi, tu auras toujours, toujours mon soutien!

Merci François pour tout. Notre amitié, depuis ce 13 Novembre 2015, n'a fait que se renforcer au cours des années. Ton soutien pendant toutes ces années à été très precieux. Je te souhaite le meilleur, surtout de trouver ta futur carrière qui te fera vibrer. Miriam, la petite dernière :) rencontrer des gens comme toi est un vrai plaisir. Il nous reste beaucoup à apprendre l'une de l'autre, mais je te dis déjà merci pour ton energie, ton dynamisme et ton intelligence qui inspire.

Merci à toute ma famille qui me soutient dans tous les choix que j'ai pu faire. Merci H pour faire partie de cette famille. Merci pour être venu me chercher à l'aéroport à chaque fois avec les pancakes qui attendent bien au chaud. Merci pour l'amour que tu donnes à ma sister. Le soutien sans limite d'une mère pour une telle aventure est la meilleure force motrice qui m'a portée jusqu'ici. Être loin de toi n'est pas facile, mais obtenir ce que j'ai obtenu, ici, n'a pas de prix. À bien des niveaux, maman, tu es une inspiration pour moi. Ton amour inconditionel, ta forte personalité, a forgé la personne que je suis aujourd'hui. Maman, ma vie me rend heureuse, merci pour le cadeau que tu m'as fait il y a de ça bientôt 29 ans. Line, il n'y a pas plus grande inspiration pour moi que toi. Tu es brillante, talentueuse, intelligente, travailleuse, créative, exigeante, etc. Tu es ma soeur et j'en suis fière! Tu m'as sauvé les fesses pour ma thèse de nombreuses fois. Je mets toute ma confiance dans tes compétences pour monter notre projet. Qui d'autre aurait la folie de me suivre dans ce délire sinon toi. Je vous aime! 16

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Abbreviations...... 25

Introduction ...... 29

I | Epigenetic ...... 31

| 1.1 | Epigenetic concept and definition ...... 31

| 1.2 | Trans- versus cis- regulation ...... 33

| 1.3 | Chromatin ...... 33

| 1.4 | Nucleosome and Post-transcriptional Modifications ...... 34

1.4.1 ...... 34

1.4.2 Histone variants ...... 36

II | Skeletal Muscle ...... 38

| 2.1 | Skeletal Muscle Myogenesis ...... 38

2.1.1 Prenatal and Adult myogenesis ...... 39

2.1.2 Myogenesis at the molecular level ...... 39

2.1.3 Muscle regeneration and adult hypertrophy ...... 41

| 2.2 | Molecular regulation of myogenic cell fusion ...... 42

2.2.1 Extracellular matrix composition ...... 42

| 2.3 | Skeletal Muscle Fiber Type and Metabolism ...... 45

2.3.1 Contractile proteins ...... 45

2.3.2 fiber type composition ...... 45

2.3.3 fiber type metabolism ...... 46

| 2.4 | Skeletal muscle pathology ...... 48

2.4.1 Skeletal muscle dystrophy ...... 48

2.4.2 Cancer related to muscle ...... 48

Table of Contents

III | PARP1 and NAD+ metabolism ...... 51

| 3.1 | PARP1 is essential for DNA repair ...... 51

3.1.1 PARP structure ...... 51

3.1.2 PARP mechanism in DNA repair ...... 52

3.1.3 PARP role in DNA repair in vivo ...... 54

| 3.2 | PARP1 physiological relevance in skeletal muscle metabolism ...... 54

| 3.3 | Cellular metabolism and NAD+ maintenance ...... 55

3.3.1 Energy production ...... 55

3.3.2 NAD is placed at central position in cellular metabolism ...... 56

3.3.3 NAD replenishment ...... 57

3.3.4 Essential for subcellular NAD+ balance ...... 58

3.3.5 Plasticity in NAD+ compartmentalization ...... 60

3.3.6 Interconnection between NAD+ and PARP1 activity ...... 60

| 3.4 | The physiological relevance of NAD+ in skeletal muscle ...... 61

IV | MacroH2A histone variant ...... 62

| 4.1 | MacroH2A structure ...... 62

4.1.1 Histone Fold Domain and Linker ...... 63

4.1.2 Macrodomain ...... 64

| 4.2 | MacroH2A function ...... 65

4.2.1 MacroH2A is a transcriptional repressor ...... 65

4.2.2 MacroH2A role in genome stability and DNA repair ...... 66

4.2.3 MacroH2A is key for the maintenance of differentiation state ...... 67

4.2.4 Role of macroH2A in cancer malignancy ...... 67

4.2.5 Mice Development and Metabolism ...... 69 19

Results ...... 71

Chapter I ...... 73

MacroH2A1s switch during myogenesis at the mRNA level ...... 74

The chromatin component macroH2A1.1 switch during myogenesis at the protein level ...... 76

MacroH2A1 reduction partially impacts muscle formation but does not impair differentiation ...... 78

MacroH2A1.1 reduction impacts cellular metabolism ...... 80

MacroH2A1.1 silencing impacts mitochondrial activity ...... 82

Mitochondrial defect is independent of macroH2A1.1 gene targets ...... 84

MacroH2A1.1 binds and inhibits PARP1 activity through its macrodomain88

PARP1 inhibition rescues mitochondrial activity ...... 90

Loss of PARP1 inhibition impairs NAD+ salvage ...... 92

Supplementation of NAD+ precursor rescue mitochondrial defect ...... 96

Conclusion Chapter I ...... 98

Chapter II ...... 99

MacroH2A1 isoforms regulate late differentiation markers in an opposite manner ...... 100

MacroH2A1 isoforms modulate myotube formation through fusion ...... 104

MacroH2A1’s dual role in gene regulation ...... 106

Opposingly regulated genes include a subset encoding extracellular matrix components ...... 110

PARP1 is not involved in the fusion phenotype ...... 114

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PARP1 does not regulate candidate genes for fusion phenotype ...... 116

Myogenic program is impaired in muscle- derived cancer ...... 118

MacroH2A1’s switch is impaired in muscle-derived cancer ...... 120

MacroH2A1.2 silencing slightly rescues myogenic marker expression, but is not enough for full fusion recovery ...... 122

Conclusion Chapter II ...... 124

Chapter III ...... 125

MacroH2A appeared at the node towards multicellularity and is conserved throughout evolution ...... 126

Generation of Caps. owc. macroH2A and PARP1 antibodies ...... 130

Capsaspora owczarzaki macroH2A inhibits PARP1 activity ...... 132

Conclusion Chapter III ...... 134

Discussion ...... 137

I | MacroH2A1.1-PARP1 axis regulates NAD+ metabolism and metabolic homeostasis ...... 139

| 1.1 | MacroH2A1.1-PARP1 axis ...... 139

| 1.2 | MacroH2A1.1-PARP1 axis regulates NAD+ pool ...... 141

| 1.3 | MacroH2A1.1-PARP1 axis in vivo ...... 142

II | Dual role of macroH2A1 isoforms on transcriptional regulation ...... 143

| 2.1 | Dual role in cancer phenotype ...... 143

| 2.2 | Dual role in transcriptional regulation ...... 145

III | Dual role of macroH2A1 isoforms in muscle development ...... 146

| 3.1 | Opposite role at the transcriptional levels ...... 146

| 3.2 |Switch of macroH2A-containing chromatin during differentiation ... 147

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| 3.3 | Is any phenotype related to C2C12 heterogeneity? ...... 148

| 3.4 | Role of macroH2A1 isoforms in fiber composition in vivo ...... 149

IV | Is ADP-ribose the ancestral role of macroH2A? ...... 150

| 4.1 | Capsaspora owczarzaki macroH2A-PARP1 axis ...... 150

| 4.2 | macroH2A role in Capsaspora owczarzaki ...... 151

| 4.3 | Ancestral role in virus and pathogen recognition ...... 152

Materials & Methods...... 155

Annex...... 199

Bibliography ...... 211

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Abbreviations

Abbreviations

DNA: deoxyribonucleic acid SWI/SNF: switch/sucrose non-fermentable RNA: ribonucleic acid SNF2-N: snf2 family proteins gDNA: genomic DNA Sec14p: sec14-like protein mRNA: messenger RNA CDK8: cyclin-dependent kinase 8 siRNA: small interfering RNA Lin28B: lin-28 Homolog B H3/H4/H2A/H2B: histone 3/4/2A/2B Ddx5/17: dead box helicase 5/17 PTMs: post-transcriptional modifications QKI: quaking homolog, KH domain RNA ac: binding me: MBNL1: muscle blind-like CENP-A: centromere protein A KO: knock-out PAX3/7: paired box protein 3/7 RFLP: restriction fragment length FOXO1: forkhead box protein O1 polymorphism bHLH: basic helix-loop-helix cDNA: complementary DNA MYOD: myogenic differentiation antigen WWE: tryptophan/glutamate-containing MYF: myogenic factor motif MEF2: myocyte Factor 2 HELICc: DEAD-like_helicase_C eMHC: embryonic myosin heavy chain Macro: macrodomain CKM: creatine kinase H2AFY: H2A Histone Family Member Y MD: muscular dystrophy H2AFY2: H2A Histone Family Member Y HDAC: 2 MLC: myosin light chain mH2A: macroH2A MYH: myosin heavy chain BRCA1: breast cancer RMS: rhabdomyosarcoma BRCT: BRCA1 C terminus domain SSBs: single strand break Zn: Zinc DSBs: double strand break HD: a-helical domain HR: homologous recombination WGR: Tryptophan-Glycine-Arginine NHEJ: non-homologous end joining domain ARTDs: ADP-ribosyltransferases AD: automodification domain PARPs: Poly(ADP-ribose) polymerases DBD: DNA binding domain PARP1: Poly(ADP-ribose) polymerase-1 NAD/NADH: nicotinamide adenosine PARP cat: PARP catalytic domain dinucleotide/ nicotinamide adenosine L1, L2: loop 1,2 dinucleotide with one hydrogen b: beta NMNAT: nicotinamide mononucleotide adenylyltransferase

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NAMPT: nicotinamide TCA: tricarboxylic acid cycle phosphoribosyltransferase ETC: electron transport chain NR: nicotinamide riboside FAD/FADH2: Flavin adenine dinucleotide/ NMN: nicotinamide mononucleotide Flavin adenine dinucleotide with 2 NAM: nicotinamide hydrogens NA: nicotinic acid CO2: carbon dioxide NADS: NAD synthase L-kin: L-kynurenine NAPRT: nicotinate 3-HAA: 3-Hydroxyanthranilic acid phosphoribosyltransferase ACMSD: α-amino-β- carboxymuconate-ε- cADPr: cyclic-ADP-ribose semialdehyde decarboxylase MAR: Mono–ADP-ribosylation QAPRT: quinolinate PAR: Poly–ADP-ribosylation phosphoribosyltransferase ADP: Adenosine diphosphate G3P: glyceraldehyde 3-phosphate ATP: Adenosine triphosphate FOXO1: forkhead factor 1 ARH3: ADP-ribosylhydrolase 3 PARG: Poly(ADP-ribose) glycohydrolase OAADPr: O-acetyl-ADP-ribose IDO: indoleamine 2,3-dioxygenase TDO: tryptophan 2,3-dioxygenase Trp: tryptophan AMPK: AMP-activated protein kinase Acetyl-CoA: acetyl coenzyme

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Introduction

Introduction

I | Epigenetic

| 1.1 | Epigenetic concept and definition

Establishment of multicellularity life was achieved by clonal division of a single cell, cell aggregation, differentiation and fusion of single cells (Sebé-Pedrós et al., 2013). Adaptation to environmental changes, maintenance of cell identity through cell division, and lineage specialization of multicellular organisms, require complex regulation of the genomic information. Although dividing and fully differentiated cells share the same genomic information (omitting somatic mutation) they manage to perform completely different functions. The classical example for this is the differentiation of totipotent cells from the zygote, which gives rise to multiple pluripotent cells forming muscle cells, neurons, epithelium, etc, of the embryo (Meissner, 2010). Such diversification in cellular function from single cell is driven by specific transcriptional programs orchestrated by epigenetic modifications. The term ‘epigenetic’ comes from the Greek prefix "epi" meaning "over" or "on top of". Thus, refers to the heritable and, most likely, inheritable information added "on top of" the genetic background without altering DNA sequence. Epigenetics events occur either during the normal organism development, or in response to environmental cues, and will affect their molecular, cellular, morphological, and physiological phenotypes (Burggren & Crews, 2014).

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In 1956, Conrad Hal Waddington discovered that development of embryo fruit flies could be manipulated by changing temperature condition or using chemical stimuli (Waddington, 1956). Characteristics acquired by the embryo fruit flies, in response to those stimuli, were found to be inherited by the next generation, which could not be explained by genomic mutation. In 1957, Waddington introduced the "epigenetic landscape" model in his book "The Strategy of the Genes" (Waddington, 1957) (Figure 1). This model summarizes the possible paths taken by totipotent cells to become terminally differentiated cells during embryonic development.

Figure 1. Epigenetic Landscape. The ball at the bottom represents a totipotent cell. This cell may cross several ridges and valleys down the slope that will shape its differentiation fate. Taken from Waddington, 1957.

Among others, Bonasio et al., define epigenetic as the information that “resides in self- propagating molecular signatures that provide a memory of previously experienced stimuli, without irreversible changes in the genetic information."(reviewed in Bonasio et al., 2010). In 1975, Riggs, Holliday and Pugh speculated that stable differentiated states were defined by cellular methylation state in the absence of genetic mutation (reviewed in Riggs, 1975; and in Holliday & Pugh, 1975). Since then, investigation of epigenetic mechanisms has largely expanded. Today, we know that epigenetic

32 mechanisms include non-coding RNA, transcription factors, ATP-

dependent remodelling proteins, DNA methylation, histone modifications and histone variants.

| 1.2 | Trans- versus cis- regulation

Epigenetic signals are divided by trans- and cis- regulation act on specific DNA sequences they previously recognized. Trans-epigenetic refers to transient self- propagating transcriptional activity (reviewed Bonasio et al., 2010) mainly sustained by transcription factors and few non-coding RNAs (reviewed in Sassone-Corsi & Borrelli, 1986; and in Kopp & Mendell, 2018). Cis-regulatory elements, such as enhancers and promoters, contain binding sites for transcriptional factors and other regulatory molecules to control transcriptional activity (reviewed in Wittkopp & Kalay, 2012). Cis-regulation includes the covalent post-modifications of DNA itself by methyl groups on the histones proteins, and the exchange of canonical histones by histone variants (reviewed in Bonasio et al., 2010).

| 1.3 | Chromatin

All epigenetic processes are regulated at the chromatin level in eukaryotic cell. Almost two meters of genomic DNA are packed in the nucleus measuring only few micrometers (reviewed in Khorasanizadeh, 2004). Two levels of packaging are necessary to achieve this feat. First, approximately 147 base pairs of DNA are wrapped around an octamer of the four negatively charged canonical histones H3, H4, H2B and H2A incorporated into DNA by specific chaperones (reviewed in Kornberg, 1977). Histones are the protein’s backbone around which DNA is wrapped. Histone proteins together with DNA form a complex called the chromatin. Dynamic reorganization of chromatin architecture during all life stages is crucial for cell fate decision and maintenance (reviewed in Meister et al., 2014). Indeed, different degrees of chromatin condensation, as well as

33 its specific localization, will determine the accessibility to transcriptional

regulators and chromatin remodelers (reviewed in Khorasanizadeh, 2004). The eukaryotic genome is compartmentalized into two chromatin categories. The first one, called euchromatin, corresponds to an open chromatin that is accessible to transcription machinery allowing gene activation. The second, heterochromatin is composed of closed chromatin structure poorly accessible to transcription factors leading to gene silencing (reviewed in Passarge, 1979).

| 1.4 | Nucleosome and Post-transcriptional Modifications

This first level of packaging forms the basic unit of chromatin is called the nucleosome (Luger et al., 1997). are found approximately every 200 base pairs (bp) in the DNA and generate what are called "beads-on-a-string" fibers of 11 nm in length. This structure is further packaged thanks to the H1 linker histones and constitutes the 30 nm fiber which promotes a higher-degree of chromatin compaction (reviewed in Kornberg, 1977).

1.4.1 Histone code

Flexible histone tails at the N-terminus, protruding out of the nucleosome, can be modified by chemical groups such as methylation, acetylation, , ubiquitination, SUMOylation, citrullination, ADP-ribosylation, etc (reviewed in Bannister & Kouzarides, 2011). The combination of those reversible post-modifications (PTMs), called the "histone code", modulates inter-nucleosomal contacts and shapes chromatin landscape by providing new binding sites for non-histone proteins (reviewed in Jenuwein & Allis, 2001). The histone code is constituted by active, repressive and bivalent histone marks. Chromatin landscape is covered by numerous histone marks that can act together by having an additive effect on each other. They can also act against

each other by antagonizing their respective function. Finally, bivalent marks

34 can act reversely at the same loci, but at distinct time points, depending on

the stimuli received (Bernstein et al., 2006; reviewed in Barth & Imhof, 2010) (Figure 2). Beyond their role in transcription, histone PTMs are also associated with DNA replication and DNA damage (reviewed in Zhao & Garcia, 2015).

Figure 2. Simplification of histone marks distribution regulating gene expression. Histone modifications are represented across the promoter region, TSS (transcriptional start site), and gene body. Active (A), repressive (B), and bivalent histone marks (C) are represented. Abbreviation is used for the nomenclature of all histone modifications. As an example, H3K9ac refers to an (ac) added to the 9 (K9) of Histone 3 (H3). Adapted from Butler & Dent, 2013.

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1.4.2 Histone variants

Another drastic modification at the nucleosome level is the exchange of replicated- coupled histone by histone variants. Replicated-coupled histones are encoded in tandem array from genes organized in clusters and their deposition into chromatin is coupled to replication. In contrast, histone variants are expressed from single-copy genes and are incorporated into DNA independently of replication (reviewed in Buschbeck & Hake, 2017). Histone variant expression and genomic deposition are spatially and temporally regulated. As for PTMs, dynamic regulation of histone variants extensively impact cellular processes such as transcription, DNA repair and DNA replication (reviewed in Biterge & Schneider, 2014). Six variants of histone H3 are described (H3.3, CENP-A (centromeric protein-A), H3.Y, H3.X, H3.1t and H3.5.); two histone H2B variants (TSH2B and H2BFWT) and eight variants were found for histone H2A (H2A.X, H2A.Bbd, H2A.Z.1, H2A.Z.2.1, H2A.Z.2.2, macroH2A1.1, macroH2A1.2 and macroH2A2) So far, no variant has been found for in mammals (reviewed in Buschbeck & Hake, 2017) (Figure 3).

Figure 3. Mammalian replication-coupled histones and histone variants. Schematic representation of human replication-coupled histones and histone variants of histone H2A (yellow), H3 (blue), H2B (orange) and H4 (green). The rectangles represent the core regions and histone tails are represented by the lines. The percentage of conservation of the protein sequence of histone variants compare to the corresponding replication-coupled histone is indicated. At the central position nucleosome structure is shown with DNA wrapped around the octameric histone core. Taken from 36 (Buschbeck & Hake, 2017).

Although many PTMs are conserved between canonical histones and histone variants, histone variants have been decorated by new PTMs throughout evolution (reviewed in Zhao & Garcia, 2015; and in Corujo & Buschbeck, 2018). As a consequence, together with their specific post-modifications, histone variants affect nucleosome stability and endow chromatin with new structural and biophysical properties.

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II | Skeletal Muscle

Skeletal muscle is the largest tissue in the human body, accounting for 40% of total body mass (reviewed in Frontera & Ochala, 2015). In humans, overall more than 600 fully differentiated skeletal muscle are found to be vital for organism survival (reviewed in Tieland et al., 2018). Indeed, skeletal muscle is essential for (1) body posture, locomotion and breathing (2), whole-body energy expenditure and metabolism, and (3) muscle hormones and cytokines secretion essential for the interconnection with other organs such as brain, liver, fat and pancreas (reviewed in Frontera & Ochala, 2015; in O’Neill, 2013; and in Pedersen & Febbraio, 2012). Muscular impairment critically affects lifespan and quality of life of the patients. In order to offer new therapy strategies, our field needs to further understand the mechanisms controlling skeletal muscle formation, degeneration, regeneration and function. Human muscles are divided into three types of muscle. Skeletal muscle is the major muscle type, the two other are cardiac and smooth muscles. Skeletal muscles are voluntary muscles controlled by the nervous system. They are composed of long striated terminally differentiated multinucleated myofibers sticking together. Those myofibers consist of elongated multinucleated cells formed by the fusion of multiple cells during a process called myogenesis.

| 2.1 | Skeletal Muscle Myogenesis

In this section, we will briefly introduce the first steps of muscle progenitor commitment to differentiation followed by fusion waves giving rise to the mature myofibers. For decades, muscle maturation has been studied in vivo using animal models (drosophila, chicken and mouse mainly) or in vitro with different cell lines (primary

38 myoblasts or C2C12 cells) recapitulating myogenic program. However,

many processes are still poorly understood, particularly in the late stages of muscle fusion, and therefore this field requires further investigation.

2.1.1 Prenatal and Adult myogenesis

Two main phases of myogenesis allow the proper formation and maintenance of skeletal muscle throughout the organism's life. The first phase is called prenatal myogenesis which forms myofibers de novo during development. The second one, called adult myogenesis occurs during postnatal growth for the maintenance of skeletal muscle mass and during regeneration upon acute muscle damage (White et al., 2010) (Figure 4).

Figure 4. Embryonic and postnatal muscle development in mouse. Prenatal myogenesis takes place from embryonic day 12 (E12) to postnatal day 0 (P0) including embryonic and fetal stages. Later on, postanal hypertrophy and regeneration maintain adult muscle. Activation of muscle progenitor cells is dependent of Pax3 and Pax7 expression. Postnatal myogenesis is initiated by the expression of Pax7 in satellite cells. Adapted from (Relaix & Zammit, 2012).

2.1.2 Myogenesis at the molecular level

During prenatal myogenesis, skeletal muscle of the trunk and limbs arises from the dorsal portion of the somites called the dermomyotome (reviewed in Relaix & Zammit, 2012a). In the somites, muscle progenitors are firstly activated in response to Wnt and Sonic hedgehog signaling pathways (reviewed in Tajbakhsh & Buckingham, 1999). Once activated, they start to express the paired box transcription factors PAX3 and PAX7, accompanied by a low expression of the basic helix–loop–helix transcription factor Myf5

(reviewed in Bentzinger et al., 2012). At late fetal stage, specification of the 39

myogenic lineage will then be established by the downregulation of PAX3 expression and up-regulation of Myf5. This drives the myogenic progenitors to migrate close to the forming muscle fibers.

Myogenesis is characterized by the sequential activation of transcription factors which drives the differentiation of precursors cells towards fully differentiated myofibers. The first skeletal muscle differentiation is driven by the sequential activation of four myogenic regulatory factors (MRF) and the myogenic enhancer factor 2 (MEF2) (reviewed in Buckingham, 1992; and in Yun & Wold, 1996). MRFs are members of a family of DNA-binding proteins containing a basic helix-loop-helix domain (bHLH) binding the E box DNA motif. Among the four MRFs, MYF5 drives the sequential activation of the three-other including MRF4 (also known as MYF6), MYOD and finally MYOG (reviewed in Buckingham, 1992; and in Braun & Gautel, 2011; Cao et al., 2011 ). Mature myofibers express myosin heavy chain expression (MHC) and late marker like the creatine kinase (CKM) (reviewed in Yun & Wold, 1996).

Figure 5. Schematic representation of skeletal muscle myogenesis Stem cells differentiate and fuse to form multinucleated myofibers through sequential expression of myogenic transcription factors. Inspired from (Hindi, Tajrishi, 40 & Kumar, 2013).

While most of these factors have redundant function, myogenin was shown to be the only gene strictly required for fetal muscle differentiation (Venuti et al., 1995; Knapp et al., 2006). MEF2 are part of a family of transcription factors that recognize A/T-rich elements present in regulatory elements of cardiac and skeletal muscle genes. At least four MEF2 members exist in vertebrates including MEF2A, MEF2B, MEF2C and MEF2D. MEF2 transcription factors interact with MRF transcription factors and activate the expression of differentiation-related genes (reviewed in Black & Olson, 1998). Lineage specification and differentiation were extensively studied thanks to different knockout approaches performed in vivo and in vitro. However, the critical event of myoblast fusion to another preexisting primary fibers is poorly studied and therefore remains elusive. In this context, many differentiation/fusion phenotypes were analyzed together without proper distinction of the two processes (reviewed in Sampath et al., 2018).

2.1.3 Muscle regeneration and adult hypertrophy

Quiescent stem cells, called satellite cells, are located beneath the basement membrane juxtaposed to myofibers. They express both PAX3 and PAX7 but no skeletal-muscle- specific markers (reviewed in Buckingham & Relaix, 2007). Once activated by injury, satellite cells re-enter the cell cycle to differentiate or self-renew, which is essential for muscle growth, muscle maintenance and regeneration (Asakura et al., 2003; reviewed in Relaix & Zammit, 2012). Injury provokes the release of cell contents into the blood which then initiate muscle repair. Muscle repair is characterized by necrosis of the damaged myofiber cell membrane activating the inflammatory response (reviewed in Rudnicki & Charge, 2004). Once activated, satellite cells differentiate either into adipocytes, osteoblasts or myoblasts (reviewed in Seale & Rudnicki, 2000; Aguiari et al., 2008). Satellite cells activation is determined by environmental stimuli transmitted to their niche. Satellite cells niche is composed of the basement membrane and the cell membrane of myofibers (also called sarcolemma).

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In adult, muscle hypertrophy may result in increased myofibers number or in size, or by a combination of both. In this case, new myonuclei are generated from the resident stem cells of skeletal muscle (White et al., 2010).

| 2.2 | Molecular regulation of myogenic cell fusion

From embryonic development to adulthood cell-cell fusion is critical for all mammalian life forms. Among others, cell fusion process includes sperm-oocyte fusion, placenta trophoblast fusion, phagocytosis, axon growth, multinucleate osteoclasts formation and skeletal muscle formation and regeneration (reviewed in Chen & Olson, 2005; and in Chen et al., 2007). So far, three main steps are commonly accepted to describe fusion of two fusion competent myoblasts (1) cell-cell recognition and adhesion (2) membrane apposition through actin protrusion and (3) formation of pores in the plasma membrane to exchange cytoplasmic material (reviewed Kim et al., 2015). Several other processes are required for those three-steps including muscle-specific transcription, cell elongation and migration, extracellular matrix and cytoskeleton remodeling, cell- cell communication and signaling, exosomes trafficking and secreted factors (reviewed in Hindi et al., 2013; and in Demonbreun et al.,2015). Although myogenesis has been widely studied, little is known about the components involved in muscle fusion. In this thesis, we will focus on extracellular matrix and cytoskeleton remodelling.

2.2.1 Extracellular matrix composition

Skeletal myofibers are coated with a layer of extracellular matrix (ECM) called the basement membrane. Focal adhesion is a protein-complex that mechanically links the cytoskeleton of myofibers to extracellular matrix essential for muscle contraction (Figure 6). Both ECM and focal adhesion are essential for contractile capacity, myogenesis and regeneration capacity of myofibers (reviewed in Thorsteinsdottir et al., 2011). In vitro, culture of primary myoblast coated on different ECM composition leads to

2 distinct myogenic features (Wilschut et al., 2010). In vivo, ECM, which 4

represents 1-10% of muscle mass, creates a supportive framework required for muscle function (reviewed in Grzelkowska-Kowalczyk, 2016). Furthermore, ECM components are involved in skeletal muscle fusion by orchestrating muscle growth, elastic and motor capacity, and cell metabolism. ECM surrounding myofibers is composed of collagens, laminins, fibronectin and proteoglycans (Figure 6). Polymerization and assembly of the matrix are controlled by transmembrane receptors. Transmembrane proteins connect the extracellular matrix (ECM) to the cytoskeleton and regulate the transduction of developmental and environmental cues from the extracellular matrix to the nucleus (reviewed in Schwartz, 2001). Focal adhesion, also known as cell-matrix adhesions, is a complex of multi-protein mainly composed of actin and myosin (Quach et al., 2010). Actin filaments are polymers of globular actin subunits. Myosins are a superfamily of ATP-dependent motor proteins that walk along actin filaments to generate muscle force (reviewed in Kneussel & Wagner, 2013) Thus, the interaction of the filamentous actin with myosin form the basic unit of the contractile apparatus in muscle (reviewed in Dominguez et al., 2011). Mechanical stress through the cytoskeleton, and intra- and extra-cellular signals are transmitted to myofibers membrane by a complex network of transmembrane proteins. This protein network is mainly composed by integrins and calcium-dependent adhesion molecules called cadherins (reviewed in Simionescu & Pavlath, 2011). Cadherins are essential for cell-cell adhesion by regulating adherens junctions. Integrins are heterodimeric receptors consisting of alpha subunits and a beta1 integrin subunit. The transmission of integrins signal goes through the activation of focal adhesion kinase which is a non-receptor tyrosine kinase localized at focal adhesions (reviewed in Grzelkowska-Kowalczyk, 2016).

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Figure 6. Simplified representation of the Extracellular Matrix and Focal adhesion. ECM is composed of collagen fibers surrounded by glycoproteins, fibronectin and connected to the actin cytoskeleton by the integrins. FAK: Focal Adhesion Kinase. Inspired by Turner, 2000 and Xue & Jackson, 2015.

ECM-Focal adhesion structures are largely involved in myofiber regeneration (reviewed in Grzelkowska-Kowalczyk, 2016). Indeed, injury provokes the release of calcium, nitric oxide synthase (NOS), growth factors like FGF and HGF (Sheehan & Allen, 1999), and inflammatory cytokines (reviewed in Smith et al., 2008). Satellite cells sense changes in stiffness of their niche thanks to the network of integrins located in the ECM. This will allow the quiescent satellite cells to get activated and differentiate towards myogenic precursor cells (reviewed in Grzelkowska-Kowalczyk, 2016). In healthy conditions, ECM composition is widely remodeled during muscle development. For instance, myoblasts favor the expression of fibronectins while myotubes favor laminin production (reviewed in Grzelkowska-Kowalczyk, 2016). The expression of α-integrins subunits is also modulated during myogenesis (Knoblauch et al., 2007). Deregulation of any of these components interferes with myoblasts differentiation and myofiber formation (reviewed in Sanes, 2003). For instance, focal adhesion kinase inhibition leads to aberrant transcriptional activity of transmembrane

44 proteins preventing proper myoblast fusion (Quach et al., 2010).

| 2.3 | Skeletal Muscle Fiber Type and Metabolism

2.3.1 Contractile proteins

Motor function of skeletal is sustained by contractile proteins that are organized in myofibrils. Myofibrils are composed of contractile proteins including actin, myosin and titin, which are found in the focal adhesion. Contractile proteins, organized in thick (myosin) and thin (actin) consist of myofilaments. Myofilaments are divided into sections called sarcomeres in a striated pattern (Gilëv, 1962). Myosins consist of one or two heavy chains and four calmodulin-related light chains (two essential light chains, and two regulatory light chains) (reviewed in Clark et al., 2007) (Figure 7).

Figure 7. Molecular organization of myosin II. Myosin II is constituted of two heavy chains in blue which bind two calmodulin-related light chains in green. Ligh chains are composed of one essential light chain and one regulatory light chain. MHCs and MLCs homodimerize and are linked to the C terminus holds a short non-helical region (red) via the long coiled-coil domain (purple). Taken from Kneussel & Wagner, 2013.

2.3.2 fiber type composition

Fiber type composition will be characterized by which myosin light chain (MLC), or myosin heavy chain (MHC) subunits, are expressed. Different fiber type will have different contractile capacity. Indeed, fiber type is classified in type I or type II which perform slow (slow-twitch) or fast contraction (fast-twitch), respectively. In mammals, myosin genes are divided into thirteen classes I, II, III, V, VI, VII, IX, X, XV, XVI, XVIII, XIX and XXXV (reviewed in Kneussel & Wagner, 2013). In skeletal muscle, type I expresses MYHC-7 (Myh7) while type II expresses MYHC-2A, 2X and 2B (Myh2, Myh1 and Myh4). In humans, hybrid fibers with mix composition exist too, such as I/IIA;

IIA/IIX; IIX/A; IID/B and IIB/IID (reviewed in Schiaffino & Reggiani, 45

2011). However, fiber type composition is driven after birth by transient expression of the embryonic myosins (MYH3), neonatal myosins (MYH8) and the light chain myosin 4 (MYL-4). Those three myosins are also re-expressed during the regeneration process after injury or in myoblast cell lines such as C2C12 upon differentiation (reviewed in Schiaffino et al., 2015).

Myosin Type Gene Expression Myosin Heavy Chain MyHC-emb MYH3 Extraocular, masticatory, MyHC-neo MYH8 laryngeal and spindles muscles MyHC-slow MYH7 Slow I fibers MyHC-2A MYH2 Fast 2A fibers MyHC-2X MYH1 Fast 2X fibers MyHC-2B MYH4 Fast B fibers Essential myosin Light Chain MLC-1fast MYL1 Fast muscles MLC-3fast MYL1 Fast muscles MLC-1emb/atrial MYL4 Atria MLC-1sb MYL3 Slow muscles & ventricles MLC-1sa MYL6B Slow muscles, not ventricles in human

Regulatory Myosin Light Chain Fast muscles MLC-2fast MYLPF Slow muscles & ventricles MLC-2slow MYL2

2.3.3 fiber type metabolism

Beside their function in motion, skeletal muscle controls whole-body thermoregulation by regulating muscle metabolism. Indeed, skeletal muscle senses and heavily consumes glucose and fatty acids (reviewed in Goody & Henry, 2018). Although

they are energetically expensive, they are also able to store large amounts 46

of energy in the form of glycogen. Stable metabolic rate is crucial for a healthy organism. In this context, muscle is an incredible plastic organ that has the ability to switch its metabolism depending on the needs, stress or nutrient availability (reviewed in Schiaffino et al., 2015). All cellular processes are controlled by metabolic pathways which will convert food to energy. Briefly, the major metabolic pathways include glycolysis, gluconeogenesis, glycogen metabolism, fatty acid metabolism, citric acid cycle, oxidative phosphorylation and amino acid metabolism. In cells, metabolic plasticity refers to their capacity to switch from one metabolic pathway to another in response to distinct stimuli. In muscle, metabolic switch occurs in three contexts: (A) during the differentiation of proliferative myoblasts to myofibers, (B) during the activation of satellite cells, (C) and during the establishment of fiber type composition that will allow muscle to perform either short or long contraction (Leary et al., 1998). (A) Myoblasts mostly rely on glycolysis to ensure the high energetic demand from nucleic acid generation during DNA replication. On the contrary, terminally differentiated myofibers mostly rely on oxidative metabolism to maintain proper cellular functions and respond massively to oxidative stress exercise, injury or aging (Oláh et al., 2015). (B) Quiescent satellite cells switch oxidative pathway towards glycolysis and glutaminolysis when they get activated (reviewed in Goody & Henry, 2018). Any of these metabolic switches require complete shift of the transcriptional regulation of metabolic- related genes (Fukada et al., 2007). (C) Oxidative myofibers are characterized by a high number of mitochondria with high degree of vascularization. They are highly resistant to fatigue due to their capacity to produce slow but long lasting contraction (slow twitch), making them ideal for long endurance exercises. On the contrary, glycolytic myofibers are characterized by low number of mitochondria and fast twitch contraction. Those myofibers handle short but intense exercises (reviewed in Schiaffino et al., 2015). Fiber composition may be modulated by exercise, acute injury and aging. Muscle plasticity is disrupted by genetic disease, chronic injury and aging.

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| 2.4 | Skeletal muscle pathology

2.4.1 Skeletal muscle dystrophy

Muscular dystrophy (MD) is a group of muscle diseases characterized by progressive skeletal muscle weakness and degeneration due to at least 30 distinct genetic disorders (Gao et al., 2016). Distinct MD will appear either during childhood or adulthood. Many patients may develop difficulties to walk or have severe respiratory or cardiac failure (reviewed in Houang et al., 2019). MD is divided into nine main categories. Among them we can find Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic dystrophy (reviewed in Messina & Vita, 2018; in LoRusso, Weiner, & Arnold, 2018; and in Lu et al., 2019). All of them are due to the mutation of either genes encoding dystrophin protein or enzymes modifying it. Dystrophin is an essential cytoplasmic protein for muscle integrity. Indeed, it drives the attachment and stabilization of the muscle cytoskeleton to the extracellular matrix of myofibers (Gao et al., 2016). So far, no cure for MD exists, but gene repair strategies will most likely offer the best therapy. While epigenetic disorders are poorly studied in MD, several findings support the implication of DNA methylation or acetylation in the MD phenotype. Thus, pharmacological inhibition of deregulated epigenetic regulators may offer an alternative, or at least reduce disease progression. For instance, pharmacological inhibition of the silencer HDACs (histone deacetylases) promotes muscle differentiation in vitro and in vivo (Iezzi et al., 2002; Minetti et al., 2006).

2.4.2 Cancer related to muscle

2.4.2.1 Rhabdomyosarcoma phenotype

Soft tissue sarcoma represents around seven percent of cancer in children and one percent in adults. Among this rare disease fifty percent comprise rhabdomyosarcoma (RMS) tumours. RMS is an aggressive and highly malignant neoplasm with

48 a 5-year survival rate of between 50 and 70% (reviewed in Hinson et al.,

2013). Although the cell origin of RMS is not well known, it is commonly accepted that most RMS arise from mesenchymal cells committed to the skeletal muscle lineage (reviewed in Marshall & Grosveld, 2012). However, RMS spread in any body parts with a preference for tissue lacking muscle such as the head, neck, extremities, trunk and genitourinary tract (reviewed in Saab at al., 2011). In line with potential skeletal muscle origin, RMS express muscle markers such as actin, myosin, myoglobin and the myogenic transcription factors PAX3, PAX7, MYOD and MYOG (Tonin et al., 1991). Although they express myogenic markers, RMS does not recapitulate the well- orchestrated transcriptional activity driving normal myogenesis. This leads RMS to undergo aberrant and incomplete differentiation and fusion processes.

2.4.2.2 Rhabdomyosarcoma classification

RMS is classified into two major histological subtypes, embryonal and alveolar RMS (reviewed in Ardnt & Crist, 1999). The most prevalent subtype is embryonal RMS which accounts for 60% of RMS, while alveolar RMS accounts for only 20%. Alveolar RMS subtype is more aggressive and is associated with poorer prognosis and higher metastasis rate (reviewed in Marshall & Grosveld, 2012).The incidence of both subtypes depends on age, gender and geographic localization (Skapek et al., 2018). Since the embryonal RMS genetic profile is not well established, this section will focus on alveolar RMS subtypes.

2.4.2.3 Alveolar rhabdomyosarcoma

Most alveolar RMS are characterized by recurrent translocation of chromosomes 1 or 2 with chromosome 13 (t(2;13)(q35;q14) or t(1;13)(p36;q14)). These two chromosomal translocations generate chimeric proteins PAX3-FOXO1 and PAX7-FOXO1, which are described as oncogenic. The PAX3-FOXO1 genetic alteration is found in 55% of alveolar RMS tumors while PAX7-FOXO1 represents 22% (Sorensen et al., 2002). As previously mentioned, the transcription factors PAX3 and PAX7 are expressed by muscle

progenitors for skeletal lineage specification. FOXO1 gene code for a 49

diversified family of forkhead transcription factors. FOXO1 is mainly associated with cell cycle and apoptosis, metabolism regulation, adipogenesis and myogenesis (Nakae et al., 2003, Bois & Grosveld, 2003). The main reason why alveolar RMS do not properly differentiate is because of the global reprogramming of the chromatin landscape due to PAX3/7-FOXO1 fusion (reviewed in Skapek et al., 2018). Indeed, the chimeric gene generate enhancers near target genes such as ALK, FGFR4, MYCN, MYOD1 and myogenin. The aberrant transcriptional regulation of those downstream target genes drives oncogenic behavior (reviewed in Skapek et al., 2018).

2.4.2.4 Fusion impairment in rhabdomyosarcoma

Interestingly, protein fusion is not detected in 23% of alveolar RMS which are similar to embryonal RMS tumors at the clinical and biological levels (reviewed in Sorensen et al., 2002; and in Skapek et al., 2018). Furthermore, germline loss of several tumor suppressors in mice seems to facilitate PAX3/7-FOXO1 occurrence (Keller et al., 2004). This suggests that PAX3/7-FOXO1 translocations are required for most of alveolar RMS cases but further disorders have to occur for alveolar RMS development (Keller et al., 2004).

2.4.2.5 Beyond genetic alteration

Beyond the genetic rearrangement several signaling pathways have been found to be deregulated in RMS. This includes mutations in key components of the RAS and STAT pathways, negative regulation of PI3K and Wnt pathways (reviewed in Skapek et al., 2018). However, very little is known about epigenetic involvement in RMS phenotype. Only recently, few studies discovered changes in global DNA methylation and histone marks at specific loci such as MYOD1 (Seki et al., 2015; Lee et al., 2011). Indeed, the histone methyltransferase KMT1A, which interacts with MYOD1, is upregulated in RMS (Lee et al., 2011). This leads to increased deposition of H3K9 methylation at MYOD1 target genes and, as a consequence, abrogate muscle differentiation. Several microRNAs

seem to be also implicated in the RMS progression (Missiaglia et al., 2017). 50

III | PARP1 and NAD+ metabolism

| 3.1 | PARP1 is essential for DNA repair

Adequate response to single strand breaks (SSBs) and double strand breaks (DSBs) require several steps: (1) detection of the DNA damage site, (2) chromatin relaxation, (3) recruitment of the repair machinery, (4) damaged DNA eviction, (5) DNA repair, (6) DNA end resection, (7) removal of the repair machinery and finally (8) chromatin condensation (reviewed Goodarzi & Jeggo, 2013). DSBs are repaired either through homologous recombination (HR) or non-homologous end joining (NHEJ). Both HR or NHEJ DNA repair mechanisms are partially executed by active ADP ribosyltransferases (ARTDs) called PARPs (Poly (ADP-ribose) Polymerase) (reviewed in D'Amours et al. 1999; Rouleau et al., 2010).

3.1.1 PARP structure

PARPs are highly conserved from bacteria to human (Citarelli et al., 2010; Daugherty, et al., 2014). Up to seventeen PARP enzymes were found in mammals (reviewed in Luo & Kraus, 2012). PARP enzymes are constituted of three domains: N-terminal DNA-binding domain (DBD); a central auto-modification domain; and a C-terminal catalytic domain (reviewed in D'Amours et al., 1999) (Figure 8). High zinc finger (F1 and F2) composition of the DBD allows PARP to act as a metalloenzyme which recognizes DNA DSBs. The capacity of PARP to strongly interacts with damaged and undamaged DNA is carried out by the two helix-turn-helix motifs (Figure 9). DNA damage, by which nuclear PARPs are activated, is generated by oxidation, alkylation, deamination, depurination, ionizing radiation and anticancerous agents (reviewed in D'Amours et al., 1999). When activated PARP transfers ADP moieties to protein targets and 51

auto-modifies itself on BRCA1 C-terminal (BRCT)-containing domain. Involvement of PARPs in DNA repair is best understood with the well characterized PARP1. PARP1 is the most abundant PARP member in eukaryotic nuclei and responsible for 85-90% of all PARP activity (reviewed in D'Amours et al., 1999; and in Vida et al., 2017)

Figure 8. Simplified representation of PARP1 structure. PARP1 is composed of six domains: zinc finger 1 (Zn1), zinc finger 2 (Zn2), zinc finger 3 (Zn3), BRCA-1 C-terminus fold (BRCT), Tryptophan-Glycine-Arginine domain (WGR) and a catalytic domain (CAT). The catalytic domain consists of an alpha-helical subdomain (HD) and an ADP- ribosyltransferase domain (ART). Taken from Steffen al., 2016.

Figure 9. PARPs remodelling and activation during DNA damage. Global PARP1 structure changes upon DNA damage. Zn1 and Zn2 bind to the ends of damage DNA. This interaction is communicated to the catalytic domain by Zn3. This leads to the complete activation of the catalytic domain, via destabilization of the HD domain, by increasing ART accessibility for NAD+. Adapted from Steffen et al., 2016.

3.1.2 PARP mechanism in DNA repair

Briefly, interaction with damaged DNA mediates conformational change of PARP1 which increases its affinity towards nicotinamide adenine dinucleotide (NAD+). Break down of NAD+ metabolite generates nicotinamide (NAM) and ADP ribose moieties (Steffen al., 2016). Newly synthesized ADP ribose are transferred to lysine, glutamate and aspartate residues of substrate proteins (Vida et al., 2017a). Acceptor

52 proteins are either mono-ribosylated (MAR), or poly-ADP-ribosylated

through the elongation of ADP-ribose branches (PAR). PAR branches are synthetized by PARP-1, PARP-2, tankyrase 1, and tankyrase 2 (reviewed in Haikarainen et al., 2014; Meehan & Chen, 2016). The main protein targeted of PARP1 PARylation are histones and PARP1 itself (reviewed in Kim, Zhang, & Kraus, 2005). Due to the negative charge of poly-ADP-ribose, PARP1 loses affinity for DNA. This creates a negative feedback loop inhibiting PARP1 activity (Zahradka & Ebisuzaki, 1982). PARP1 eviction attracts repair proteins to the damaged DNA site. Poly(ADP-ribose) has a short half-life. This is due to its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG) and ADP- ribose hydrolase 3 (ARH3) to generate ADP-ribose molecules and free poly-ADP-ribose (reviewed in Rouleau et al., 2010) (Figure 10).

Figure 10. PARP1 mechanism in DNA repair. (a) Structure of PARP1 sequence with the DBD (DNA Binding Domain; in pink and purple), the AD (Automodification Domain; in grey) and the Catalytic domain (in green and blue). (b) PARP1 recognized DNA damage, hydrolyses NAD+ and cover DNA breaks by poly-ADP-riboylation. PARG and ARH3 metabolize ADP-ribose and activate AMPK. WGR: Tryptophan-Glycine-Arginine motif. Zn1 (zinc finger); Zn2 (zinc finger 2); NLS (Nuclear Localization Signal); BRCT (BRCA1 C-terminal domain); NUDIX (NUDIX hydrolase); Residues essential for nicotinamide adenine dinucleotide binding (histidine, H*; and tyrosine; Y*) and for polymerase activity (glutamic acid; E*) are indicated. Taken from Meehan & Chen, 2016.

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3.1.3 PARP role in DNA repair in vivo

In support with PARP1 role in DNA repair, mice PARP1-/- were hypersensitive to genotoxic agents or γ-irradiation (de Murcia, 1997; Wang et al., 1997). Pharmacologic PARP1 inhibition was first used as a strategy for cancer therapy in BRCA1 deficient breast tumours (Combs-cantrell et al., 2009). Indeed, by inhibiting PARP1 activity, BRCA1 deficient tumour have no more functional repair machinery leading to their cytotoxic lethality. Today synthetic inhibitors (PJ34, Olaparib, ABT-888) or natural (NAM) PARP1 inhibitors are widely tested in combination with DNA damaging chemotherapeutic drugs (Farmer et al., 2005).

| 3.2 | PARP1 physiological relevance in skeletal muscle metabolism

Beyond DNA repair, PARP enzymes were also associated with transcription, and chromatin relaxation (reviewed in Kraus & Lis, 2003; Kozlowski et al., 2018). Furthermore, PARPs regulate homeostatic processes including apoptosis, proliferation and differentiation, circadian rhythm, inflammation and metabolism (reviewed Ryu et al., 2015; Vida et al., 2017). Thus, PARP inhibitors may have additional benefits in other diseases like cardiovascular or metabolic disorders (reviewed in Pacher & Szabo, 2007; Shevalye et al., 2010).

Type 1 diabetes is a chronic metabolic disorder resulting from the destruction of pancreatic ß-cells. Interesting work determined that PARP-1 KO or inhibition improved ß-cell regeneration making PARP-1 -/- mice resistant to streptozotocin-induced diabetes (Yonemura et al., 1984; Burkkart et al., 1999; Szabó et al., 2006). PARP-1 KO or inhibition improved several obesity parameters and rendered mice protected against high fat diet-induced obesity (Bai et al., 2011; Pirinen et al., 2014; Erener et al., 2012; Lehmann et al., 2015). However, PARP-related phenotypes in mice are

54 more complex than expected (reviewed in Hurtado-Bagès, 2018). In this

context, PARP-related phenotypes were shown to be tissue specific (Erener et al., 2012; Lehmann et al., 2015).

Overall, PARP1 inhibition was generally associated with beneficial outcomes. For instance, Pirinen et al., demonstrated the beneficial effect of PARP1 inhibition for the fitness and mitochondrial activity of mice skeletal muscle (Pirinen et al., 2014). This was explained by an increase in the amount of mitochondrial respiratory complexes and enhancement of mitochondrial respiratory capacity. Another example is given by the increase of the deacetylase Sirtuin 1 activity during exercise. Sirtuin 1 enhancement protected mice from prolonged PARP1 activity and NAD+ depletion (Mohamed et al., 2014).

| 3.3 | Cellular metabolism and NAD+ maintenance

3.3.1 Energy production

Dietary nutrients are metabolized to adenosine triphosphate (ATP) which is the organic chemical providing cellular energy in all living organisms. Briefly, ATP is generated via anaerobic (fermentation) and aerobic (respiration) pathways. Anaerobic glycolysis transforms glucose source to lactate generating two ATP molecules. In the presence of oxygen, pyruvate, which is a product of glycolysis, enters into mitochondria to be metabolized into Acetyl-CoA (Reece et al., 2011). Acetyl-CoA produced from glycolysis, fatty-acid β-oxidation or protein catabolism, delivers acetyl group to the citric acid cycle (Krebs cycle also called TCA for tricarboxylic acid cycle). Series of reactions, via TCA, oxidize Acetyl-CoA in mitochondrial redox reactions. Within the mitochondrial inner membrane, proton gradient across the electron transport chain (ETC) generates massive number of ATPs. Proton gradient is initiated by the electron donor NADH and FADH2 (reduced forms of nicotinamide adenine dinucleotide (NAD)) and flavin adenine dinucleotide (FAD), respectively), previously generated by the TCA. Thus, NAD/NADH and FAD/FADH2 ratios have a central role in ATP production in the oxidative pathway.

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Figure 11. Energy production in eukaryotic cell. Glucose is used either for glycogen formation, fatty acids synthesis, or is converted to pyruvate through the glycolysis pathway. The final products of glycolysis are two ATP, two NADH and two Pyruvate. Pyruvate crosses mitochondrial membrane to be decarboxylated into Acetyl-CoA. To note, Acetyl-CoA is also generated from circulating fatty acids oxidation (not appearing on the schema). Through the citric acid cycle (TCA), Acetyl-CoA produces NADH and FADH2 and two ATP molecules. Accumulation of NADH and FADH2 generated by glycolysis, pyruvate oxidation, citric acid cycle drives oxidative phosphorylation through the electron transport chain. The total amount of ATP obtained by the oxidative pathway approaches 26 to 28 molecules. Taken from Reece et al., 2011.

3.3.2 NAD is placed at central position in cellular metabolism

NAD is an essential cofactor involved in mitochondrial respiration, transcriptional regulation, calcium signaling, DNA repair and cell death, circadian rhythms and lifespan (reviewed in Cantó, 2015). NADH, the reduced form of NAD+, transfers an electron from a molecule to another (Warburg et al., 1935). Through the citric acid cycle, NADH transfers electrons to the mitochondrial electron transport chain (ETC). This allows oxidative phosphorylation which oxidizes nutrients into CO2 and energy in the form of ATP. Thus, NAD+/NADH ratio plays a crucial role in energy homeostasis within eukaryotic cells (reviewed in Cantó, 2015). NAD+ consists of adenine nucleobase and nicotinamide connected by phosphate groups. It also acts as a donor of ADP-ribose moieties, a precursor of the second messenger molecule cyclic ADP-ribose acting as a calcium-mobilizing agents, and as a substrate for sirtuins deacetylases and

PARP enzymes (reviewed in Nikiforov et al., 2015). 56

3.3.3 NAD replenishment

NAD+ pool can be restored either by synthesis of NAD+ de novo or by NAD+ salvage pathway (reviewed in Cantó et al., 2015) (Figure 11). NAD+ can be replenished from dietary sources including nicotinic acid (NA), nicotinamide (NAM), tryptophan (TRP), and nicotinamide riboside (NR). Through the Preiss-Handler pathway, three enzymatic reactions are necessary for NA to generate NAD+ de novo (Figure 11). The key enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) converts nicotinic acid mononucleotide (NAMN) into nicotinic acid adenine dinucleotide (NAAD). Through the kynurenine pathway, dietary tryptophan is transformed to N- formylkynurenine by indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO). Several enzymatic reactions lead to the conversion of N-formylkynurenine into quinolinate entering in the Preiss-Handler pathway by the formation of NAMN (Figure 11). NAD+ salvage pathway allows the recycling of its product, NAM, generated by NAD+- consuming enzymes such as sirtuins and PARPs. NAM and NR from dietary sources are then converted into nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT) enzymes (reviewed Verdin, 2015). As in the Preiss- Handler pathway, NMN is transformed to NAD+ by NMNATs (Figure 11).

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Figure 11. NAD+ synthesis de novo and NAD+ salvage pathway. Three independent pathways maintain NAD+ levels. Both (1) Preiss-Handler pathway using nicotinic acid as source, and (2) Kynurenine pathway using tryptophan, enable NAD+ biosynthesis de novo. (3) NAD+ recycling is sustained by the conversion of NAM and NMN into NAD+. NMN as a central role in such NAD+ salvage pathway. NAD+/NADH ratio balances redox metabolism in the mitochondria. NAD+ is mainly consumed by sirtuins, PARPs, and cADPR synthases (CD38 and CD157) in the nucleus. Enzymatic reactions initiated by specific enzymes (white bubbles) are shown by arrows. Nicotinate Phosphoribosyltransferase (NAPRT); NAD+ synthase (NADS), nicotinamide mononucleotide adenylyltransferase (NMNAT1-3), L-kynurenine (L-kin), 3- hydroxyanthranilic acid (3-HAA), ACMS decarboxylase (ACMSD), Quinolinate phosphoribosyltransferase (QAPRT), nicotinamide phosphoribosyltransferase (NAMPT). Taken from Verdin, 2015.

3.3.4 Essential enzymes for subcellular NAD+ balance

The three NMNAT (NMNAT1-3) enzymes have distinct subcellular distribution. NMNAT1 is localized in the nucleus, NMNAT2 is found in the cytoplasm and at the surface of the Golgi apparatus, while NMNAT3 is mainly located within the mitochondria (Berger et al., 2005). In skeletal muscle, NMNAT1 and NMNAT2 are highly expressed, while NMNAT3 is barely detectable (Berger et al., 2005; reviewed in Cantó et al., 2015) (Figure 12). While those organelle-specific enzymes catalyze the conversion of NMN to NAD+, NAD+ salvage pathway is also rate-limited by the conversion of NAM into NMN by NAMPT enzymes (Verdin, 2015). Therefore, NMN is placed at a central position in the NAD+ salvage pathway in all cell compartments (reviewed in Cantó et al., 2015) (Figure 12). 58

An imbalance of NAD+ levels between subcellular compartments may have severe effects, such as deregulation of signal-dependent transcriptional programs (Ryu et al., 2018), mitochondrial defects (Virág et al., 1998; reviewed Cipriani et al., 2005), oxidative stress and cell death (Virág et al., 1998; Zong et al., 2004). NAD+ levels can be modulated in response to glucose deprivation (Fulco et al., 2009), fasting, caloric restriction and exercise (Costford et al., 2009; Vaquero & Reinberg, 2009; Cantó et al., 2010).

Figure 12. Central role of NAD+ salvage pathway in eukaryotes. NAD+ can be synthesized de novo through three different paths in the cytoplasm. NR, from the blood, crosses the plasma membrane through specific transporters and generates NMN thanks to NRK1 and NRK2 enzymes. NA enters the cells and gives rise to NAD+ through successive enzymatic reactions leading to the formation of NAMN by NAPRT transformed to NAAD by NMNAT and finally NAD+ by NADSYN. NAMPT catalyzes the reaction producing NMN from NAM. NAD+ is maintained in the nucleus, cytoplasm and mitochondria through NAD+ salvage involving NMNAT1, NMNAT2 and NMNAT3 enzymes. NAD+/NADH ratio will be regulated by the equilibrium state of each compartment. The flow of NAD+ and NADH between cytosol and mitochondria is regulated by malate/aspartate (M/A) and glyceraldehyde 3-phosphate (G3P) shuttles. NADH is used as an electron donor in the electron transport chain (ETC) of the mitochondria to generate ATP through the TCA cycle. In the nucleus NAD+ is used as a substrate by sirtuins and PARP

enzymes. Taken from Cantó et al., 2015. 59

3.3.5 Plasticity in NAD+ compartmentalization

Interestingly, in proliferative cells and cancer cells, the formation of building blocks of nucleic acids is mainly ensured by glycolysis. On the other hand, mitochondrial activity sustains tissue function of fully differentiated cells (Leary et al., 1998). This suggests that the differentiation process, among others, requires a certain plasticity in NAD+ balance between subcellular compartments. Measurement of NAD+ levels in different cellular compartments is technically challenging to overcome. Recently, molecular tools have been developed to monitor free NAD+ (Cambronne et al., 2016) and ATP (Imamura et al., 2009) in those compartments. Today, those tools enable monitoring of how the imbalance of NAD+ and ATP from one organelle impacts another (Wright et al., 2016; Ryu et al., 2018).

3.3.6 Interconnection between NAD+ and PARP1 activity

More than 50 years ago, NMN was shown to induce PARylation when added to liver nuclear extract. This was the first hint that NAD+ salvage, PARP activity and PAR metabolism where linked to each other (Chambon et al., 1963). In eukaryotic cells, PARP1 is the major NAD+ consumer in the nucleus (reviewed in Fouquerel & Sobol 2014). As a consequence, PARP1 hyperactivation occurring during prolonged stress, does not only deplete nuclear NAD+ and ATP but rather impair the whole energetic balance in cells (reviewed in Hassa et al., 2006). NAD+ depletion triggers cell death mainly mediated by energy depletion and finally apoptosis (Fouquerel et al., 2014). In summary, cellular homeostasis is governed by proper balance between PARP1 activity for DNA repair mainly, and subcellular NAD+ pool for energy maintenance.

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| 3.4 | The physiological relevance of NAD+ in skeletal muscle

The hallmarks of skeletal muscle aging are increased inflammation, oxidative stress, and decreased regenerative ability. During aging, decline in mitochondrial function leads to degenerative diseases and cancer (reviewed in Wallace, 2017). Loss of skeletal mass is called sarcopenia, which is accompanied by metabolic disorders and a dramatic decrease of quality of life (reviewed in Goody & Henry, 2018). In a zebrafish model of muscular dystrophy, NAD+ supplementation improved extracellular matrix organization, regeneration capacity and motility ability (Goody et al., 2012). NAD+ is a major regulator of glucose and fatty acids metabolism, mitochondrial biogenesis, transcription and ECM organization in skeletal muscle (reviewed in Goody & Henri, 2018). Muscle from aged mice contains lower NAD+ levels, lower NAD+-dependent histone deacetylase sirtuin 1 activity and higher PARP1 activity (Mohamed et al., 2014). In the mdx mouse model of Duchenne muscular dystrophy, NAD+ reduction was also observed, accompanied by reduced expression of nicotinamide phosphoribosyltransferase (NAMPT) (Ryu et al., 2016). NAD+ decrease during aging is associated with impaired muscle development and regeneration, and promotes metabolic diseases, muscular myopathies and dystrophies (reviewed in Goody & Henry, 2018). Strikingly, NAD+ levels were boosted in Duchenne muscular dystrophy-mice treated with nicotinamide ribose (NR) which increase satellite cells number and therefore regeneration capacity (Ryu et al., 2016). In conclusion, NAD+ has a major role in muscle physiology and pathology. Thus, the supplementation of NAD+ precursors may improve patient condition and could be used in addition to other treatments.

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IV | MacroH2A histone variant

| 4.1 | MacroH2A structure

Among all histone variants, macroH2A have an atypical structure of three distinct domains consisting of the histone fold H2A-like, an unstructured linker and a globular macrodomain (Chakravarthy et al., 2005; Kustatscher et al., 2005). In mammals, macroH2A1 and macroH2A2 are encoded by the two distinct H2AFY and H2AFY2 genes, respectively. MacroH2A1 and macroH2A2 histone fold domains share approximately 60% similarity to H2A amino acid sequence. Splicing event further generates macroH2A1.1 and macroH2A1.2 isoforms that differ by 26 amino acids in their macrodomain sequence (Pehrson et al., 1997)

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Figure 13. MacroH2A gene structures and exclusive exon splicing of macroH2A1 gene. (A) MacroH2A tripartite structures, macroH2A HFD (in orange), linker (flexible line), macrodomains (in blue) and macroH2A1.2 exclusive exon (in green). (B) Exclusive splicing of exons six and seven from H2AFY gene giving rise to macroH2A1.1 and macroH2A1.2 isoforms in mouse. Amino acid composition of macroH2A1.1 and macroH2A1.2 are shown below. Adapted from Kustatscher et al., 2005.

4.1.1 Histone Fold Domain and Linker

The domains that confer the physiological relevance of macroH2A are still elusive. In eukaryotes, histones are constituted by a histone fold domain and unstructured NH2- terminal domain. Histone fold domains are comprised of three α helices connected by two short loops, Loop1 (L1) and Loop2 (L2). Histone heterodimers are generated thanks to the assembly of three α helices interleaved as a 'handshake motif' and the juxtaposition of L1 and L2 forming parallel β-bridges (Arents et al., 1991; Luger et al., 1997). Four amino-acid changing in the L1 of the HFD, compared to H2A histone, confers new structural and ionic characteristics to macroH2A-containing nucleosome. MacroH2A-containing nucleosomes assemble and stabilize with lower high-salt concentrations than the replicated-coupled one in vitro (Chakravarthy & Luger, 2006). This suggests that MacroH2A-containing nucleosomes are more stable. Increased macroH2A-containing nucleosome stability may be reinforced by the unstructured linker. Indeed, amino acid composition with rich lysine region of the macroH2A linker is similar to the unstructured region of the histone H1 (John R. Pehrson & Fried, 1992). MacroH2A linker increases nucleosome compaction via oligomerization of chromatin fibers and decreases extra-nucleosomal accessibility at the entry/exit site nucleosome (Chakravarthy et al., 2012). Generally, macroH2A HFD and linker regulates nucleosome stability and chromatin accessibility. Additionally, interconnection between linker and macrodomain was found. Indeed, macroH2A linker capacity to increase cellular chromatin fiber-fiber interaction was abolished by the presence of the macroH2A1.2 macrodomain in vitro (Muthurajan et al., 2011). On the contrary, macroH2A linker capacity to reduce chromatin capacity is either promoted by the presence of

macroH2A1.1 macrodomain or unchanged with macroH2A2 macrodomain 63

in cellulo (Kozlowski et al., 2018). Thus, macrodomains of the three distinct macroH2As may have different effects on chromatin compaction.

4.1.2 Macrodomain

Macrodomains are 25 kDa globular domain composed of six-stranded β-sheet and five α-helices and its crystal structure revealed binding pocket (Kustatscher et al., 2005). They are highly conserved, being found in prokaryotes and eukaryotes as well as in some viruses (Kraus, 2009). Initially identified in murine coronavirus by DNA sequencing, macrodomains were later found to be fused to H2A-like domain creating the histone variant macroH2A (Pehrson & Fried, 1992). Identification of this histone variant (the largest of histone variants) gave macrodomains their name (Rack et al., 2016) So far, 12 proteins containing 16 different macrodomains have been discovered in humans. These proteins are classified into five groups: MacroH2A-like, MacroPARPs-like, MacroD- type, PARG-like (enzyme PAR glycohydrolase) and ALC1-like (Snf2-type chromatin remodeler-like) (Rack et al., 2016; Figure14).

Figure 14. Domain organization of macrodomain-containing proteins from human. The amino acid length is taken from SMART/Pfam database. Abbreviations: core histone H2A- like domain (H2A); helicase conserved C-terminal domain (HELICc); macrodomain (Macro); poly(ADP-ribose) polymerase catalytic domain (PARP cat); RNA recognition motif (RRM); yeast secretory protein 14 (Sec14p) (SEC14); SNF2 helicase family N- terminal domain (SNF2_N); domain with conserved tryptophan/glutamate-containing motif (WWE). Adapted from Rack et al., 2016.

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The binding pocket of all macroH2A macrodomains, as revealed by their crystal structures, differ in size and hydrophobicity (Kustatscher, Hothorn et al., 2005). Interestingly, only the macrodomain of macroH2A1.1 was shown to bind NAD+ derived metabolites, including ADP-ribose and O-acetyl-ADP-ribose (Kustatscher et al., 2005). The direct recognition of ADP-ribose permit macroH2A1.1 to sense PARP1 enzymes to DNA repair sites (Timinszky et al., 2009).

Figure 15. Structures of macroH2A binding pockets. On the left, the crystal structure of macroH2A-containing nucleosome (H3 in blue; H4 in green; H2B in red and macroH2A fold domain in yellow) surrounded by the DNA helix (in grey). MacroH2A macrodomain (in purple) protrudes out of the nucleosome. MacroH2A linker is represented by the dashed line. On the right, the three macroH2A macrodomains are cut open. Protein surface in grey, protein interior in black, basic amino acid in blue, acid amino acid in red and neutral amino acids in white. Only macroH2A1.1 macrodomain accommodates ADP-ribose forming a binding pocket for Poly-ADP-ribose (PAR) and O-acyl-ADP-ribose (OAADPr). Taken from Posavec et al., 2013.

| 4.2 | MacroH2A function

4.2.1 MacroH2A is a transcriptional repressor

The first clue regarding macroH2A function came from the observation that macroH2A was enriched on the inactive X chromosome (Costanzi & Pehrson, 1998; (Mermoud et al., 1999). In mammals, transcriptional silencing of one female X chromosome is necessary for two reasons. First, X-inactivation prevents the female genome to encode double dose of X-related genes which could be toxic. Secondly, its allows to equilibrate gene dosage in males carrying only one X copy. Enrichment of macroH2A on thr inactive X chromosome led to the hypothesis that macroH2A acts as a

transcriptional repressor. Later on, several studies supported the 65

repressive role of macroH2A targeting subset of autosomal genes and repetitive elements by establishing repressing chromatin landscape (Changolkar & Pehrson, 2006).This may be due to the abolition of transcription factors binding in the macroH2A-containing nucleosome, which prevents chromatin remodeler such as SWI/SNF (Angelov et al., 2003). MacroH2A is broadly distributed across the genome and comprises approximately 1% of the total H2A pool (reviewed in Buschbeck & Hake, 2017). MacroH2A is enriched on large chromatin domains marked by H3K27me3 and H3K9me3 (Douet et al., 2017; Sun et al., 2018). H3K27me3 is associated with facultative heterochromatin, and H3K9me3 is associated with constitutive heterochromatin. Recently, MacroH2A1 incorporation was also found in H2B-acetylated chromatin (Chen et al., 2014). However, the machinery behind macroH2A incorporation remains unknown, although the histone fold domain was shown to be required (Ruiz & Gamble, 2018). In conclusion, macroH2A is mainly associated with repressive marks and transcriptional silencing.

4.2.2 MacroH2A role in genome stability and DNA repair

During DNA damage response, extensive chromatin remodeling occurs to give access to DNA repair machinery. The transient role of macroH2A in DNA repair related to chromatin plasticity has been recently reported. Loss of macroH2A1 impaired BRCA1 retention to DNA damage sites by preventing chromatin condensation (Khurana et al., 2014). Upon microirradiation macroH2A1.1 is recruited to DNA damage site by sensing PARP1 activation. This leads to PARP1 inhibition, release of both macroH2A1.1 and PARP1, and finally chromatin condensation (Timinszky et al., 2009). The role of macroH2A in chromatin rearrangement is not restricted to macroH2A1.1 macrodomain since macroH2A linker also prevents chromatin expansion during double strand breaks (Kozlowski et al., 2018).

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4.2.3 MacroH2A is key for the maintenance of differentiation state

Loss-of-function experiments demonstrated that macroH2A promotes differentiation of embryonic and adult stem cells (Pasque et al., 2012). In human NT2 teratocarcinoma cells, macroH2A cooperates with the polycomb complex 2 to repress key development genes such as homeobox A cluster genes. Upon activation of neuronal differentiation by retinoic acid macroH2A occupancy drop off leading to the transcriptional activation of homeobox A genes (Buschbeck et al., 2009). Furthermore, macroH2A restricts reprogramming of somatic cells undergoing nuclear transfer by Xenopus oocyte ( Pasque 2011). Altogether, macroH2A is associated with transcriptional repression and differentiated state maintenance. This is further supported by the role of macroH2A in suppressing transcriptional noise, contributing to the maintenance of heterochromatin architecture and nuclear organization (Lavigne et al., 2015; Fu et al., 2015; Douet et al., 2017).

However, most of the studies previously mentioned did not distinguish the function of different macroH2A isoforms beyond gene repression. First of all, macroH2A is found in other species that do not undergo X inactivation and is equally expressed in male and female ( Pehrson & Fuji, 1998; Rasmussen et al., 1999). Secondly, macroH2A1 knockout mouse did not show X inactivation defect (Changolkar et al., 2007). Finally, positive regulation of a large subset of genes was also reported in macroH2A1-containing chromatin in breast cancer cells and lung fibroblasts (Gamble et al., 2010). This is in contrast to the transcriptional repressor role attributed to macroH2A.

4.2.4 Role of macroH2A in cancer malignancy

MacroH2A is generally associated with tumor suppressive function. For example, macroH2A decreases melanoma progression in colorectal cancer through repression of the oncogenic CDK8 (mediator complex component) transcriptional regulator (Kapoor et al., 2010). Depletion of macroH2A1 and macroH2A2 leads to significant increased

invasion of bladder cancer cells and anal neoplasms, respectively (Kim et 67

al., 2013; Hu et al., 2016). This may be caused by the loss of the RNA binding protein Lin28B repression that promotes stem-like properties in bladder cancer (Park et al., 2016). However, implication of macroH2A isoforms shall be distinguished between different cancer outcomes. While macroH2A1.1 and macroH2A2 generally act as tumor suppressors, the role of macroH2A1.2 seems to be context-dependent (reviewed in Cantariño et al., 2013). MacroH2A1.1 and macroH2A2 expressions were inversely correlated with the proliferation of lung cancer, hinting at their potential use as a biomarker, (Sporn et al., 2009). Chen et al., observed a drastic reduction of macroH2A1.1 but increased macroH2A1.2 in several cancer cell lines versus primary cells (Chen et al., 2014). Interestingly, several cancer cells do not express macroH2A1.1 at all (H. Chen et al., 2015). Furthermore, overexpression of macroH2A1.1 reduces proliferation of lung and cervical cancer cells (Novikov et al., 2011). When macroH2A1.2 was found to be up- regulated in primary colorectal cancer samples, loss of macroH2A1.1 correlated with poorer outcome in colon cancer patients (Sporn & Jung, 2012). While RNA helicases Ddx5 and Ddx17 favor macroH2A1.2 expression, macroH2A1.1 expression is promoted by splicing factors such as QKI and MBNL1 (Novikov et al., 2011; Dardenne et al., 2012). In invasive breast cancer cells, depletion of Ddx5 or Ddx17 inhibited both cell migration and invasion (Dardenne et al., 2012). This was partially due to increased expression of extracellular superoxide dismutase 3 which followed up- regulation of macroH2A1.1 (Dardenne et al., 2012). Jerónimo group recently demonstrated that lower expression of macroH2A1.1 and its splicing factor QKI was correlated with a malignant phenotype of prostate cancer (Vieira-Silva et al., 2019). This is supported by the reintroduction of QKI expression in gastric cancer which inhibits cell proliferation, migration, and invasion (Li et al., 2016). Therefore, macroH2A1.1 and macroH2A2 are mainly associated with decreased cell proliferation, while macroH2A1.2 tends to promote proliferation, although the role of macroH2A1.2 is cell-context dependent.

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4.2.5 Mice Development and Metabolism

Zebrafish macroH2A-/- showed developmental defects. In mice lacking one macroH2A were viable and fertile (Changolkar et al., 2007; Buschbeck et al., 2009, Boulard et al., 2010). However, mice lacking both macroH2A-encoding genes had growth defects (Pehrson et al., 2014). Discrepancies were observed regarding systemic metabolic alterations in macroH2A knockout (KO) mice (reviewed in Hurtado-Bagès et al., 2018). Indeed, macroH2A1 KO mice displayed a pre-diabetic phenotype when fed with high-fat diet (Changolkar et al., 2007). On the contrary, macroH2A1 KO mice shown increased leanness (Sheedfar et al., 2015). While a pre-diabetic phenotype was associated with impaired clearance of bolus glucose injections and increased insulin resistance, leanness was associated with increased energy expenditure and lower accumulation of fat (Changolkar et al., 2007; Sheedfar et al., 2015). In conclusion, these two studies contradict each other. In liver, genes involved in lipid metabolism were deregulated (Changolkar et al., 2007; Boulard et al., 2010). This led to fat accumulation in the liver only in female mice with 50% penetrance (Boulard et al., 2010). Finally, while exogenous macroH2A1.1 expression protected liver cancer cell lines from lipid accumulation (Pazienza et al., 2014), exogenous macroH2A1.2 expression in mice enhanced leanness by reducing adipogenesis (Pazienza et al., 2016). In conclusion, further work is required to understand the role of macroH2A isoforms in metabolic outcomes.

To summarize, macroH2A has been shown to play a role in genomic stability, cancer development, differentiated state maintenance, and seems to control metabolic homeostasis. Overall macroH2A1.1 depletion was associated with poor outcome

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Results

Results

Chapter I

The knowledge about the function of macroH2A1 isoforms was mainly limited to transcriptional regulation, particularly their role as gene repressors. Expression of macroH2A isoforms is tissue- and cell- specific. * Do both macroH2A1 isoforms perform the same function? * In what cells are they relevant? * Is the macrodomain involved? In this chapter, we will see that macroH2A1.1 has an essential metabolic role in muscle cells. This function is isoform-specific and largely independent of gene regulation.

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MacroH2A1s switch during myogenesis at the mRNA level

Since the expression of both macroH2A1 was known to be tissue-specific, we first determined the abundance of macroH2A1 spliced variant in a panel of mouse tissue. Initially, antibodies for specific macroH2A1 isoforms were not available. Therefore, we performed a restriction-fragment-length polymorphism (RFLP) experiment. This technique allows to distinguish mRNAs of both isoforms by enzyme digestion (Figure 18A). In mouse, HpaII/MspI enzyme cut macroH2A1.1 cDNA from RNA into two bands of 131 and 38bps (Figure 18A). MacroH2A1.2 is cut into two bands of 89 bp each. In human, cut macroH2A1.1 gives one band at 191 bp while macroH2A1.2 digestion gives two bands at 121 and 79 bp (Figure 18A, right panel). Complementary DNA of macroH2A1, amplified from RNA, were expressed in all tissues tested but shown distinct profile between the two isoforms. MacroH2A1.2 is highly expressed in the liver, brain, spleen, small and large intestine compare to macroH2A1.1 (Figure 18B, upper panel). Similar expressions between both isoforms were found in testis, kidney and lung. Strikingly, mature skeletal muscle express only macroH2A1.1 (Figure 18B, upper panel). This leads us to pursue our investigation on primary myoblasts from human and mouse, as well as the commonly used murine C2C12 myoblasts cell line. During differentiation of these three models, we observed a splicing switch occurring between the two macroH2A1 isoforms giving rise to mature myotubes expressing only macroH2A1.1 isoform. (Figure 18B, lower panel). MacroH2A1.2 tends to decrease upon differentiation while macroH2A1.1 massively increases at the mRNA level in C2C12, human and mouse primary myoblasts (Figure 18C, 18D). MacroH2A2 expression was found to be negligible in C2C12, human and mouse primary myoblasts. On the other hand, expression of early and late myogenic markers such as Myog and Ckm increased normally during myogenesis (Figure 18C, 18D).

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A metabolite binding mouse human

mH2A1.1 mH2A1.1

mH2A1.2 mH2A1.2 macro domain

C2C12 B C mH2A1.2 mH2A1.1 Myog 2 2 mouse tissue * *

sk.muscleliver brain testis kidneyspleensmall. big.int intlung * 1 * 1

131 bp mH2A1.1 89 bp (2x) mH2A1.2

RFLP 0 0

2 total mH2A1 mH2A2mH2A 2 Ckm * primary myoblasts myoblats cell line level mRNA rel.

human mouse C2C12 * * 1 1 * * Days [D] Prol D2 D4 D6 Prol D1 D2 D4 Prol D1 D4 mH2A1.1 mH2A1.2 0 0 RFLP 0.50 2 41 0.50 2 41 0.50 2 41 Day of differentiation [D] D

mouse primary myoblasts

mH2A1.2 mH2A1.1 total mH2A1 mH2A2 Myog Ckm

1.2 1.2 4 50

0.6 0.6 2 25 rel. mRNA level mRNA rel. level mRNA rel.

0 0 0 0

prol D1 D2 D4 prol D1 D2 D4 prol D1 D2 D4 prol D1 D2 D4 prol D1 D2 D4 prol D1 D2 D4

human healthy donor

mH2A1.2 mH2A1.1 total mH2A1 mH2A2 Myog Ckm 2.6 3.4 1.4 1.4

1.3 1.7 0.7 0.7 rel.level mRNA rel.level mRNA

0 0 0 0

prol D1 D4 prol D1 D4 prol D1 D4 prol D1 D4 prol D1 D4 prol D1 D4 –

Figure 18. MacroH2A1s splicing switch during myogenesis. (A) On the left, schematic representation of splicing event of the two alternative macroH2A1 exons (6 and 7) giving rise to macroH2A1.1 and macroH2A1.2 isoforms in mouse. On the right, scheme of the RFLP experiment. Lightning lines show which part macroH2A1 cDNA was cut by HpaII/MspI enzymes. In human, the two alternative macroH2A1 exons are 7 and 8 exon s. (B) On the top, RFLP experiment performed on mouse tissues. At the bottom, RFLP experiment performed75 on human and mouse primary myoblasts. Length of the digested fragments (bp) are shown for all RFLP. 2X refers to two bands of the same size (C-D). Relative mRNA levels of macroH2A1.1, macroH2A1.2, total macroH2A1 and macroH2A2 in C2C12, human and mouse primary myoblasts. Relative mRNA levels of differentiation markers as myogenin and Ckm were used as reference samples. prol: proliferative cells, D1, D2, D4 and D6: Day 1, 2, 4 and 6 of differentiation. Data in C-D are the mean +s.d of n=3 independent experiments; *p < 0.05; Student's t-test.

The chromatin component macroH2A1.1 switch during myogenesis at the protein level

We tested the specificity of newly available mH2As antibodies. Expression of endogenous macroH2A was tested in C2C12 transfected either with si Ctrl or si macroH2A1.1 (Figure 19A). The specificity of the antibodies was assessed by exogenous expression of flag-macroH2A1.1, flag-macroH2A1.2 and flag macroH2A2 in HepG2 DKD (Double knock-down for macroH2A1 and macroH2A2 (previously described in (Douet et al., 2017)). Immunoblot analysis reveals strong specificity of each macroH2A antibodies used (Figure 19A). High expression of macroH2A1.1 protein was confirmed in skeletal muscle samples (Figure XB). The previously observed switch of macroH2A1 at the mRNA level was confirmed at the protein level in C2C12 and mouse primary myoblasts (Figure 19C). Interestingly, the increase in macroH2A1.1 was more pronounced at the protein level. Furthermore, macroH2A1.2 protein did not disappear similar to the decrease observed at mRNA levels (Figure 19C). As expected from a chromatin component, macroH2A1.1 was found to be located in the nucleus of human muscle tissue and C2C12 (Figure 19D, 19E). In this cell line, the macroH2A1.1 fraction is found to be incorporated and enriched into C2C12 chromatin (Figure 19F). ChIP-seq analysis shown a coverage of macroH2A1 between 13,9 and 12,9% in proliferative and differentiated (day 5) C2C12 chromatin (Figure 19G, table). Most of macroH2A1 peaks were found to be equally distributed between proliferative and differentiated C2C12 (Figure 19G).

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A BC

C2C12 HEPG2 DKD HEPG2 DKD D4 FLAG FLAG C2C12 mouse myoblasts

si macroH2A1.1 +- mH2A1.1mH2A1.2mH2A2 sk. muscle mH2A1.1mH2A1.2mH2A2 prol D2 D4 prol D1 D2 D4 mH2A1.1 si macroH2A1.1 mH2A1.1 mH2A1.2

si macroH2A1.2 FLAG eMHC

myogenin si macroH2A2 Histone H3

si macroH2A2 long exposure D human muscle section Flag

H3

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C2C12 E F C2C12 [D4]

si Ctrl cytosoltotal nucleinucleosolchromatin mH2A1.1

D1.5 Tubulin si mH2A1.1 NPM1

Histone H3

DAPI mH2A1.1

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prol Prol D5 Supplementary Figure 2 D5 (332 Mb) (310 Mb)

mH2A1 SICER peaks Nuclear localization and chromatin association of mH2A1 ChIP-seq (a) Cross-sections of snap-frozen human muscle Input Supplementary Figure 2 rrowheads indicate the positive signal of laterally UCSC genes (b) C2C12 cells were fixed at day 1.5 of differen Nuclear localization and chromatin association of macroH2A1.1. was stained with DAPI. Scale bar is 20 μm. (a) Cross-sections of snap-frozen human muscle were fixed in cold 100%(c) acetonImmunoblot and immunostained analysis with after anti-macroH2A1.1 cellular fractionatioantibody. rrowheadsSupplementary indicate the Figure positive 2 signal of laterally located myonuclei in the muscle fibers. Scale bar is 50 μm. (b) C2C12 cells were fixed at day 1.5 of differentiation and analyzed Databy immunofluorescence Set 1. with anti-macroH2A1.1 antibody. DNA Figure 19. wasSplicing stainedNuclear with switchlocalization DAPI. Scale of and barthe chromatin is 20chromatinμm. association ofcomponent macroH2A1.1.(d) macroH2A1 Overview table protein. of peaks (A) called Immunoblot by SICER analysis in pr Supplementary(c) Immunoblot analysis Figure after cellular 2 fractionation of differentiated C2C12 cells. Uncropped blot images are shown in Supplementary comparing express(a) Cross-sectionsion of endogenous of snap-frozen human and muscle exogenous were fixed in macroH2A cold(e) 100%UCSC aceton genome isoforms and immunostained browser in C2C12 with window anti-macroH2A1.1 and of HepG2a represen antibody DKD expressing DataFlag Set-taggedrrowheads 1. macroH2A.indicate the positive MacroH2A1 signal of laterally isoforms,located myonucl macroH2A2,ei in the muscle fibers. Flag Scale proteins bar is 50 μ,m. and histone H3 used as a loading control, are shown (B) Immunoblot of macroH2A1.1 and Flag expression in skeletal muscle and HepG2 DKD expressing Flag-tagged macroH2A. (C) Immunoblot of C2C12 and mouse primary myoblasts during the indicated differentiation time course. Expression of macroH2A1.1, of macroH2A1.2, of the late myogenic marker eMHC (embryonic myosin heavy chain), of the early myogenic marker myogenin, and of the histone H3 are shown. (D) Cross section of human muscle was stained with anti- macroH2A1.1 antibody. Positive expression is indicated by an arrow. Scale is 50 µM (E)

Immunofluorescence of macroH2A1.1 in C2C12 at day 1.5 of differentiation. DNA was stained by the DNA

intercalant DAPI.77 Scale is 20 µM. (F) Immunoblot analysis of C2C12 fractionation. Good cell fractionation was checked by the protein expression of the cytoplasmic tubulin, the nucleolus nucleophosmin 1 (NPM1), the chromatin histone (H3) and macroH2A1.1. (G) At the top, overlap of SICER peaks of macroH2A1 in proliferation and after 5 day of differentiation in C2C12. Below, the table shows an overview of SICER peaks and macroH2A1 coverage. On the right, UCSC genome browser screenshot of representative 3Mb from chromosome 8 (8:121.500.000-124.500.000). Black rectangle represents SICER peaks.

MacroH2A1 reduction partially impacts muscle formation but does not impair differentiation

Since macroH2A1 isoforms are dynamically switched during muscle differentiation, we wondered if their modulation would impact the proper myogenic program. In order to respond to this question, we performed loss-of-function analysis using specific siRNAs directed against macroH2A1.1 and macroH2A1.2 at several time points during C2C12 differentiation (Figure 20A). Samples were collected at day 0 of differentiation (when growth medium (GM) was changed for differentiation medium), at days 2 and 4 after medium change (DM2, DM4). Successful macroH2A1.1 and macroH2A1.2 silencing was tested during C2C12 differentiation (Figure 20B). Interestingly, macroH2A1.2 silencing (si macroH2A1.2) increased macroH2A1.1 expression at day 2 of differentiation (D2). On the contrary, macroH2A1.1 silencing (si macroH2A1.1) increased macroH2A1.2 all along differentiation (Figure 20B). This suggests that transcriptional regulation of both macroH2A1 isoforms are interconnected. At day four of differentiation (D4), morphological changes could be observed with the three conditions. Myotubes were slightly less elongated myotubes in si macroH2A1.1. On the contrary, myotubes seemed bigger and more elongated in si macroH2A1.2, respectively (Figure 20C). However, no drastic impairment in differentiation was observed since multinucleated myotubes were formed in all conditions (si Ctrl, si macroH2A1.1, and si macroH2A1.2). Expression of the early transcription factors MyoD1 and Myog were unchanged. On the contrary, the late myogenic marker, creatin kinase Ckm, was significantly increased with si macroH2A1.1 and inversely decreased with si macroH2A1.2 at day 2 and 4 of differentiation (D2, D4) (Figure 20D). Therefore, macroH2A1 modulation seems to slightly impact late phase of myogenesis in a reversed manner.

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Figure 20. MacroH2A1 manipulation impacts partially muscle development. (A) Schematic representation of the small interfering RNA protocol we used. C2C12 cells were transfected with siRNAs three times: one day after plating C2C12, the day of growth medium change for differentiation medium and two days later. For different analysis, C2C12 was collected at day 0 of differentiation (prol), two and four days after differentiation (D2, D4). (B) Relative mRNA levels of macroH2A1.1 and macroH2A1.2 during differentiation in C2C12 treated with si Ctrl, si macroH2A1.1 and si macroH2A1.2. (C) Immunofluorescence of the differentiation marker eMHC in C2C12 at day 4 of differentiation transfected with siRNAs. DNA was stained by the intercalant DAPI. (D) Relative mRNA levels of the early myogenic markers MyoD1 and Myog, as well as the late marker Ckm, during C2C12 differentiation transfected with siRNAs. Data in B-D are the mean +s.d of79 n=5 independent experiments; *p < 0.05; Student's t-test.

MacroH2A1.1 reduction impacts cellular metabolism

We previously saw that macroH2A1.1 is highly expressed in myotubes compared to other mouse tissues (Figure 20). Strikingly, silencing of macroH2A1.1 provokes color change of the cell culture media of C2C12 due to pH change (Figure 21A). We hypothesised that this could be due to changes in cellular metabolism in culture. Thus, we decided to measure both glucose consumption and lactate production. At day four of differentiation, glucose concentration in the media increased significantly with si macroH2A1.1 (Figure 21B). This suggests that C2C12 treated with si macroH2A1.1 consumed less glucose compared to control. In this cell line, lactate, a glucose product, was decreased without affecting total protein levels (Figure 21B). This suggests that macroH2A1.1 silencing decreases the glycolysis capacity of C2C12 cells. The glycolysis pathway can be monitored by an ECAR (Extracellular Acidification Rate) experiment. After a period of starvation, supplementation by glucose forces cells to favor glycolysis. Glycolysis capacity is reduced in si macroH2A1.1 treated cells compared to the control (Figure 21C). A reduction in glycolysis activity remains after the addition the glucose analogue 2-deoxyglucose (2-DG) used to inhibit glycolysis. Cellular respiration is monitored via OCR (Oxygen-Consumption rate) analysis through addition of several inhibitors and stimulators of the mitochondrial activity. Following the addition of the mitochondrial uncoupler carbonyl cyanide p- trifluoromethoxyphenylhydrazone (FCCP), macroH2A1.1 silencing strongly reduced maximal mitochondrial respiration (Figure 21C). In conclusion, silencing of macroH2A1.1 impairs whole cellular metabolism.

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B C Glucose Lactate Total protein Glycolysis Mitochondrial oxidation ** ns 15 1.2 OFAA 30 si Ctrl Glc 2-DG 50 100 si mH2A1.1 40 80 10 0.8 20 30 60

20 40 5 10 0.4 Conc. [mM] Conc. [mM] 20 Relative level 10 OCR (pmol/min) ECAR ECAR (mpH/min) 0 0 0 0 0 si Ctrl si Ctrl si mH2A1.1 si mH2A1.1

Figure 21. MacroH2A1.1 silencing reduced glycolytic and oxidative C2C12 capacity. (A) Color change of the cell culture medium in C2C12 treated with si Ctrl or si macroH2A1.1. (B) Extracellular glucose, lactate concentration, and total protein level. (C) On the left, extracellular acidification rate (ECAR) analysis. C2C12 cells treated with si Ctrl and si macroH2A1.1 were glucose starved overnight. Glycolysis was boosted by glucose (Glc) addition and stopped by 2-deoxyglucose (2-DG) (n=5, *p < 0.05; Student's t-test). Results were normalized by genomic DNA content. On the right, oxygen- consumption rate (OCR) experiment. Mitochondrial respiratory capacity was first reduced by the addition of the ATPase inhibitor oligomycin (O). Maximal mitochondrial capacity was monitored after the addition of the uncoupling compound (FCCP). Mitochondrial activity was finally shut down by the electron transport chain inhibitors rotenone and antimycin A (AA). Data in B are the mean +s.d of n=3-4 independent experiments +s.d.; *p < 0.05; Student's t-test. n.s. not significant.

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MacroH2A1.1 silencing impacts mitochondrial activity

Besides glycolysis, oxidation of fatty acids is used by mitochondria to generate massive amount of energy. In this context, we tested C2C12 capacity to use fatty-acid as a unique source of energy with either si macroH2A1.1 or si macroH2A1.2 compared to the control. C2C12 treated with si macroH2A1.2 and primed with fatty-acid does not show any change in OCR (Figure 22A). On the contrary, macroH2A1.1 silencing provokes 40% OCR reduction. Reduced mitochondrial activity was recapitulated in C2C12 isolated mitochondria (Figure 22A). From now on, only maximal respiratory, occurring after fatty acid addition, will be presented (Figure 22A, right). A gain-of-function experiment was performed in the human HepG2 hepatoma cells having been knocked-down for macroH2A1 and macroH2A2 (described in Douet et al., 2017). Similar amount of macroH2A1 isoforms were expressed in HepG2 DKD (double KD) cells (Figure 22C). While exogenous macroH2A1.2 expression did not impact mitochondrial activity, macroH2A1.1 overexpression significantly boosted the maximal respiratory capacity of DKD cells (Figure 22B). Interestingly, maximal respiratory capacity was equally impaired by almost 50% in isolated mitochondria from C2C12 with si macroH2A1.1 (Figure 22D).

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Starved, fatty acid Max. resp. capacity primed cells

OFAA 100 si Ctrl 150 si Ctrl * si mH2A1.1 80 si mH2A1.1 * si mH2A1.2 si mH2A1.2 100 60

40 50 50 OCR (pmol/min) OCR Percent of si Ctrl si of Percent 0 0

B C Max. resp. capacity HepG2 DKD HepG2 DKD

* p=0.062 150 Ctrl F-mH2A1.1 HepG2ControlYFP-mH2A1.1YFP-G224EF-mH2A1.1F-mH2A1.2 F-mH2A1.2 100 mH2A1.2

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Percent of F-mH2A1.1 of Percent 0 FLAG (F)

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150 si Ctrl * si mH2A1.1 100

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Figure 22. MacroH2A1.1, but not macroH2A1.2, regulates mitochondrial activity. (A) On the left, OCR analysis of starved C2C12 treated with siRNAs, and primed with fatty acids. On the right, maximal respiratory capacity is calculated between the addition of uncoupling compound (FCCP) and rotenone with antimycin A (AA) is shown. Oligomycin (O). (B) Maximal respiratory capacity of HepG2 double knock-down expressing exogenous Flag-tagged macroH2A isoforms. (C) Immunoblot of macroH2A1 isoforms, Flag and the loading control NPM1 (Nucleophosmin 1). YFP constructs were used as control for macroH2A1 isoforms expression. (D) Maximal respiratory capacity in isolated mitochondria from C2C12 cells treated with si macroH2A1.1 compared to control. Data in A-D are the mean of n=6 independent experiments. Data in B are the mean of n=9 independent experiments + s.d.; * p<0.05, Student's t-test.

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Mitochondrial defect is independent of macroH2A1.1 gene targets

First of all, mitochondrial defects could not be explained by deregulation of mitochondrial transcriptional misbalance since no significant variation was observed in the ratio between mitochondrial DNA (mtDNA) and nuclear genomic DNA (gDNA) (Figure 23A, left panel). Similarly, no change was observed concerning transcriptional expression of complexes forming the electron transport chain (ETC) (Figure 23A, right panel). Since macroH2As are known to regulate gene transcription, we assessed if macroH2A1.1 gene targets are involved in a metabolic phenotype. To do so, we performed transcriptomic analysis from a micro-array comparing C2C12 control versus si macroH2A1.1 at day four of differentiation (Figure 23B). Only 797 genes were found to be deregulated (DEGs: deregulated genes) by the silencing of macroH2A1.1. Among them, 49 genes were up- or down-regulated with more than a two fold-change (Figure 23B, red numbers). Gene Ontology analysis (GO) of the deregulated genes showed association with cell adhesion and migration, signaling and extracellular organization (data not shown). Therefore, no obvious candidate genes could explain the observed metabolic disorder (Figure 23C). The macrodomain of macroH2A1.1 does not play a direct role on macroH2A1.1 transcriptional function. Indeed, stable C2C12 cell lines expressing either macroH2A1.1 wild-type (WT) or its binding pocket mutant (G224E), equally rescued macroH2A1.1 target genes expression (Figure 23D, 23E). The list of genes tested includes three top deregulation genes (Itga11, Cdhr1, and Tmem171) in C2C12 treated with si macroH2A1.1. Additionally, Mstn and Igf1, were also tested based on the fact they play a role in muscle growth. Although no obvious candidate genes for metabolism defects were found,

we tested the si macroH2A1.1 effect on genes coding for the five complexes 84

of the electron transport chain (ETC) (Figure 23F). Among them, only Cox4i1 shown a slight increase associated with macroH2A1.1 silencing (Figure 23F, left panel). It is interesting to note that the expression of all ETC genes significantly increased during differentiation. This suggests that differentiated cells favor mitochondrial biogenesis. We further looked at several key metabolic genes for glycolysis and fatty acid oxidation (Figure 23F, right panel). None of them were deregulated except for the fatty-acid transporter Fabp3. Decrease of Fabp3 could partially explain the decline in mitochondrial activity. Nonetheless, Fabp3 was instead increased by macroH2A1.1 silencing (Figure 23F, right panel).

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A C si si Mito. DNA content nuclear / mito. mRNA Ctrl mH2A1.1 Tmem171 Itga11 1.5 Tspan8 1.5 Cdhr1 si Ctrl [D4] Rgcc Slc1a3 si mH2A1.1 [D4] Pi16 1.0 Myh7 1.0 Olfml3 F3 Cilp 0.5 H2afy 0.5 Casq1 Itga10 Camk1g mtDNA / gDNA / mtDNA mRNA ratio [AU] ratio mRNA Olfml3 0 Ptn 0 Ptpn5 IIVETC complex Pla2g2e Dio2 Plin4 B Itm2a Sparcl1 Ttn BM022387 DEGs with si macroH2A1.1 Ddc Bex1 Ms4a8a Sdsl Fap > 2 Myh2 8 Acp5 > 1.5 Serpina1a Serpina1c 6 > 1.2 Acp5 Myh1 4 Gm13889 Fabp3 Asb2 2 Fabp3 Cxcl13 0 Tceal3 Ddc

Fold-change Crym -2 Tceal7 Tceal6 -4 Myf6 Atp9a si macroH2A1-1 vs si Ctrl si vs macroH2A1-1 si Gm10639

D E updown

Rescue Itga11 Cdhr1 Tmem171Mstn Igf1 e-mH2A1.1 1.5 -- G224E * * * * * WT ns ns ns ns ns +---si Ctrl * * * * *

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Histone H3 0 si Ctrl + - - - + - - - + - - - + - - - + - - - si mH2A1.1 - + + + - + + + - + + + - + + + - + + + e-mH2A1.1 ------F WT WT WT WT WT G224E G224E G224E G224E G224E

ETC component genes Glycolysis regulation Fatty acid oxidation I ComplexI II III IV V

1.5 GM * si Ctrl [D4] 1.5 * * * * * si mH2A1.1 [D4] 1.0

1.0

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0 0 Cs Hk2 Cytc Sdha Glut1 Glut4 Pdk4 CD36 Cpt1b Mcad UCP2 Uqcrc2 Cox4i1 Atp5a1 Fabp3 CoxVb Ndufa9 Pdh1Ea

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Figure 23. MacroH2A1.1 decreases mitochondrial activity independently of gene regulation. (A) On the left, mitochondrial DNA (mtDNA) was normalized by nuclear DNA (gDNA, genomic DNA) by measuring the expression of the mitochondrial Ndufv1 and nuclear mt-Nd2 genes. On the right, transcriptional proper expression of electron transport chain complexes was tested. For this, we calculated the mRNA ratio of the expression of nuclear (mt-Nd3, mt-Co1) genes divided by the expression of mitochondrial genes (Ndufa9, Cox4i) coding for ETC complexes (I and IV). (B) Transcriptomic analysis of the deregulated genes, at day four of differentiation, comparing si macroH2A1.1 versus si Ctrl. The numbers in brackets refer to the number of genes inside each interval of indicated fold change (>1.2; >1.5 and >2). The genes on the left are downregulated, while the ones on the right are up-regulated. (C) Heat map of the top deregulated genes (fold change >2; p<0.05; blue, down-regulated; red, up-regulated). (D) Immunoblot of stable C2C12 cell lines, at day four, which express the Flag-tagged macroH2A1.1 or the mutant G224E. (E) Relative mRNA levels of macroH2A1.1-target genes in C2C12 cell lines described in (D). (F) Relative mRNA levels of ETC component genes, glycolysis and fatty acid oxidation- related genes. Data A-F are the mean of n=4-6 and n=3-4 independent experiments, respectively; +s.d. * p < 0.05; Student's t-test. n.s. not significant.

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MacroH2A1.1 binds and inhibits PARP1 activity through its macrodomain

Although the macrodomain of macroH2A1.1 is not involved in macroH2A1.1 gene regulation, we assessed if it was involved in the metabolic phenotype. Maximal mitochondrial activity was almost fully rescued by re-expression of wild-type macroH2A1.1 (from 20 to 80%, approximately) (Figure 24A). However, the mutant G224E rescued only partially mitochondrial activity (from 20 to 50% approximately) (Figure 24A). This suggests that the binding pocket, able to bind ADP-ribose, is implicated in the metabolic phenotype. Since several findings suggested that macroH2A1.1 inhibits PARP1 activity through ADP-ribose interaction we assessed the inhibitory capacity of both macroH2A1.1 WT and mutant in vitro (Figure 24B). As expected, increasing concentration of macroH2A1.1 macrodomain inhibits PARP1 auto- PARylation. On the contrary, G224E did not affect PARylation levels (Figure 24B). When transfected into human kidney HEK293T cells, binding to PARylated-PARP1 was observed with macroH2A1.1, but neither with G224E nor with macroH2A1.2 (Figure 24C). Similarly, macroH2A1.1, but not G224E, could precipitate PARP1 protein in C2C12 (Figure 24D). To further demonstrate the PARP1 inhibitory capacity is restricted to macroH2A1.1 WT we measured stress-induced PARylation in HepG2 DKD cells. As expected, the addition of hydrogen peroxide (H2O2) during 10min largely increased nuclear PARylation in response to PARP1 activation. This increase was recapitulated in control conditions and in cells expressing G224E (Figure 24E, 24F). On the contrary, PARP1 activation was decreased in cells expressing the WT form of macroH2A1.1 which confirms its specific inhibitory effect on PARP1 enzyme (Figure 24E, 24F).

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Figure 24. MacroH2A1.1 macrodomain binds and inhibits PARP1 activity. (A) Maximal respiratory capacity of C2C12 treated with si macroH2A1.1 expressing either macroH2A1.1 wild-type (WT) or G224E mutant (n=6, *p >0.05, Student's t-test). (B) Immunoblot of PARP1 auto-PARylation in presence of increasing macrodomain WT or mutant concentrations (0, 10, 25 and 50uM). Naphthol blue was used as a loading control. (C) Immunoblot of immunoprecipitated GFP (IP) and input from HEK293T transfected with macroH2A1 isoforms and mutant G224E. (D) Immunoblot of immunoprecipitated Flag

(IP) and input from C2C12 transfected with macroH2A1 WT or G224E. (E) PARylation immunofluorescence89 of HepG2 DKD, transfected with macroH2A1 WT or G224E, after H2O2 induction (0.1 mM, 10 min). YFP autofluorescence was used as infection control and DAPI stained nuclei. Maximal and minimal intensity are scaled by color. (F) Quantification of PARylation intensity comparing untreated to treated (H2O2) HepG2 DKD cells described in (E). Boxplot based on the median of n=40 cells per condition + s.d.; *p<0.05 Wilcoxon test.

PARP1 inhibition rescues mitochondrial activity

Based on our previous results we know that macroH2A1.1 binds and inhibits PARP1 activity in a binding pocket manner. Loss of macroH2A1.1 leads to metabolic defects. Therefore, we hypothesized that mitochondrial defect could be due to the loss of PARP1 inhibition which is known to be deleterious for metabolic outcomes. Thus, we investigated the role of PARP1 activity in metabolic regulation of C2C12 cells. PARP1 expression decreased during C2C12 differentiation at the mRNA levels while PARP2 was unchanged (Figure 25A). PARP1 reduction during differentiation was confirmed at the protein levels (Figure 25B). This would suggest that PARP1 expression is favored in proliferative cells. At day four of differentiation macroH2A1.1 protein was slightly more abundant than PARP1 (Figure 25C). Strikingly, mitochondrial activity was fully rescued by the pharmacological inhibition of PARP1 activity or its genetic reduction in si macroH2A1.1 treated C2C12 cells (Figure 25D). This was confirmed in C2C12 isolated mitochondria as well (Figure 25E).

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Figure 25. PARP inhibition rescues mitochondrial activity. (A) Relative mRNA levels of PARP1 and PARP2 upon differentiation in C2C12 (n=4; *p >0.05; Student’s t-test). (B) Immunoblot of PARP1 protein in proliferative (prol) and differentiated C2C12 cells (D4). (C) Immunoblot of endogenous PARP1 and macroH2A1.1 expression compared to purified PARP1 and macroH2A1.1 proteins (full PARP1 length (trevigen) and His-tagged macroH2A1.1 macrodomain were used). (D) Rescue of maximal respiratory capacity by

genetic (siRNA) or pharmacological Supplementary inhibition Figure of 4 PARP1 (PARP inhibitor: ABT888, 100nM for 16hs). (E) On the left, immunoblot of mitochondrial purification. The MacroH2A1.1 sensitive genes are not rescued by NMN and PARP inhibition. mitochondrial Ndufa9, coding for ETC(a) The complex purity of isolated I, mitochondria was was used checked as by western mitochondrial blot. control. Histone H3 was used as a nuclear control.(b) The On mRNA the levels ofright, Nmnat3 inrescue siRNA-treated of C2C12 mitochondrial cells was analyzed by activity RT-qPCR. Data is the independent experiments (*p<0.05). in isolated mitochondria by PARP inhibitors(c) No rescue (ABT888, of gene expression. 100nM The mRNA levels for of si16hs). macroH2A1.1-sensitive Data in genes D were-E analyzed are at day 4 control and rescue conditions with PARP inhibitor (100 nM PARP inhibitor ABT-888 for 16 hours as in Figure 6g) the mean of n=6 independent experiments24 hours + ass.d.; in Figure *p 7e) by>0.05, RT-qPCR. DataStudent is represented t- test).as mean + s.d. of three independent experiments. macroH2A1.1 alone and co-treatments were not significant.

1

91

Loss of PARP1 inhibition impairs NAD+ salvage

PARP1 is the main nicotinamide adenine dinucleotide (NAD+) consumer in eukaryotic nucleus and its hyperactivation leads to drastic NAD+ depletion. NAD+ cofactor and its salvage are essential for the redox reactions generating cellular energy (Figure 26A). Thus, we wondered if the mitochondrial phenotype could be explained by impaired NAD+ salvage pathway downstream of PARP1 inhibition loss. Therefore, we measured NAD+ abundance in proliferative and differentiated C2C12 cells treated or not with PARP inhibitors (Figure 26B). NAD+ levels rose with PARP1 inhibition in proliferative cells but were unchanged in terminally differentiated C2C12 (Figure 26B). This supports previous studies showing that myotubes are resistant to oxidative stress generated by PARP1 activity. In total cellular extract, NAD+ and its product, nicotinamide (NAM) were unchanged when modulating macroH2A1.1 expression (Figure 26C, left panel). However, increase of the NAD+ precursor nicotinamide mononucleotide (NMN) during differentiation was abolished with si macroH2A1.1 (Figure 26C, left panel). Since the levels of metabolites involved in NAD+ salvage pathway may differ between different subcellular compartments, we measured NAD+, NMN, and NAM in isolated mitochondria. Strikingly, NMN and NAD+ were significantly reduced in si macroH2A1.1 mitochondria, while NAM was increased (Figure 26C, right). This suggests that the mitochondrial NAD+ salvage is perturbed in C2C12 cells treated with si macroH2A1.1 (Figure 26A). Decrease of NAD+ and NMN led us to assess the regulation of key genes known to regulate NAD+ salvage (Figure 26D). Nicotinamide phosphoribosyltransferase (NAMPT) enzyme recycles NAM into NMN. On the other hand, nicotinamide mononucleotide adenylyltransferase (NMNATs) enzymes transform NMN towards NAD+. NAMPT is ubiquitously expressed in all cellular compartments. NMNAT1, NMNAT2 and NMNAT3 are located in the nucleus, cytoplasm and golgi apparatus,

mitochondria, respectively. Upon differentiation, NAMPT expression 92

remained stable (Figure 26D). On the contrary, different NMNATs were either up- or downregulated during C2C12 differentiation. Indeed, the nuclear and the mitochondrial NMNAT1 and NMNAT3, respectively, were increased at day four of differentiation (Figure 26D). Interestingly, NMNAT2 shown an opposite profile, being repressed during myogenesis (Figure 26D). This suggests a switch in the transcriptional program of NMNATs during muscle development. While NMNAT1 and NMNAT3 were unchanged in si macroH2A1.1 treated cells, NMNAT2 shown a slight increase (Figure 26D). Finally, none of the top deregulated genes with si macroH2A1.1 were rescued by PARP1 inhibition or NMN addition (Figure 26E). In conclusion, PARP1 and NMN are not involved in the regulation of macroH2A1.1-target genes.

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NAD+ recycling pathway * 1.6 untreated Nucleus Cytosol Mitochondria PARP inh. 1.2 NAM NAM consumers incl. PARP-1 NAM consumers 0.8 NAD+ Nampt NAD+

Nmnat1 NMN Nmnat3 0.4 NMN NMN Other NAD+ abundance 0 prol D4 C

Isolated mitochondria Total metabolite levels metabolite levels prol * 1.5 1.5 D4 si Ctrl * * * * D4 si mH2A1.1

1.0 1.0

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D

NAD+ recycling enzymes

3 prol * D4 si Ctrl 2 * D4 si mH2A1.1 * * 1 rel. mRNA level mRNA rel. 0 Nampt Nmnat1 Nmnat2 Nmnat3 Cytosol Nucleus Golgi Mito. Cytosol

E D4

4 si Ctrl si mH2A1.1 3 si Ctrl + PARP inh. si mH2A1.1 + PARP inh. 2 si Ctrl + NMN si mH2A1.1 + NMN

rel. mRNA level mRNA rel. 1

0 mH2A1.1 Itga11 Cdhr1 Igf1 Mstn Fabp3 Nmnat2

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Figure 26. Silencing of macroH2A1.1 leads to NAD+ depletion in a gene-independent manner. (A) Schematic representation of the NAD+ salvage pathway in the three subcellular compartments. (B) NAD+ levels were measured in proliferative (prol) and differentiated (DM=D4) C2C12 treated or untreated with PARP inhibitor (ABT888: 100nM, 16hs). (C) Relative levels of NMN, NAD+ and NAM metabolites in proliferative (prol) and differentiated (D4) C2C12. Total nuclei extract and isolated mitochondria from C2C12 treated with si Ctrl versus si macroH2A1.1 were used. (D) Relative mRNA level of enzymes regulating NAD+ salvage, during differentiation in C2C12 treated with si Ctrl versus si macroH2A1.1 (E) Relative mRNA levels of macroH2A1.1 top-regulated genes in C2C12 untreated or treated with PARP inhibitor or NMN addition (ABT888: 100nM, 16hs; NMN: 500nM, 24hs) at day 4. Data in B, C, D, E are the mean of n=4 independent experiments + s.d.; *p < 0.05; Student's t-test.

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Supplementation of NAD+ precursor rescue mitochondrial defect

Implication of the mitochondrial NAD+ salvage pathway in the metabolic phenotype was assessed using siRNA directed against the mitochondrial Nmnat3 (Figure 27A). Nmnat3 is the enzyme transforming NMN into NAD+. As expected, Nmnat3 reduction leads to mitochondrial deficiency (Figure 27B). This implies that NMN is placed at a central position in the NAD+ balance between nucleus, cytoplasm and mitochondria. Therefore, we attempted to rescue mitochondrial activity in C2C12 treated with simacroH2A1.1 by adding NMN in the medium. Impressively, NMN supplementation rescued mitochondrial activity of C2C12 treated with si macroH2A1.1 (Figure 27B). Rescue by NMN supplementation, of mitochondrial activity was recapitulated in isolated mitochondria (Figure 27C). On the other hand, NMN addition could not rescue C2C12 silenced with both si macroH2A1.1 and si Nmnat3 (Figure 27B). This confirms that loss of NMN replenishment explains mitochondrial defect when macroH2A1.1 is silenced. In summary, macroH2A1.1 maintains NAD+ salvage pathway, and therefore mitochondrial activity, by its capacity to inhibit PARP1 activity (Figure 27D).

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A

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mRNA 50 Percent of siCtrl of Percent

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0 0 sisi Ctrl Ctrl + - - - - si Ctrl Ctrl - -+si sisi mH2A1.1mH2A1.1 - + + + - si mH2A1.1 mH2A1.1 - + +si NMN M N + - + +N - NMN -+ +NMN sisi NMNAT3NMNAT3 - - - + +

hibition.D

was analyzed by RT-qPCR. Data is the mean + s.d. of three Nucleus 1.1-sensitive genesmH2A1.1 were analyzedPARP at day 4 of differentiation unde ibitor ABT-888 for 16 hours as in Figure 6g) and NMN (500 nM fo n + s.d. of threeNAD+ independent consumption experiments. Differences between si

NMN

NAD+ pool

Optimal Ox. Phos.

Mitochondria

Figure 27. MacroH2A1.1 indirectly influences the key NMN pool to maintain mitochondrial functions. (A) Relative mRNA level of the Nmnat3 gene represented in percent compared to si Ctrl (n=3; *p < 0.05, Student´s t-test). (B) Maximal respiratory capacity rescued by NMN supplementation (NMN: 500nM; 24hs) in C2C12 cells treated with si macroH2A1.1 or si Nmnat3. (C) As in (B), rescue of the maximal respiratory capacity in isolated mitochondria by NMN addition (500nM; 24hs). (D) Schematic summary of macroH2A1.1 maintaining NNM and NAD+ pools for proper mitochondrial activity (oxphos phosphorylation (Ox. Phos)) via PARP1 inhibition. Data from B-C are the mean of n=6 independent experiments, * p< 0.05; Student's t-test.

97

Conclusion Chapter I

MacroH2A1 isoforms during muscle differentiation. MacroH2A1.1, which is predominantly expressed in differentiated C2C12 cells, regulates energy homeostasis. We showed that macroH2A1.1 is able to bind and inhibit PARP1 activity. In this context, macroH2A1.1 prevents consumption of nuclear NAD+ by PARP1 enzyme. Thus, the balance in NAD+ pool is maintained between nuclear and mitochondrial compartments. When macroH2A1.1 is silenced, C2C12 cells lose this balance and mitochondrial capacity.

Note: This project was initiated by Melanija Posavec Marjanovic, a former PhD student in the lab. Many collaborators were involved. Monica Suelves and Pura Muñoz kindly provided us primary myoblast pellet or antibodies. Furthermore, as muscle experts, Monica Suelves and Pura Muñoz guided us during the entire project. Maximillian Lassi and Raffeaele Teperino performed most of the seahorse experiments. Along with Andrew Pospisilik, they also offered their expertise in cell metabolism. Miriam Navarro and Oscar Yanes performed and supervised all target metabolomics experiments. Ivan Ahel and Andreas Ladurner advised us for PARP assay and help us with their expertise in macrodomain field. Roberto Malinverni did all statistical analysis from Microarray and ChIP-seq experiments. David Corujo performed the PARylation immunofluorescence. Iva Guberovic performed PARP1 auto-PARylation assay. Many experiments were done in the lab or repeated by Vanesa Valero. Marcus Buschbeck, Melanija Posavec Marjanovic and myself conceived and supervised this project. I performed or guided several experiments. I supervised all final metabolic assays including last seahorse experiments and targeted metabolomics. Marcus Buschbeck, Melanija Posavec Marjanovic and myself prepared the revised manuscript. This work was published in Nature Structural Molecular Biology in 2017 (Posavec Marjanovic & Hurtado-Bagès, 2017. http://doi.org/10.1038/nsmb.3481).

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Chapter II

Metabolic phenotype is independent of gene regulation. However, several genes related to adhesion-, extracellular structure, migration, and fiber type-encoding genes were found to be deregulated in C2C12 treated with si macroH2A1.1. Furthermore, small morphological alterations were observed with si macroH2A1.1 and si macroH2A1.2. Interestingly, si macroH2A1.2 seemed to boost cell elongation while si macroH2A1.1 decreased it. Based on this observation we attempt to respond to those questions in this chapter:

*Are macroH2A1 isoforms involved in fusion process of muscle cells?

* Is this phenotype gene-related?

*If yes, do macroH2A1 isoforms act on gene regulation in an opposite manner?

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MacroH2A1 isoforms regulate late differentiation markers in an opposite manner

A silencing RNA protocol to reduce either macroH2A1.1 or macroH2A1.2 was used as previously described in chapter one (Figure 18A; Figure 28A, left). As expected, in si Ctrl, macroH2A1.1 expression increased at day four of differentiation (D4) in C2C12 (Figure 28A, right). While proliferative C2C12 cells highly express macroH2A1.2, its mRNA expression decreased during myogenesis and abolished in mature myotubes (D4) (Figure 28A, right). We successfully knocked-down macroH2A1 isoforms at the transcriptional and protein levels (Figure 28A, right; 28B). Interestingly, macroH2A1.1 expression was increased at D2 by macroH2A1.2 silencing (Figure 28A, right), This increase was conserved at protein level at D4 (Figure 28B). Additionally, si macroH2A1.1 significantly increased macroH2A1.2 at the mRNA level at D4 and subtly increased at the protein level (Figure 28A, right; 28B). Thus, transcriptional regulation of both macroH2A1s seems to be interconnected. Early myogenic markers such as Myod1 and Myog normally increased upon differentiation in all conditions (Figure 28C). However, two observations called our attention. First, the late myogenic marker creatine kinase (Ckm) was oppositely regulated by macroH2A1 isoforms. Indeed, Ckm was up- or down-regulated in si macroH2A1.1 and si macroH2A1.2, respectively (Figure 28C). During muscle development, competent cells for fusion expressed eMHC (embryonic myosin heavy chain) contractile protein. Strikingly, embryonic myosin heavy chain that was up-regulated in C2C12 cells silenced for macroH2A1.2 (Figure 28B). Since those data were strikingly consistent as the results obtained in chapter one (Figure 20D), we decided to further investigate macroH2A1 function in the late maturation process of C2C12 myogenesis. Control C2C12 myotubes were thin but long and well organized as well as closely aligned to each other (Figure 28D). Visually on the microscope, silenced-macroH2A1.1 myotubes were less elongated and disorganized and only a few big fiber were observed compared to the control (Figure 28D). On the contrary, silenced-

100 macroH2A1.2 myotubes were well organized, much bigger, more elongated

with huge "hand-like" structure characterized by myotubes with a huge number of nuclei (Figure 28D). Immunofluorescence of eMHC in the three conditions allows us to analyse total nuclei number and differentiation index (Figure 28E). The opposite fusion phenotype could not be explained by proliferation or differentiation changes. Indeed, total nuclei number (DAPI) was unchanged. Although an increase in differentiation index (nuclei number in eMHC positive cells / total nuclei number) was found with si macroH2A1.2, no change was observed with si macroH2A1.1 (Figure 28E).

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Figure 28. MacroH2A1 isoforms oppositely regulate cell fusion and myotube formation. (A) On the left, schematic representation of the silencing RNA protocol, as used in Chapter I (Figure 18A). On the right, relative mRNA levels of macroH2A1 isoforms in si Ctrl, si macroH2A1.1 and si macroH2A1.2 condition during differentiation. (B) Immunoblot analysis at day 4 of macroH2A1 isoforms, the late myogenic marker eMHC, and histone H3 in the three siRNA conditions. (C) Relative mRNA levels of early (Myod1, Myog) and late (Ckm) myogenic markers through differentiation time points. (D) At the top, phase contrast image of C2C12 with the three-siRNA condition at D4. At the bottom, eMHC immunofluorescence at D4. Nuclear DNA was stained by DAPI. (E) On the left, total nuclei number at D4. On the right, percent of differentiated eMHC positive cells at D4. (n=4 photos containing between 100 and 200 myotubes from 2 biological replicates; * p < 0.05; Student’s t-test). Data in A and C are the mean + s.d. or n=4; * p < 0.05; Student- test. n.s. not significant.

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MacroH2A1 isoforms modulate myotube formation through fusion We further analysed morphological change by assessing length and perimeter of 600 myotubes (Figure 29A, scheme). For this, we measured myotube length and perimeter from eMHC cells, and analyzed the distribution of nuclei (DAPI) per positive eMHC cells. Both myotube length and perimeter were significantly decreased or increased with si-macroH2A1.1 or macroH2A1.2, respectively (Figure 29A). Similarly, macroH2A1.1 silencing significantly diminished the number of nuclei per myotube. On the contrary, nuclei distribution per myotubes was strongly augmented with si macroH2A1.2 (Figure 29B). More precisely, the majority of si control and si macroH2A1.1 myotubes contains between 2 and 49 nuclei per fiber (Figure 29C). Interestingly, 70% and 80% of fiber contained between 2 and 14 nuclei in si-control and si-macroH2A1.1, respectively. On the other hand, 25% and 15% contained between 15 and 49 nuclei in si-control and si- macroH2A1.1, respectively (Figure 29C). This suggests that si-macroH2A1.1 disfavours late fusion events. This is supported by the slight enrichment of si macroH2A1.1 myotubes in the fraction composed by mononucleated C2C12 positive for eMHC (around 6%) (Figure 29C). Strikingly, si macroH2A1.2 myotubes were found to be enriched in the fractions from intermediate (from 15 to 49 nuclei/myotubes) to large myotubes (more than 50 nuclei/myotubes) (Figure 29C). With si macroH2A1.2, no mononucleated C2C12 were found, and myotubes containing from 2 to 14 nuclei/fiber represented only 40% of the total si macroH2A1.2 myotubes compared to 70% in control. Clearly, the silencing of both macroH2A1 isoforms impacts C2C12 fusion capacity in a reverse manner. MacroH2A1.1 KD significantly declines secondary fusion, which as a consequence, prevents the formation of large multinucleated myotubes and gives rise to disorganized small myotubes (Figure 29D). Conversely, si macroH2A1.2 boosted secondary fusion generating large and well organized myotubes (Figure 29D).

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Figure 29. MacroH2A1 isoforms oppositely regulate fusion. (A) On the left, schematic representation of length and perimeter parameters. On the right, violin graph showing myotube length and perimeter in pixels from a total number of 600 myotubes in si ctrl, si macroH2A1.1 and si macroH2A1.2 at D4. (B) Violin graph of the nuclei distribution per myotubes. (C) Percent of myotube distribution from four photos of two biological replicates. Myotubes were

distinguished in four groups: myotubes containing less than two, between two and 14, between 15 and 49, and more than 50 nuclei. Data in A-B are the median of 600 myotubes from two biological replicates, * p < 0.05, Wilcoxon test. Data 105 in C are the median of four photos from two biological replicates containing between 100 and 200 myotubes each, * p < 0.05, Student's t-test.

MacroH2A1’s dual role in gene regulation

While the metabolic phenotype was independent of gene regulation, we wondered if fusion-related genes would explain the fusion phenotype observed. In fact, the previous microarray comparing si macroH2A1.1 versus si Ctrl revealed deregulated genes involved in cell adhesion and migration, signaling and extracellular organization. Thus, we performed RNA-seq analysis comparing C2C12 transfected with si-Control, si- macroH2A1.1 or si-macroH2A1.2 at day four of differentiation. PCA analysis demonstrated that the three biological replicates clustered together (Figure 30A). Furthermore, the three siRNA conditions were located in distinct compartments with a high PC1 variance of 71% and a PC2 variance of 11%. Variance accounts for the variability found in the our data set. This suggests that all three conditions behave differently and independently of each other. Differential expression analysis was performed comparing si macroH2A1.1 versus si Ctrl, si macroH2A1.2 versus si Ctrl and si macroH2A1.2 versus si macroH2A1.1 (Figure 30B). Significant deregulated genes (DEGs) were found (red) and several ones were deregulated with a fold change greater than 2 particularly in si macroH2A1.1 compare to the si Control. Interestingly, the number of deregulated genes was increased when comparing si macroH2A1.2 versus si macroH2A1.1 (Figure 30B). This could be due to reversely regulated genes which did not appear significant with the comparison to the control. The silencing of both macroH2A1s leads to the deregulation of 1269 genes in total (padj <0.01; FC (<-1.6 & >1.6)) (Figure 30C). Among them, 995 were deregulated with the si macroH2A1.1 and 372 with the si macroH2A1.2. In total, 98 deregulated were shared by both si macroH2A1.1 and si macroH2A1.2 (Figure 30C). Although macroH2A was mainly associated with gene repression, we and studies from other suggest a dual role of

macroH2A1 regarding transcriptional regulation. In line with repressor 106

function, macroH2A1s silencing led to the up-regulation 61,7% (784) of all DEGs (1269). However, 33,2% (421) were down-regulated. From the 98 genes deregulated by both si macroH2A1s, 2,5% (34/1269) were deregulated in the same direction and 5,0% (64/1269) were deregulated in the opposite direction (the complete list is found in Annexe 1). MacroH2A1.1 silencing led to the deregulation of 995 genes which represents three times the number of DEGs in cells silenced for macroH2A1.2. This data suggests that macroH2A1.1 has a key role in the transcriptional program of C2C12 cells during myogenesis. Gene ontology (GO) analysis showed that up-regulated genes with si macroH2A1.1 belong to immune response, including response to virus and interferon-β (Figure 30D). Down-regulated genes with si macroH2A1.2 were enriched in the GO of metabolic and muscle processes. Interestingly, several biological processes related to muscle development were associated with genes up-regulated with si macroH2A1.2 and down- regulated by si macroH2A1.1 (Figure 30D, grey blocks). Among those genes, several code for proteins essential for extracellular matrix formation of the basement membrane, and cell-cell adhesion (Figure 30D, grey blocks). For instance, genes encoding integrin complexes, collagen and fibril were found to be oppositely deregulated. This was in line with the GO analysis obtained from the microarray of si macroH2A1.1 in chapter one (Figure 23C). Silencing of macroH2A1.2 leads to the opposite regulation of several of these genes (Figure 30D, grey blocks).

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Figure 30. MacroH2A1s deregulate several genes involved in muscle basement membrane. (A) Principal Component Analysis (PCA) of the RNA-seq experiment comparing three biological replicates of si Ctrl, si macroH2A1.1 and si macroH2A1.2 at D4. (B) MA plot of differential expression analysis found between the three conditions is shown. Log fold change scale was restricted from -6 to 6. Significant (p < 0.05) deregulated genes are colored in red. (C) Venn diagram of deregulated genes (p < 0.01; log2 fold change [-0.8; +0.8]. (D) Gene ontology analysis separating up- to down-regulated genes in the three siRNA conditions (-10 x log (pvalue <0.05)). Grey boxes highlight opposite GO.

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Opposingly regulated genes include a subset encoding extracellular matrix components

Since the fusion phenotype was found to be opposite between the silencing of both macroH2A1 isoforms, we mainly focus our attention on the 64 genes showing opposite profile. From now, the 64 reversely regulated genes will be called opDEGs (opposite deregulated genes) (Figure 31A, left). Interestingly, 46 genes were also found in the opposite GO categories related to extracellular matrix formation (ECM) and skeletal muscle development (Figure 30D; Figure 31D). Paintomics3 analysis performed on those 46 opDEGs demonstrated their involvement in several pathways associated with ECM formation such as ECM-receptor interaction, focal adhesion, proteoglycans, regulation of actin cytoskeleton, PI3K-Akt and Rap1 signaling pathways (Figure 31A, right). More importantly, several of the 46 opDEGs had a central position in ECM formation and focal adhesion such as fibronectin-1 (Fn1), Thrombospondin 1 (Thbs1) and collagen type I alpha 1 chain (Col1a1) (Figure 31B). Remarkably, we found several of the 46 genes in the same interaction nodes by string analysis, which predicts protein- protein interaction (Figure 31C). This was the case for Fn1, fibromodulin (Fmod), insulin like growth Factor 1 (Igf1), integrin subunit alpha 11 (Itga11), and several collagen (Col1a1, Col1a2, Col12a1 and Col8a2) among other (Figure 31C).

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Figure 31. The 46 opDEGs inversely regulates the same pathways. (A) On the left, representation of gene distribution of the 98 genes that are regulated by both macroH2A1 isoforms. Two main gene categories can be distinguished, (i) the genes deregulated in the same direction (34) and (ii) those that are going in the opposite direction (64) caused by the silencing of both macroH2A1 isoforms. The two previous categories are then divided by up-regulated versus down-regulated genes. On the right, pathway analysis of the 46 genes selected as candidates for the fusion phenotype by Paintomics3 web server. The list of the 46 genes was compared to the input list from the RNA-seq analysis comparing si macroH2A1.2 versus si macroH2A1.1 (Figure 30B). (B) Screenshot of one pathway that is significantly deregulated in the Paintomics3 analysis. ECM- and Focal

adhesion-related pathways is shown. (C) String analysis of predictable protein-protein interaction from our list of 46 genes is shown. 111

We further validated the opposite profile of few of the 46 opDEGs (Figure 31A). Five opDEGs up-regulated in si macroH2A1.1 were validated by qRT-PCR. Through differentiation, myosin heavy chain 1 and 4 (Myh1 and Myh4) 'were highly up-regulated at day four of differentiation in si control (Figure 31B). While this up-regulation was enhanced by si macroH2A1.1, it was abolished by si macroH2A1.2 (Figure 31B). A similar trend was observed for the fatty acid binding protein 3 (Fabp3) and ATPase phospholipid transporting 9A (Atp9a) genes. Nevertheless, decrease in si macroH2A1.2 did not reach significance (Figure 31B). Six of the ten opDEGs up-regulated with si- macroH2A1.2 increased during differentiation including Fn1, Col1a1, Itga11, Igf1, and Cilp (Figure 31C). Another two of the ten opDEGs were decreased upon differentiation including the transmembrane protein 171 (Tmem171), and Fmod. Finally, the regulator of cell cycle (Rgcc) and the Calcium/Calmodulin Dependent Protein Kinase IG (Camk1g) were initially up-regulated and later down-regulated at day 4 (Figure 31C). The last two genes were selected based on the fact that they were found to be downregulated in both microarray and RNA-seq analysis with a pvalue < 0.05. Briefly, for most of opDEGs, up- or down-regulation provoked by si macroH2A1.2 or si macroH2A1.1, respectively, was validated at day 4 (Figure 31C). This was true already at day 2 for a few genes such as Col1a1, Cilp, Tmem171, Fmod, Rgcc and Camk1g (Figure 31C).

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Figure 31. MacroH2A1 isoforms deregulated subset of genes in an opposite way. (A) table of the top 10 opDEGs. (B) Relative mRNA levels of opDEGs up-regulated with si macroH2A1.1 and down-regulated with si macroH2A1.2. (C) Relative mRNA levels of opDEGs up-regulated with si macroH2A1.2 and down-regulated with si macroH2A1.1. Data shown in B-C are the mean of n=4 independent experiments + s.d.; *p < 0.05; Student's t-test.

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PARP1 is not involved in the fusion phenotype

We previously showed that macroH2A1.1 maintained the NAD+ pool through PARP1 inhibition. Therefore, we wondered if a similar mechanism could explain the reduced fusion capacity observed in C2C12 treated with si macroH2A1.1. Through C2C12 differentiation PARP1 expression decreased, particularly between proliferative cells and D2 (Figure 32A). Silencing of both macroH2A1 did not affect the level of PARP1 mRNA during differentiation (Figure 32A). To assess a potential role of PARP1 in myogenesis, we reduced its expression with specific siRNA (Figure 32B). Interestingly, the PARP2 mRNA level was slightly increased by PARP1 reduction (Figure 32B). No obvious morphological change was observed with the loss of PARP1 at day four (Figure 32C). Neither proliferation or differentiation were affected by PARP1 loss (Figure 32D). Surprisingly, myotube length and perimeter were significantly reduced with si PARP1 (Figure 32E). However, the relative proportion of myotubes with different ranges of nuclei numbers was not affected (Figure 32F). In conclusion, PARP1 repression has only a minor influence on myogenic differentiation and myotube formation.

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Figure 32. PARP1 does not affect drastically the fusion capacity of C2C12 cells. (A) Relative mRNA levels of PARP1 during differentiation in si Ctrl, si macroH2A1.1 and si macroH2A1.2 conditions. (B) successful PARP1 silencing by siRNA. Relative mRNA levels of PARP1 and PARP2 at day 4. (C) eMHC immunofluorescence at D4 of si Ctrl versus si PARP1 conditions. Nuclear DNA was stained by DAPI. (D) On the left, total nuclei number at D4. On the right, percent of differentiated eMHC positive cells. (E) violin graph showing myotube length and perimeter in pixels from a total number of 600 myotubes

in si ctrl, si PARP1 at D4. (F) Percent of myotube distribution of five photos from three biological replicates. Myotubes were distinguished in four groups: myotubes containing less than two, between two and 14, between115 15 and 99, and more than 100 nuclei. Data in D-F are the median of n=5 photos containing between 100 and 150 myotubes from 3 biological replicates; * p < 0.05; Student's t-test). Data in D-F are the median of n=3 independent experiments; * p < 0.05; student-test. n.s. not significant. Data in E are the median of 600 myotubes from 3 biological replicates, * p < 0.05, Wilcoxon test.

PARP1 does not regulate candidate genes for fusion phenotype

MacroH2A1.1 and PARP1 can cooperate in the activation and repression of genes (REF). Although PARP1 silencing did not have a major effect on cell fusion, we tested its impact on opDEGs. However, in the context of opDEGs, we found that several opDEGs up- regulated by si macroH2A1.1 and down-regulated in si macroH2A1.2 were downregulated by si PARP1 (Figure 33A, left). These included Myh1, Myh4, and Myf6. OpDEGs up-regulated by si macroH2A1.1 (down-regulated in si macroH2A1.2) were largely unaffected by PARP1 silencing, (Figure 33A, right). As this includes those genes likely to be relevant for cell fusion processes, we conclude that the influence of macroH2A1 isoforms on fusion is most likely independent of PARP1 regulation. Nonetheless, we cannot discard any involvement of Myh1, Myh4 or Myf6 or any other genes that could accentuate indirectly fusion capacity. It is interesting to note that PARP1 silencing has the tendency to increase the expression of all macroH2A, as well as the cytoplasmic NMNAT2 enzyme (enzyme converting NMN to NAD) (Figure 33B). This suggests that PARP1 activity and macroH2A gene regulation are interconnected.

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Figure 33. PARP1 does not affect the fusion candidate genes. (A) Relative mRNA levels of opDEGs at day 4 in si Ctrl, si macroH2A1.1, si macroH2A1.2 and si PARP1 conditions. (B) Relative mRNA levels of macroH2A isoforms and genes coding for NAD+ salvage previously introduced in chapter one (Figure 26A-D). All data are the mean of n=4 independent experiments + s.d.. * p < 0.05. Student's t-test. n.s: not significant.

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Myogenic program is impaired in muscle- derived cancer

Rhabdomyosarcoma is a soft-tissue sarcoma that is considered to derive from muscle precursor cells. Rhabdomyosarcoma cells express myogenic markers such as MYOD1 and MYOG but do not fully differentiate due to an aberrant transcriptional program. We decided to study the role of macroH2A1 in rhabdomyosarcoma. For this study, we used the Rh30 cell line to investigate its differentiation capacity under the same conditions used for C2C12 cells (change of growth medium to differentiation medium). In line with the inability to achieve terminal differentiation, Rh30 cells did not express the differentiation marker eMHC at day 4 (Figure 34A). Additionally, the expression profile of the earlier markers, MYOD1 and MYOG, does not follow the profile of myogenesis in C2C12 (Figure 34B). In C2C12 cells, Myod1 levels continuously increased during myogenesis and Myog levels started to increase from D2 (Figure 34B). On the contrary, Myod1 decreases during Rh30 upon "differentiation" (Figure 34B). Myogenin is aberrantly expressed in proliferative Rh30 and decrease during Rh30 "differentiation" (Figure 34B). Nonetheless, in the case of ckm, a late myogenic marker, it is aberrantly expressed in proliferative cells but recover a normal profile at D4 with a drastic increase (Figure 34B). Interestingly, opDEGs up-regulated in C2C12 with si macroH2A1.1 did not shown strong deregulation in Rh30. Although we cannot compare the absolute levels of MYH1, MYH4 and MYF6 between the two cell lines, they followed the same trend as for C2C12 cells, with an increase at day 4 (Figure 34C, right). This would suggest that the transcription state of MYH1, MYH4 and MYF6 is not affected in Rh30. In contrast, opDEGs up- regulated in si macroH2A1.2 in C2C12 cells were largely unaffected when switching Rh30 cells to differentiation medium (Figure 34C, left). This reinforces our hypothesis that opDEGs up-regulated in si-macroH2A1.2 are essential in the fusion phenotype observed in C2C12. The lack of their regulation might contribute to fusion-deficiency in rhabdomyosarcoma.

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Figure 34. Aberrant transcriptional program and fusion capacity in rhabdomyosarcoma. (A) Immunofluorescence of the late myogenic marker eMHC, at day 4, in C2C12 and Rh30 cell lines. Nuclear DNA was stained by DAPI. (B) Relative mRNA levels of early and late myogenic markers in C2C12 and Rh30 during differentiation. (C) Relative mRNA levels of opDEGs genes. The colored balls refers to the normal profile of the opDEGS in C2C12 with si Ctrl (Figure 31B-C). Data in B-C are the mean of n=4-5 independent experiments +s.d.; p < 0.05; Student's t-test.

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MacroH2A1’s switch is impaired in muscle-derived cancer

Based on the impaired transcriptional regulation in RMS, we analysed the expression profile of macroH2A1 isoforms in RMS cell lines and myoblasts from healthy donors. We observed that, in healthy samples, both macroH2A1 isoforms are expressed in proliferative cells (Figure 35A), while macroH2A1.1 became the predominantly expressed isoform after inducing differentiation (Figure 35A). This reconfirmed the switch in macroH2A1 isoform expression described in chapter one (Figure 18). Such a macroH2A1 isoform switch did not properly occur in rhabdomyosarcoma cell lines, Rh30 and Rh4 (Figure 35A). While macroH2A1.1 levels increased slightly in Rh4 and Rh30 upon differentiation, macroH2A1.2 mRNA levels remain high even after several days of differentiation (Figure 35A). This was further supported at the mRNA and protein levels (Figure 35B). In C2C12 cells, macroH2A1 switch is very apparent from day 2 to day 4. In Rh30 cells, the macroH2A1 isoform switch is only subtle (Figure 35B, left). Indeed, Rh30 express similar levels of macroH2A1.1 in proliferative versus "differentiated" cells at day 4. On the other hand, loss of macroH2A1.2 is much weaker at day 4 in Rh30 compared to C2C12 (Figure 35B, left). Impaired macroH2A1 switch was recapitulated at the protein level (Figure 35B, right). Surprisingly, both macroH2A1 isoforms, but in particular macroH2A1.2, were more abundant in Rh30 cells when compared to C2C12 (Figure 35B, right). Aberrant overexpression of macroH2A1 proteins is accompanied by aberrant overexpression of myogenin compared to C2C12 (Figure 35B, right; Figure 34B). If increased myogenin expression is related to macroH2A1 splicing switch deregulation, further investigation is required.

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Figure 35. MacroH2A1 isoform switch is impaired in rhabdomyosarcoma cell lines. (A) On the left, schematic representation of RFLP experiment, as described in Chapter I (Figure 18A). On the right, RFLP experiment performed on human rhabdomyosarcoma cell lines and healthy myoblast donor. Digested cDNA from macroH2A1.1 by HpaII/MspI enzymes, gives rise to the upper band at 191bp. MacroH2A1.2 digestion generated two bands at 121 and 79 bp. (B) On the left, relative mRNA of impaired macroH2A1 isoforms splincing switch in Rh30 compared to C2C12 during differentiation. On the right, immunoblot analysis of impaired macroH2A1 isoforms switch during myogenesis. eMHC and Myog were used as myogenic markers while Histone H3 was used as a loading control. Data in B (left) are the mean of n=4-5 independent experiments +s.d.; * p < 0.05; Student's t-test.

121

MacroH2A1.2 silencing slightly rescues myogenic marker expression, but is not enough for full fusion recovery

High levels of macroH2A1.2 expression is generally associated with cell proliferation, but its role in cancer development is mainly context-dependent (Gamble et al., 2010). We observed that macroH2A1.2 silencing enhanced fusion in C2C12 cells. Therefore, we speculated that the modulation of macroH2A1 isoforms could be used as new therapeutic approaches to induce fusion of RMS. For this purpose, we investigated if the reduction of macroH2A1.2 would be able to induce differentiation and fusion of Rh30 after changing growth medium to differentiation medium. When analyzed after four days of “differentiation”, siRNA directed against macroH2A1.2 had successfully reduced its expression by 60% without having affected macroH2A1.1 expression (Figure 36A, right). Intensity of eMHC expression seemed to have slightly increased with the si macroH2A1.2 in Rh30. This was associated with an increase in elongated cells (Figure 36A, left). Interestingly, early and late myogenic markers including Myod1, Myog and Ckm were significantly up-regulated in Rh30 silenced for macroH2A1.2 (Figure 36B). However, expression of most opDEGs was not affected on the mRNA level (Figure 36C). We could only observe a slight increase in the mRNA of integrin subunit alpha 11 (Itga11). Altogether, increase in Myod1, Myog, Ckm and Itga11 expression will have to be confirmed at the protein level. Additionally, to know if their up-regulation is involved in the slight increase in myoblast elongation observed in Figure 36A, further investigation will be required. In summary, macroH2A1.2 enhances myogenic marker expression associated with an increase in elongated cells. However, increase of Rh30 differentiation is not as apparent. This suggests that other factors would need to be manipulated, in addition to macroH2A1.2, to push proper differentiation and fusion of rhabdomyosarcoma cells.

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Figure 36. MacroH2A1.2 slightly improves rhabdomyosarcoma phenotype. (A) On the left, immunofluorescence of the late myogenic marker eMHC, at day 4 in Rh30 cell lines treated with si Ctrl or si macroH2A1.2. Nuclear DNA was stained with DAPI. Below, a zoom-out in greyscale of a square section from the upper image is shown. The white arrows indicate elongated cells. On the right, knock-down efficiency of the siRNA directed against macroH2A1.2 (relative mRNA level) (B) Relative mRNA levels of myogenic markers at day 4 with si Ctrl or si macroH2A1.2. (C) Relative mRNA levels of the opDEGs selected from C2C12 experiment, at day four with si Ctrl and si macroH2A1.2 conditions. Data from B-C are the mean of n=4 independent experiments +s.d.; * p < 0.05; Student's t-test.

123

Conclusion Chapter II

MacroH2A1 isoforms regulate muscle fusion at different time points. Proliferative cells favour macroH2A1.2, while differentiated muscle favours macroH2A1.1 expression. MacroH2A1.1 promotes fusion during late differentiation while macroH2A1.2 inhibits it. This is accompanied by opposing regulation of genes encoding relevant proteins of basal membrane, extracellular matrix formation, cell-cell adhesion, integrin complexes, and collagen fibrils. PARP1 is not involved in the regulation of macroH2A1-target genes potentially involved in the fusion phenotype. Further work will be needed in order to understand the molecular mechanism by which both macroH2A1 isoforms affect this subset of genes. MacroH2A1 is impaired in Rhabdomyosarcoma cell lines. Silencing of macroH2A1.2 slightly boosted cell elongation but did not drive complete differentiation process. This suggests, that macroH2A1.2 prevents muscle differentiation/fusion processes. Thus, its silencing seems beneficial as a differentiation-based therapy approach. However, si macroH2A1.2 on its own has so far been observed to be enough to recapitulate full differentiation and fusion processes.

Note: I initiated this project after the former PhD student, Melanija Posavec Marjanovic, shared some of her ideas and data with me. Later on, Roberto Malinverni was involved in all RNA-seq analysis. David Corujo wrote the script allowing to analyse myotubes. The technician Vanesa Valero supported me by performing many immunoblots and qRT-PCR. Finally, the master student, Johanna Albert, was involved in the generation of the PARP1 data. I supervised all aspects of this project (with the support of my thesis director, Marcus

Buschbeck) and generated the largest part of the results and performed most of the

124 data analysis.

Chapter III

The unique difference between the two macroH2A1 isoforms in vertebrates is located in the use of mutually exclusive exons. Both proteins differ by 32 amino acids in their macrodomain. Several of these amino acids are known to be crucial for PAR and ADP-ribose binding (D203, G224) by macroH2A1.1. Beyond vertebrate lineage, macroH2A sequence has been recently found in the unicellular eukaryote Capsaspora owczarzaki (Rivera-Casas et al., 2016). Two initial studies investigated the evolution of macrodomain-containing proteins (Pehrson & Fuji, 1998; Rack et al., 2016b). However, we are the first to focus on the phylogenetic history and diversification of macroH2A macrodomain throughout evolution. This intrigued us to ask questions regarding the functions of ancestral macroH2A: * Is ADP-ribose binding the ancestral and original function of macroH2A? * Is a metabolic role of macroH2A conserved in Capsaspora owczarzaki?

125

MacroH2A appeared at the node towards multicellularity and is conserved throughout evolution

We traced back the origin of macroH2A at the node towards multicellularity, right before the appearance of the metazoan lineage. Indeed, the macroH2A sequence was found in the unicellular amoeba Capsaspora owczarzaki (Caps. owc.) (Figure 37A). So far, we could not find the macroH2A sequence in other groups close to Caps. owc. such as Choanoflagellata, Ichthyosporea or Fungi. This suggests that macroH2A appeared in the holozoa lineage. Interestingly, we could detect only one macroH2A variant in all protostomes and early deuterostomes (non-vertebrate chordates, hemichordates and echinoderms) (Figure 37A). Ancestral macroH2A is most similar to the mammal macroH2A1.1 sequence (Annex 2, 6). It is believed that the whole-genome has been duplicated in vertebrate ancestors (Dehal & Boore, 2005). In this context, a new version of macroH2A arose by duplication and most likely diversification, giving rise to the macroH2A2 isoform (Figure 37A). The second mutually exclusive exon of macroH2A1 encoding macroH2A1.2 splicing variant appeared later in vertebrate evolution (Figure 37A) (All amino acids alignment can be found in Annex 2, 3, and 4). It is interesting to note that all three macroH2A isoforms undergo strong diversification in the fish lineage (Annex 2, 3, and 4) Since the major difference between macroH2A isoforms is located in their macrodomain sequences, we then decided to analyse the rates of evolution of each exon 5 in vertebrates compared to macrodomain full length (Figure 37B). All macroH2A had a rate of evolution in between fast-evolving histone (H2A.Bbd) and slow-evolving histone (H2B) (Figure 37B). The most recent isoform, macroH2A1.2, showed the lowest levels of variation, followed by macroH2A1.1 and macroH2A2 (Figure 37B). MacroH2A1.2 macrodomain was also the most conserved since it appeared in vertebrates. The key residues in the macrodomain for ADP-ribose binding are found in the exon 5 of Caps. owc.. Thus, we analysed the amino acid and nucleotide p-distances (Figure 37C, Annex 5), as well as positive and negative selective pressure (Annex 5).

126 Calculation of the p-distances refers to the estimation of the divergence of

each exon. Conservation of the exon 5 of macroH2A1.2 macrodomain is even more pronounced when comparing the rates of evolution with the two other macroH2A isoforms (Figure 37B-C, Annex 5). All macrodomains shown selective pressure (Annex 5). Overall, the exon 5 of Caps. owc. of all macroH2A macrodomain was found to be well conserved throughout evolution (Figure 37D). Strikingly, exon 5 of Caps. owc. is highly similar to the protein sequence of the macrodomain of macroH2A1.1. Furthermore, the known amino acids essential for ADP-ribose binding (D203 and G224), present only in macroH2A1.1 macrodomain, were also conserved in the Caps. owc. sequence (Figure XD).

127

A B Vertebrates

mH2A1.1 mH2A1.2 mH2A2

mH2A.1 mH2A.2

mH2A Amino acid substitutions per 100per substitutions Aminoacid sites

Divergence Time (MYA)

mH2A mH2A1.1

mH2A1.2

mH2A2 C Global aa distances Global nt distances Exon 5 p-distancestd. error p-distance std. error mH2A1.1 0.123 0.03 0.100 0.01 mH2A2 0.161 0.03 0.181 0.02 mH2A1.2 0.062 0.02 0.067 0.01

D

mH2A1.2

mH2A2 Evolutionary time

mH2A1.1

*** known residues essential for ADP-ribose non.verteb. (D203, G224) mH2A

128

Figure 37. The histone macroH2A1.1 is the oldest isoform appearing before metazoan kingdom. (A) Tree of life based on the estimated divergence times of selected species. MacroH2A origin (thunderbolt), gene duplication (X2), and splicing isoform emergence (+1) are depicted in the tree. Red crosses refer to macroH2A loss events. Estimated divergence times were retrieved from the TimeTree database (www.timetree.org). (B) The estimation rates of evolution for macroH2A proteins in vertebrates is shown. Based on the species divergence times, defined in the TimeTree database, the Pairwise amino acid identities are represented. For each macroH2A, the macro domain are in dashed lines, and the exons 5 are in solid line. As reference, we used the evolutionary rates for fast-evolving histone H2A.Bbd, slow-evolving histones H2B, ans histone H1 (González-Romero et al., 2015). (C) p-distance of the amino acid (aa) and nucleotide (nt) of the exon 5 from all macroH2A in mammals, birds and reptiles. (std error: standard error). (D) Logo plots comparing the exon 5 of all macroH2A in vertebrate versus Caps. owc. (non-vertebrate). Residue colors are based on their biochemical properties: positively charged in blue, negatively charged in red, hydrophobic in black and polar residues in grey. Grey square shows conservation of the most abundant residue. Lighter grey squares are shown when the conservation is uncertain. White squares represent amino acid sharing similar electrostatic properties.

129

Generation of Caps. owc. macroH2A and PARP1 antibodies

In order to further study the role of macroH2A in unicellular species such as Capsaspora, we successfully generated specific antibodies against macroH2A and PARP1. For this, we followed two approaches. For the Caps. owc. macroH2A antibody, we used recombinant Caps. owc. macroH2A macrodomain as the antigen (Figure 38A). After injection with macroH2A macrodomain, rabbits were boosted for immunization four times. The serum from rabbit was collected and antibody efficiency was tested by immunoblot. In HEK293T cells transfected with the construct GFP- Caps.owc.macroH2A, we successfully detected a specific band for macroH2A (Figure 38A). For PARP1 antibody production, we used synthetic peptides as antigens. Peptides were designed from the N-terminal tail of PARP1 and injected into rabbits for serum production (Figure 38B). In vitro, we successfully obtained a specific band for His- Caps.owc.PARP1 produced protein (Figure 38B). Nucleotide alignment between mouse and Caps.owz. macroH2A and PARP1 are in Annex 6.

130

A MacroH2A Caps. owc. antibody production

H2A-like fold domain Linker Macrodomain HEK293T

N C Caps. owc. macroH2A + GFP

1 116 183 368 Caps.owc. Protein Production (His-tag) Caps.owc.

adherent aggregative cystic

adherent Injection aggregative cystic

O GFP_Caps.owc.mH2A

Caps.owc. mH2A

H3 Antibody Antibody production (Serum)

B

PARP1 Caps. owc. antibody production

DBD AD Catalytic Domain

N C Caps. owc. PARP1 1 316 600 968

+ His

Protein production

Peptide synthesis

amino acids PET28-vectornon-inducedinduced His-PARP His-PARP 103-114 132-142 299-310 Caps.owc. PARP1

Injection

Antibody Antibody production (Serum)

Figure 38. Capsaspora owczarzaki macroH2A and PARP1 antibodies production. (A) Schematic representation of Caps. owc. macroH2A antibody production and its validation. Caps. owc. macrodomain was produced in bacterial culture and then injected into rabbits. Serum from immunized rabbits was collected, and tested by immunoblot of HEK293T transfected cells with GFP-tagged macroH2A (Casp. owc.). Histone H3 was used as a loading control. (B) Schematic representation of Caps. owc. PARP1 antibody production and its validation. Three peptides from the N-ter of PARP1 (numbers indicated) were used as antigens. Serum from immunized rabbits was collected. The specificity of PARP1 antibody was tested by immunoblot with His-PARP1 protein produced in

bacteria into pET-28a(+) vector (Casp. owc.). Bacterial lysate with no

131 induction of PARP1 was used as a negative control.

Capsaspora owczarzaki macroH2A inhibits PARP1 activity

Capsaspora owczarzaki is nowadays used as a model to study the origin of metazoan multicellularity thanks to its large genetic repertoire, as well as its proteomic complexity (Sebé-Pedrós et al., 2011). Under specific culture conditions, Caps. owc. may differentiate in three distinct life cycles (Figure 39A). In the filopodial stage, the unicellular Caps. owc. amoebas proliferate, grow and develop filopodia to crawl over its substrate. This may be followed, upon stimuli, by the multicellular stage formed by the aggregation of Caps. owc. expressing cohesive extracellular material to gather together. Finally, Caps. owc. resistant form, called cystic stage, has drastic drop regarding transcriptional activity and represents a quiescent cycle life (Sebé-Pedrós et al., 2013) (Figure 39A). We could successfully detect Caps. owc. expression of macroH2A and PARP1. While macroH2A is similarly expressed in all stages, PARP1 is absent in the cystic stage (Figure 39B). Using an in vitro (biochemical) assay for PARP1 autoPARylation, we could demonstrate that, similar to the mouse macroH2A1.1 macrodomain, Caps. owz. macrodomain is able to inhibit PARP-1 activity (Figure 39C). In fact, its inhibitory capacity was even stronger compared to mouse macroH2A1.1 macrodomain.

132

Figure 39. Capsaspora owczarzaki macroH2A inhibits human PARP1 activity. Scheme of Capsaspora life cycle with the adherent, aggregative and cystic stages (Adapted from Sebé-Pedrós et al., 2013). (B) Immunoblot analysis of Caps. owc. PARP1 and macroH2A expression through Caps. owc. life stages. (C) Immunoblot of human PARP1 auto- PARylation in presence of increasing macrodomain from mouse or Caps. owc. Two concentrations of the macrodomain from mouse macroH2A1.1 were used as reference for inhibitory capacity (10, 50uM). The macrodomains of Caps. owc. macroH2A WT or mutant (G224E, G314E) were added at four different concentrations (0, 10, 25 and 50uM). Naphtol blue was used as a loading control.

133

Conclusion Chapter III

The ancestral macroH2A appeared right before the emergence of the metazoan lineage and is present in Capsaspora owczarzaki. It is similar to the macroH2A1.1 isoform. Gene duplication gave rise to macroH2A2. Later on, diversification of the H2FAY/macroH2A1 gene generated macroH2A1.2 with a differently composed exon 5. This leads to loss of ADP-ribose binding capacity of macroH2A1.2. It is also interesting to note that macroH2A has been sporadically lost in some species or lineages. For instance, macroH2A sequence is absent in invertebrate genomes such as Drosophila spp., the nematode Caenorhabditis elegans or the tunicate Ciona intestinalis. Strong diversification of all macroH2A isoforms was observed in fish lineage. It would be interesting to investigate what factors trigger such gene loss or diversification throughout evolution. Capsaspora owczarzaki macroH2A is expressed in all life stages and inhibits the human PARP1 activity. Further investigation is required to test if the role of macroH2A, related to metabolism and cell- cell fusion, is conserved in Capsaspora owczarzaki species.

Note: For this project, we collaborated with the chromatin biologist Juan Ausió and his team (University of Victoria, Canada) for antibody production. We also collaborated with the computational chromatin evolution group of Jose Maria Eirin-Lopez (Florida International University, USA). Many of their data are shown in this chapter including all computational analysis. Finally, we collaborated with the group of Iñaki Ruiz-Trillo (CSIC and Universitat Pompeu Fabra, Barcelona, Spain) who kindly provided us with Capsaspora material for immunoblot analysis. Finally, my PhD colleague, Iva Guberovic, is also involved in this project. She did excellent work and some of her data have been incorporated here (PARP1-assay; antibody production). I initiated this project after a few months of intense reading on macroH2A evolutionary history. Marcus Buschbeck, Iva Guberovic and myself supervised all of this project and performed all data and final

analysis. 134

Discussion

Discussion

I | MacroH2A1.1-PARP1 axis regulates NAD+ metabolism and metabolic homeostasis

The large distribution of macrodomains, PARPs and NAD+ signaling among all domains of life, as well as their diversification across vertebrates, further strengthen their essential role in cellular processes (reviewed in Perina et al., 2014; Gossmann & Ziegler, 2014). Proteins containing macrodomains might have co-evolved with enzymes consuming NAD+ such as PARP and Sirtuins (De Souza & Aravind, 2012; Gossmann & Ziegler, 2014)

| 1.1 | MacroH2A1.1-PARP1 axis

MacroH2A1.1 and PARP1 have been shown to act on transcriptional activity by two ways. (1) Activated PARP1 is recruited to chromatin by macroH2A1.1. Together they act in concert to regulate gene expression. (2) On the other hand, macroH2A1.1 binds and inhibits PARP1 activity which affect whole cellular metabolism.

1. Together macroH2A1.1 and PARP1 have been shown to regulate a subset of genes. For instance, overexpression of macroH2A1.1 leads to the repression of the inducible Hsp70.1 gene via PARP1 recruitment and inhibition of its activity (Ouararhni et al., 2006). Activation of Hsp70.1 promoter upon heat hock requires release of both macroH2A1.1 and PARP1 from Hsp70.1 promoter due to PARP1 hyperactivation (Ouararhni et al., 2006). This is an example showing how macroH2A1.1 and PARP1 may act together to regulate inducible genes.

This was supported in fibroblasts, where macroH2A1.1-bound PARP1 139

recruits the histone acetyltransferase CBP. This leads to H2B K12 and K120 acetylation, which either positively or negatively regulates the expression of macroH2A1-target genes (Chen et al., 2014). Changes in energy homeostasis may impact gene expression and therefore muscle plasticity. For this, energy-sensing molecules senses energetic and metabolites variation including NAD+ pool (reviewed in Freyssenet, 2005). For this reason, we tested to see if PARP1 inhibition or NMN supplementation impacted macroH2A1.1 target genes. Neither PARP1 inhibition or NMN addition had an effect on macroH2A1.1 target genes. However, both were clearly involved in the metabolism phenotype. Therefore, we conclude that the metabolic phenotype observed in si macroH2A1.1 was mainly independent of gene regulation. The role of macroH2A1.1 in metabolism was further supported in preadipocyte differentiation by inhibiting regulatory genes such as Wnt10b (Wan et al., 2017) Nonetheless, we need to comment that mitochondrial activity was partially rescued by the binding mutant (Figure 24A). This suggests, that the macrodomain of macroH2A1.1 is indeed involved in the maintenance of mitochondrial NAD+ pool, but other mechanisms may play a role in a macrodomain-independent manner.

2. Since the fusion phenotype seemed to be related to gene regulation, we tested the impact of PARP1 inhibition over candidate genes. PARP1 silencing did not explain the opposite deregulated genes in the observed fusion phenotype (Figure 33A, right). Furthermore, no major changes were observed related to fusion capacity in C2C12 cells silenced for PARP1 (Figure 32). This suggests that PARP1 is not a major contributor for muscle fusion, and PARP1 recruitment is not required for the regulation of those macroH2A1.1 target genes. However, Myh1, Myh4 and Myf6 were down-regulated in si PARP1, following the same trend as si macroH2A1.2 (Figure 33A, left). In this case, it is interesting to note that si PARP1 leads to opposite gene regulation compared to si macroH2A1.1. This could suggest that macroH2A1.1 antagonizes PARP1 regulation on specific

subset of genes. This would be in line with the identified direct inhibition 140

of PARP1 by macroH2A1.1 binding (Figure 24B). To clarify this, we would need to test the requirement of the binding pocket for the regulation of these genes.

| 1.2 | MacroH2A1.1-PARP1 axis regulates NAD+ pool

Proliferative cells mainly rely on glycolysis for the rapid synthesis of nucleic acid during DNA replication and replication-associated DNA repair. On the contrary, differentiated tissues have a high demand of ATP, mainly sustained by mitochondria for muscle contraction. To replenish the NAD+ pool, cells favor the salvage pathway due to its high enzymatic rate requiring only a few reactions compared to NAD+ synthesis de novo (Verdin, 2015). Imbalance in the subcellular NAD+ pool may have drastic outcomes on whole energy metabolism in the cells. In this cell line, hyperactivation of PARP1 leads to depletion of cellular NAD+ (Hassa et al., 2006). This leads to mitochondrial dysfunction, and cell death (Virág et al., 1998). Thus, imbalance of nuclear NAD+ metabolism could affect the function of other organelles through the NAD+ pool.

PARP1 was shown to protect differentiated myotubes from oxidative stress (Oláh et al., 2015). We showed that macroH2A1.1 silencing provoked mitochondrial disorder due to the loss of PARP1 inhibition in differentiated cells. This mechanism was mainly due to the capacity of the macroH2A1.1 macrodomain to interact with PARP1 via ADP-ribose binding (Figure 24C, D). Therefore, we conclude that macroH2A1.1 and PARP1 form an interconnected axis controlling metabolic outcomes. In this context, we speculate that during differentiation, muscle cells change their chromatin composition to switch their metabolic activity. In this case, macroH2A1.2 expression is replaced by macroH2A1.1 to favor NAD+ consumption in the mitochondria rather than in the nucleus.

Interestingly, several findings suggest that PARP and sirtuins may compete for NAD+ accessibility (Verdin, 2015). As an example, the drastic decrease of NAD+ observed during prolonged PARP1 activation leads to reduced sirtuin 1 activity (Bai et al., 2011). Interestingly, macroH2A1.1 was proposed to act as a 141

recruitment platform for HDAC1 deacetylase (Chakravarthy et al., 2005). In this context, further work is required to determine if a macroH2A1.1-sirtuin axis exists.

| 1.3 | MacroH2A1.1-PARP1 axis in vivo

Controversial phenotypes was observed in macroH2A-/- and PARP1 -/- mice (reviewed in Hurtado-Bagès et al., 2018). In the context of metabolic processes, loss of PARP1 activity was mainly related with beneficial outcomes while macroH2A1 depletion was deleterious. Removal of all macroH2As, without distinguishing between isoforms, may explain the contradictory results obtained in mice. In our hands, we demonstrated that macroH2A1.1 depletion in C2C12 had a dramatic impact on mitochondrial activity. However, supplementation with NMN rescued mitochondrial function. This encourages the usage of dietary supplementation for metabolic-related disease, already tested in mice (Yeung et al., 2016). Inhibition of PARP1 enhances mitochondrial function (Pirinen et al., 2014). Therefore, we wonder if the combination of both NMN supplementation, and PARP1 inhibition could be used to prevent metabolic alterations occurring in metabolic-related disease or during aging. In collaboration with Raffaele Teperino (Environmental Epigenetics, Munich, Germany), mice lacking macroH2A1.1 have been generated. We assessed a complete physiological screening analysis (in collaboration with the German Mouse Clinic, Munich, Germany), including metabolic parameters. We are unable to share the results in my thesis for confidentiality reasons, but we can confirm that the results look promising.

142

II | Dual role of macroH2A1 isoforms on transcriptional regulation

Contradictory reports in the last two decades suggest that macroH2A1 isoforms may play a more complex role in gene regulation than a simple repressive function that was initially postulated by Pehrson and colleagues (Costanzi & Pehrson, 1998). Positive or negative transcription regulation by macroH2A1 isoforms is largely context-dependent (reviewed in Gamble & Kraus, 2010).

| 2.1 | Dual role in cancer phenotype

Interestingly, macroH2A1 splicing is repetitively impaired in cancer (Novikov et al., 2011; Sporn et al., 2009). MacroH2A1.1 and macroH2A2 are associated with tumor suppressor features and differentiated cell states, while the function of macroH2A1.2 was dependent on the cancer context by acting as pro-cancerous in some cases and as tumor- suppressive in others (reviewed in Cantariño et al., 2013). It has recently been shown that macroH2A1.2 reduces prostate cancer-induced osteoclastogenesis (Kim et al., 2018). A tumour suppressive role was also attributed to macroH2A1.2 in lung metastasis and bladder cancer (Kapoor et al. 2010; Park et al. 2016). In contradiction to tumour suppressor features, macroH2A1.1 expression was positively correlated with epithelial-mesenchymal transition and poor survival of triple- negative breast cancer (Lavigne et al., 2014). In murine breast cancer cells, macroH2A1 isoforms were shown to have a differential impact on expression of a subset of genes (Dardenne et al., 2012). 143

In rhabdomyosarcoma cells, both macroH2A1 isoforms were more abundant at the protein levels compared to C2C12 cells (Figure 35B, right panel). Similarly, myogenin was expressed at higher levels than in C2C12 (Figure 35B, right panel). In summary, rhabdomyosarcoma cells were able to express key myogenic genes and macroH2A1 isoforms. However, the timing of their transcriptional activity was impaired and gave rise to aberrant protein levels. Since the splicing switch of the macroH2A1 transcript was impaired in rhabdomyosarcoma, we speculated that enforcing the downregulation of macroH2A1.2 or the increase of macroH2A1.1 might be able to induce rhabdomyosarcoma differentiation. Although, macroH2A1.2 silencing seemed to increase cell elongation (Figure 36A), rhabdomyosarcoma fusion was not successfully achieved. Investigating the expression of both isoforms in an accessible data set from rhabdomyosarcoma transcriptomics is not feasible, since published data sets do not allow to distinguish between the two isoforms. The knockdown of macroH2A1.2 increased the expression of the key myogenic regulators MYOD1, MYOG and CKM in Rh30 (Figure 36B). This was surprising since no change was observed in C2C12 when macroH2A1 isoforms were silenced (Figure 28B). Global reprogramming of the chromatin landscape is occurring in RMS (reviewed in Skapek et al., 2018). Thus, changes in the macroH2A1 gene target could be due to reorganization of the chromatin in rhabdomyosarcoma. For instance, nucleosome positioning, looping, PTMs, cis- and trans-regulation, and therefore macroH2A enrichment may differ between RMS and healthy cells. MacroH2A was previously shown to maintain nuclear architecture in HepG2 cells (Douet et al., 2017). In this context, it would be interesting to study the role of macroH2A in RMS nuclear architecture Taken all together, modulation of macroH2A isoforms may have a beneficial outcome against cancer development in both an isoform and cancer-specific manner.

144

| 2.2 | Dual role in transcriptional regulation

It is commonly accepted that, in most contexts, macroH2A contributes to transcriptional repression. Generally, macroH2As creates large chromatin domains enriched mostly by the repressive mark H3K27me3 or by nine active marks (H2BK15ac, H2BK20ac, H3K4ac, H3K14ac, H3K18ac, H4K91ac, H2AK5ac) (Ruiz & Gamble, 2018). In this chromatin environment, positive regulation by macroH2A1 on target genes was related to its presence in gene body, rather than in the promoter region. This was the case for transcribed regions encoding for key genes in cell–cell signaling, or genes responsive to serum starvation (Gamble et al., 2010). This was supported in IMR90 primary human lung fibroblasts where macroH2A1.1 promotes the acetylation of H2B K12 and H2B K20. As a consequence, macroH2A1-target genes are either positively or negatively regulated (Chen et al., 2014). Creppe et al. suggesting that the ambivalent role of macroH2A could be explained by its capacity to stabilize chromatin states that are repressed but highly sensitive to signal-induced activation (discussed in Creppe et al., 2012).

145

III | Dual role of macroH2A1 isoforms in muscle development

| 3.1 | Opposite role at the transcriptional levels

In the context of muscle development, Stefania Dell’Orso et al. have shown that macroH2A1.2 is essential for the proper activation of muscle enhancers through the interaction with the transcription factor Pbx1 (Orso et al., 2016). In their hands, macroH2A1.2 was expressed all along myoblast differentiation in culture. While macroH2A1.1 reached equal gene expression at the latest stages of differentiation, they did not study its potential implication in the myogenic process. In our hands, we have shown that both isoforms affect fusion. Most interestingly, silencing of both isoforms did not only up-regulate genes as a suppressor protein would, but down-regulate several genes too. Down-regulation of genes could be downstream of general transcriptional deregulation. However, we demonstrated that macroH2A1 isoforms regulated a subset of genes (opDEGs) potentially involved in the fusion phenotype in an opposite manner. A few of these opDEGS have already been shown to have an essential role in muscle fusion. For instance, Fmod was shown to regulate myogenic markers, collagen and myostatin genes (Lee et al., 2016). The same study showed that loss of Fmod in C2C12 leads to the down-regulation of Myod, Myog, Myl2 and Col1a1 genes. This leads to a reduced fusion index in combination with up-regulation of the negative regulator of muscle growth Mstn gene. Similarly, Itga11 knockdown inhibits the fusion capacity of muscle satellite cells and therefore myotube formation (Grassot et al., 2014). Thus, we plan to perform a “rescue experiment” by silencing either Fmod or Itga11 in C2C12 cells transfected with si macroH2A1.2. In this way, we will assess 146

if the fusion phenotype is indeed due to the deregulation of the opDEGs. Deregulation of cytoskeleton attachment to extracellular matrix is observed in muscle dystrophy carrying a dystrophin mutation. It would be interesting to test if macroH2A1 modulation could improve the outcome of MD-related diseases.

| 3.2 |Switch of macroH2A-containing chromatin during differentiation

The reversed fusion phenotype observed between the two macroH2A1 knock downs rise key questions. Are macroH2A1.1 and macroH2A1.2 located at shared genomic loci? Are the macroH2A1 isoforms able to replace each other in order to maintain differentiation state? The former PhD students in our lab performed ChIP-seq analysis of macroH2A1 in C2C12 myoblasts and myotubes. Enrichment of both macroH2A1 isoforms did not massively change during differentiation. This suggests that, indeed, macroH2A1.1 and macroH2A1.2 could occupy common chromatin regions. This could be explained by three distinct mechanisms. First, during differentiation, nucleosomes containing both isoforms could be formed. Although we cannot discard this possibility, it is most unlikely since it was demonstrated that macroH2A preferentially forms hybrid with the canonical H2A (Chakravarthy & Luger, 2006). Two other hypotheses could explain the opposite regulation made by macroH2A1 isoforms. On the one hand, macroH2A1 isoforms could be exchanged by one another at the nucleosome level. On the other hand, macroH2A1.1 enrichment during differentiation could mask macroH2A1.2 regulation on a specific subset of target genes. In both cases, increased macroH2A1.1 during differentiation is accompanied with transcriptional switch. To challenged those hypotheses, we prepared a new ChIP-seq analysis in proliferative and differentiated C2C12 cells with antibodies that are isoform-specific. Those samples are currently being sequenced at the genomics unit of CRG (in collaboration with Jochen Hecht, Barcelona). This will allow us to distinguish the dynamic of both isoforms during differentiation at the chromatin level. The initial analysis will focus on the opDEGs determined in Chapter II (Figure 31).

47 Furthermore, we plan to determine if such macroH2A1 dynamic is 1

conserved at a larger scale in the genome. This will be analysed in parallel with the ChIP- seq of macroH2A1.2, performed in proliferative and differentiated C2C12 cells by Hossein Zare’s group (Orso et al., 2016; GEO: GSE76010).

| 3.3 | Is any phenotype related to C2C12 heterogeneity?

Interestingly, cell heterogeneity is observed when C2C12 cells. Indeed, others have shown that a small portion (1 to 5%) of C2C12 cells are delayed in their capacity to differentiate and were considered as a reserve of myoblasts (Yoshida et al., 1998; Benchaouir et al., 2004). In our hands, we believe that the non-differentiated population is largely underestimated. Thus, we wondered if the fusion phenotype was partially caused by this reserve fraction. In vertebrates, the principal source of ECM components known so far are fibroblasts, endothelial cells, immune cells, smooth muscle, and epidermis. In planarians, muscle cells were capable of producing ECM components such as collagen proteins (Cote et al., 2019). Muscle from Japanese quail grown in culture does not manage to produce complete basement membrane, but produced and realized collagen proteins (Cote et al., 2019). Similarly, in chicken, muscle cells are able to synthesize type I collagen (Lipton, 1977). In conclusion, myoblasts were shown to be able to express ECM components. Although, we could not distinguish both population, we believe that the fusion phenotype is occuring due to internal deregulation of the myotubes rather than other cell population. This was also supported by the deregulation of genes coding for proteins essential for cell-cell adhesion located inside the myofiber such as focal adhesion components.

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| 3.4 | Role of macroH2A1 isoforms in fiber composition in vivo

The splicing switch leading to the expression of the macroH2A1.1 isoform in differentiated C2C12 cells was recapitulated in both human and mouse primary muscle cells (Figure 18B). In order to assess the relevance of macroH2A1.1 in vivo, we will soon perform an analysis of the fiber type composition and fiber size in hind limb muscles of mice lacking macroH2A1.1. For this we have initiated a collaboration with molecular exercise physiologist expert, Henning Wackerhage (Faculty of Sport and Health Science, Technical University, Munich). Preliminary data generated by the former PhD student Melanija Posavec Marjanovic, in collaboration with Philippe Bouvet (ENS Lyon, France), suggested that a fiber type composition of Tibialis and Gastrocnemius was altered in macroH2A1 KO mice compared to control mice. We also plan to check the level of opDEGs in collected macroH2A1.1 KO and control muscles.

How are metabolic and fusion phenotypes interconnected is a major question. Distinguishing the deregulation of processes from one phenotype to one another is still challenging. We showed that metabolic phenotype was mainly independent of gene regulation. On the contrary, we showed that the fusion phenotype was most likely due to the opposite deregulated of a specific subset of genes. Nevertheless, NAD+ was shown to improve ECM organization in zebrafish (Goody et al. 2012). Decrease of NAD+ was observed in Duchenne muscular dystrophy and could be rescued by NR supplementation (Ryu et al., 2016). Therefore, we wondered if NMN supplementation could improve fusion reduction in si macroH2A1.1. We also believe that both metabolism switch and change of the expression ECM components are necessary for proper differentiation. Thus, impairment of one process most likely affects the other to a certain extent.

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IV | Is ADP-ribose the ancestral role of macroH2A?

Until recently, macroH2A was thought to be widespread but restricted to vertebrate lineages. However, Rivera-Casas et al., characterized macroH2A function in the non- vertebrate mussel Mytilus. Furthermore, macroH2A proteins are highly conserved throughout evolution from the emergence of multicellular life (Rivera-Casas et al., 2016). Comparative genomic analysis described macroH2A1 as the oldest form appearing before macroH2A2 (Rack et al., 2016; Rivera-Casas et al., 2016). Our data demonstrated that among both macroH2A1s, macroH2A1.1 is the ancestral isoform that appeared right at the node towards metazoans lineage. Therefore, we wondered if the ancestral function of macroH2A was related to macroH2A1.1-related functions. Whether the youngest macroH2A1.2 and macroH2A2 acquired new metabolite binding capacity or lost its binding function will require further investigation. However, based on their strong conservation since they appeared, we speculate that their binding pocket may indeed acquire new binding capacity.

| 4.1 | Capsaspora owczarzaki macroH2A-PARP1 axis

As shown in the Chapter III (Figure 39C), the macrodomain of Capsaspora owczarzaki macroH2A inhibits human PARP1 activity. In fact, the inhibitory capacity of Capsaspora owczarzaki macrodomain was stronger than the one of murine macroH2A1.1 macrodomain. Did macroH2A macrodomain gradually reduce its capacity to inhibit PARP1 activity throughout evolution? Could it be due to rapid PARP1 diversification in order to escape from macroH2A inhibition? On the contrary, if both would co-evolve together, could it be explained by lower activity of PARP1, due to the diversification of other PARPs during evolution? To respond to some of those questions we

are currently investigating the structure of Capsaspora owczarzaki 150

macrodomain and its capacity to bind and inhibit PARP1. First, Iva Guberovic, a PhD student in the lab, will soon focus on the capacity of Capsaspora owczarzaki macrodomain to bind ADP-ribose. For this, she will use biophysical methods to determine ADP-ribose binding affinity. Furthermore, she has just solved the crystal structure of Capsaspora owczarzaki macrodomain in collaboration with Andreas Ladurner (Physiological Chemistry, LMU, Munich, Germany). Secondly, using Seahorse experiments, we are planning to monitor mitochondrial capacity of HepG2 DKD cells expressing Capsaspora owczarzaki macroH2A WT and mutant. This will allow us to determine if Capsaspora owczarzaki macrodomain has a similar capacity to affect metabolic function as the murine protein (Results Chapter I). Finally, we are collaborating with Iñaki Ruiz-Trillo and Michelle Léger (Institut de Biologia Evolutiva, CSIC UPF, Barcelona, Spain) to assess the capacity of Capsaspora owczarzaki macroH2A to bind PARP1 in vivo by co-.

| 4.2 | macroH2A role in Capsaspora owczarzaki

In Ruiz-Trillo’s laboratory, they manage to culture the three stages of Capsaspora owczarzaki. The three stages are classified by quiescent, filopodial, and adherent stages. Since methods for genetic perturbation have not been established yet in this model, we will expand our descriptive analysis. Although we already know in which stage macroH2A and PARP1 are expressed (Figure 39B), it will be interesting to monitor their localisation in the three stages of Capsaspora owczarzaki life by performing immunofluorescence. In the future, it would be interesting to figure out the role of the ADP-ribose-macroH2A-PARP1 axis in Capsaspora owczarzaki. Capsaspora owczarzaki has a complex transcriptional repertoire. Several proteins in Capsaspora owczarzaki essential for the emergence of metazoan multicellularity include cell adhesion molecules such as integrins and cadherins (Sebé-Pedrós et al., 2010; Nichols et al., 2012). Since several genes coding for integrins and cadherins were deregulated in our muscle model, we speculate that ancestral macroH2A may have similar impact on adhesion

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regulating genes in Capsaspora owczarzaki. This could be relevant for regulating the cell aggregation.

| 4.3 | Ancestral role in virus and pathogen recognition

Structural and functional diversification of the macrodomain increased with organism complexification throughout evolution (reviewed in Rack et al., 2016). Most macrodomains bind ADP-ribose, O-acyl-ADP-ribose and ADP-ribosylated proteins such as histones in most species (Karras et al., 2005; Posavec et al., 2013). During evolution, pathogens develop mechanisms in order to escape host immune response. Disruption of host phosphorylation and acetylation signaling are one of the strategies commonly used by pathogens to replicate in their host (Daugherty et al., 2014). In response to this adaptive strategy, hosts have evolved their innate immunity. Involvement of ADP-ribose modification in such host-pathogen conflict is still unclear.

Interestingly, Daugherty et al. have shown that the macrodomains of PARP9, 14 and 15 evolved under strong positive selection with ADP-ribosylation and host-viral conflict (Daugherty et al., 2014). In response to virus infection, silencing of macroH2A induces upregulation of the key inflammatory signal IL-8, in human B cells (Agelopoulos & Thanos, 2006). It is interesting to note that macroH2A1.1 silencing leads to up-regulation of genes involved in cellular response to interferon-ß and to virus (Figure 30, Chapter II). Altogether, we speculate that the macroH2A macrodomain may intervene in immune and virus response. This line of research has been recently funded in the lab. We will test the response of mice lacking macroH2A to acute inflammatory signals by LPS, which mimics bacterial infection.

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Materials & Methods

Materials & Methods

Several Materials and Methods sections were inspired or taken from (Posavec Marjanovic & Hurtado-Bagès, 2017) or from Melanija Posavec Marjanovic's thesis, written in 2014 (The Role of histone variant macroH2A1 in muscle physiology and pathophysiology: https://www.tesisenred.net/handle/10803/316789).

Cell cultures

C2C12 were obtained from Monica Suelves (Associate investigator, Epigenetics of cell differentiation and cancer, IGTP, Badalona, Barcelona) and Pura Muñoz-Cánoves (ICREA Senior Research Professor at Universitat Pompeu Fabra). Proliferative C2C12 grow in growth medium (GM) composed of DMEM (Gibco) with 20% FBS (Invitrogen) supplemented with 1% Pyruvate, 1% Glutamate and 1% Penicillin- Streptomycin (Gibco). Differentiation of C2C12 cells was achieved by replacing GM to differentiation media (DM) were FBS was exchanged by 2% of horse serum (Gibco). Fresh medium was added every two days. Proliferative C2C12 and mature myotubes were collected by scraping after PBS washing. Rh4 and RH30 cells were obtained from Oscar Martinez Tirado (Group leader, Sarcoma Research group, IBIDELL, Hospitalet de Llobregat, Barcelona) and Sonia Forcales (Lecturer Professor of Immunology. Dept. of Pathology and Experimental Therapeutics. Bellvitge, University of Barcelona). Proliferative Rh30 were cultured in DMEM implemented with 10% FBS (Invitrogen) supplemented with 1% Pyruvate, 1% Glutamate and 1% Penicillin-Streptomycin (Gibco). Proliferative Rh4 were cultured in RPMI implemented with 10% FBS (Invitrogen) supplemented with 1% Pyruvate, 1% Glutamate and 1% Penicillin-Streptomycin (Gibco). Both Rh30 and Rh4 were differentiated in DM as for C2C12. HepG2 cells (HB-0865), HEK293T cells (ATCC, CRL-3216) and GP2 packaging cells (Clontech; 631458) were cultured in DMEM containing 10% FBS, supplemented with 1% Glutamate and 1% Penicillin-Streptomycin (Gibco).

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Mouse primary myoblasts were maintained on collagen-coated dishes in Ham’s F10 medium complemented with 20% FBS (Invitrogen), 10ng/ml of bFGF (Invitrogen), 0.1% Fungizone (Invitrogen), penicillin- streptomycin. For maintenance, mouse primary myoblasts were seeded on plates coated with rat tail collagen I (BD Biosciences). For differentiation, they were cultured on plates coated with Matrigel™ (BD Biosciences) basement membrane matrix (as described in Perdiguero et al., 2007). Differentiation of primary myoblasts was performed by medium change, as described for the C2C12 cells. Human primary myoblasts were kindly provided by Eduard Gallardo (Institut de Recerca Hospital de la Santa Creu i Sant Pau). They were maintained in growth medium DMEM (Biowhittaker), complemented with 10% FBS (Hyclone), 15ng/ml of bFGF (Invitrogen), 10ng/ml EGF and 1% insulin at 1mg/ml (Sigma-Aldrich)). They were plated on 0.1% gelatin coated plates (Sigma-Aldrich). Differentiation of human primary myoblasts was obtained by changing the growth medium for differentiation medium (DMEM containing 2% of Horse serum) supplemented with 1% insulin at 1mg/ml (Sigma-Aldrich).

Plasmids

For macroH2A1.1 plasmid construction, standard cloning techniques were used. The cDNA of mouse macroH2A1.1 was amplified from C2C12 cell and inserted into a retroviral pBabe.puro backbone. The G224E pocket-binding mutant was generated using Stratagene site-directed mutagenesis QuikChange protocol. Silent mutations were introduced in macroH2A1.1 construct in order to prevent siRNA recognition site. Plasmids encoding GFP and His-tagged macro domains (corresponding to amino acids 155-369 of macroH2A1.1 and 155-372 of macroH2A1.2) were described previously (Timinszky et al., 2009). For Capsaspora owczarzaki construct, Gibson cloning was used (reference “DNA library construction using Gibson Assembly” prepared at the centre de Regulació Genòmica (CRG) Biomolecular Screening & Protein Technologies Unit). The Capsaspora owczarzaki G224E and G314E pocket-binding mutants were generated using Stratagene site-directed mutagenesis QuikChange protocol. All constructs were inserted into a retroviral pBabe.puro backbone (Lablife) (Figure 40). For protein

158

production, pET-28a(+) vector with His-tag was used (Novagen) (Figure 40).

pBabe-puro (Lablife)

pET-28a-c( + ) VectpET-28a-c( or s + ) Vect or s TB074 12/98 TB074 12/98

Cat. No. The pET-28a-c(+) vectors carryThe an pET-28a-c(+) N-terminal His• vectors Tag® /thrombin/T7•carry an N-terminal Tag® confHis• Tagguration®/thrombin/T7• plus Tag® conf guration plus pET-28a DNA 69864-3 an optional C-terminal His• Tagan sequence. optional C-terminal Unique sites His• are Tag shown sequence. on the Unique circle map.sites areNote shown that the on the circle map. Note that the pET-28b DNA 69865-3 sequence is numbered by the sequencepBR322 convention, is numbered so by the the T7 pBR322 expression convention, region is so reversed the T7 expressionon the region is reversed on the pET-28c DNA 69866-3 circular map. The cloning/expressioncircularpET-28a map.region The of thecloning/expression coding (+) strand Vector transcribed region of the by T7coding RNA strand poly- transcribed by T7 RNA poly- merase is shown below. The f1merase origin is is shown oriented below. so that The infection f1 origin with is oriented helper phageso that will infection produce with helper phage will produce virions containing single-strandedvirions DNA containing that (Novagen)corresponds single-stranded to the codingDNA that strand. corresponds Therefore, to thesingle- coding strand. Therefore, single- stranded sequencing should bestranded performed sequencing using the should T7 terminator be performed primer using (Cat. the No. T7 69337-3). terminator primer (Cat. No. 69337-3).

Xho I(158) Not I(166) pET-28a(+) sequence landmarks Eag I(166) pET-28a(+) sequence landmarks Hind III(173) T7 promoter 370-386T7 promoter 370-386 Sal I(179) T7 transcription start 369 Sac I(190) T7 transcription start 369 EcoR I(192) His• Tag coding sequence 270-287His• Tag coding sequence 270-287 BamH I(198) T7• Tag coding sequence 207-239 Bpu1102 I(80) Nhe I(231) T7• Tag coding sequence 207-239 Nde I(238) Multiple cloning sites Multiple cloning sites Dra III(5127) Nco I(296) (BamH I - Xho I) 158-203(BamH I - Xho I) 158-203 Xba I(335) Bgl II(401) His• Tag coding sequence 140-157His• Tag coding sequence 140-157 03-5358) SgrA I(442) T7 terminator 26-72 (49 T7 terminator 26-72 igin Sph I(598) or lacI coding sequence 773-1852lacI coding sequence 773-1852 f1 pBR322 origin 3286pBR322 origin 3286 ) 7 Kan coding sequence 3995-4807Kan coding sequencePvu 3995-4807 I(4426) 0 Sgf I(4426) 8 4 f1 origin 4903-5358f1 origin 4903-5358 - 5 Sma I(4300) 9 9 Mlu I(1123) 3 l The maps for pET-28b(+) andThe pET-28c(+) maps for pET-28b(+) and pET-28c(+) ( a c n Bcl I(1137) I are the same as pET-28a(+) (shown)are the samewith as pET-28a(+) (shown) witha Cla I(4117) ( K 7 the following exceptions: pET-28b(+)the following is a exceptions:Nru pET-28b(+) I(4083) is a 7 BstE II(1304)

3 -

5368bp plasmid; subtract 1bp 5368bpfrom each plasmid; site subtract 1bp from each site 1

pET-28a(+) 8 Apa I(1334)

beyond BamH I at 198. pET-28c(+)beyond is Bama H I at 198. pET-28c(+) is a 5 (5369bp) 2

5367bp plasmid; subtract 2bp from each site ) 5367bp plasmid; subtract 2bp from each site BssH II(1534) beyond BamH I at 198. beyond BamH I at 198. Eco57 I(3772) EcoR V(1573) Hpa I(1629) AlwN I(3640) o r i ( 32 86 BssS I(3397) ) PshA I(1968)

BspLU11 I(3224) Bgl I(2187) Sap I(3108) Fsp I(2205) Bst1107 I(2995) Psp5 II(2230) Tth111 I(2969)

T7 promoter primer #69348-3 pET upstream primer #69214-3 T7 promoter Bgl II lac operator Xba I rbs

Nco I His•Tag Nde I Nhe I T7•Tag

Eag I thrombin BamH I EcoR I Sac I Sal I HindIII Not I Xho I His•Tag

Bpu1102 I T7 terminator

T7 terminator primer #69337-3 pET-28a-c(+) cloning/expressionpET-28a-c(+) region cloning/expression region

Figure 40. Map of pBabe and pET-28a(+) sequences.

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Transfection and retroviral infection

GP2 cells were initially seeded 24 hrs prior to transfection in P10 plates with 8ml of fresh medium. The next day, GP2 cells were transfected with the mixture of 4-8ug of retroviral plasmid of interest, 3.6ug of VSVG packaging plasmid, 125mM of CaCl2, 1X HBS. HBS was prepared as the following: 272 mM NaCl, 2.8 mM Na2HPO4, 55mM HEPES, pH 7. Transfection efficiency was checked the day after by autofluorescence of the pRETRO Super-GFP construct used as positive control. The following later, C2C12 or HepG2 cells were seeded the day prior the infection onto 6-well plates at the density of 10.000 cells per well. Virus supernatant was collected and filtered (0.45um filters, Milipore), mixed with 8 µg/ml polybrene (Sigma-Aldrich). Cells were incubated with the virus supernatant either by centrifugation at 1000 g at 32ºC for 45 min and left 1.5 extra hour in the incubator, or left overnight. Fresh medium was added to the cells the day after. After 48hs, 1ug/ml of puromycin was added to the medium to select infected cells only. After selection, cells were directly used or stored at -80ºC. HEK293T cells were transiently transfected using calcium-phosphate method as previously described for GP2 cells. siRNAs transfections

For the knock-down of macroH2A1, isoforms we used siRNAs (Resource table, ordered from Invitrogen) previously described. (Dardenne et al., 2012). Their sequences are found in (Resource table, Invitrogen). While si NMNAT3 was ordered from (Resource table, Invitrogen), si PARP1 was designed in our lab. For all siRNAs, 10nM was found to be the optimal concentration to use. Briefly, in a 6-well plate, 20 000 C2C12 cells were seeded. C2C12 cells were then transfected with the transfection reaction composed of 500ul of OptiMEM (Gibco) 3,12 ul of lipofectamine RNAiMAX Transfection Reagent (Invitrogen) and 10nM of siRNA. For knockdown in myotubes, siRNA was repeatedly delivered at day +1 after seed (-1 of differentiation), the day of medium change for differentiation (D0) and 2 days after (D2) (see the scheme in Chapter I Figure 18A). In

the case of p15 plate, 300 000 C2C12 were seeded and transfected with 160

7,5ml Optimem, 45ul lipofectamine and siRNA at a final concentration of 10nM.

Antibodies

The list of all antibodies used is found in the resources table, at the end of the materials and methods section.

Protein production

Sequence encoding for recombinant Caps.owc. macroH2A macrodomain (amino acid sequence 177-368) was cloned into pET28a expression vector. All protein coding sequence (Human and Caps.owc. macroH2A1.1 wild-type, G224E Residues, PARP1) were inserted into pET28a and all of them had the same purification protocol. Briefly, proteins were produced in DE3 (BL21) strain of E.coli. Protein production was induced with 500uM IPTG and bacterial culture grown overnight at 20ºC. The next day, the bacteria pellet was recovered by centrifugation for 30 min at 4ºC and 20,000g. The bacterial pellet was lysed with lysis/wash buffer (50mM Tris-HCl, 300mM NaCl, 10mM imidazole, pH 8, protease inhibitors and lysozyme) and sonicated (30’’ON/ 30’’OFF, 10 cycles). Once sonicated, the lysate was incubated with Ni-NTA beads (Qiagen) for 2 hs. After being washed with 10mM imidazole-containing buffer, proteins were eluted with elution buffer (containing 50mM Tris-HCl, 300mM NaCl, and 200mM imidazole). In a big graduated cylinder, proteins were dialyzed overnight against dialysis buffer (1x PBS, 5mM β-mercaptoethanol and 10% glycogen). The day after, proteins were recovered gently in several tubes. Protein concentration was checked by Nanodrop (Thermoscientific) and stored at -80ºC.

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Anti-macroH2A and PARP1 antibodies production (Caps. owc.)

Caps.owc. anti-macroH2A antibodies were raised in rabbits using recombinant Caps.owc. His-macroH2A macrodomain as antigen. His-tagged protein was sent to the Servei de Producció d'Anticossos (Estabulari CID-CSIC, Jordi Girona 18-26, 08034 - Barcelona). Two rabbits were inoculated with the recombinant Caps.owc. His-macroH2A macrodomain for antibody production. Efficiency of Caps. owc. macroH2A antibody was first tested on pre-immunization serum from both animals. After 4 rounds of immunization, the polyclonal antibody was obtained from final exsanguination. For the generation of PARP1 antibody, its protein sequence (XM_004363900.1), three peptides from the N-terminal domain of PARP1 were conjugated with KLH (sequences of the peptides used: KLH-C S A A G N D E D E D A G; KLH-C E E P A E A P K K A

A; KLH-C S S Q S N A A A E D D N). Peptides were ordered by GL Biochem from Synpeptide: (Company name: Synpeptide, Co., Ltd, Shanghai China. Web page: http://www.synpeptide.com). The three peptides were mixed together and injected in two rabbits at the animal care facility unit of the University of Victoria (Canada). After three immunization boost, the specificity of the serum containing PARP1 antibody was tested by immunoblot as for macroH2A antibody. Recombinant Caps. owc. His-PARP1 (AA1- 350) was used as a control.

PARP1 activity assay in vitro

PAP1 auto-PARylation assay was performed in the buffer containing 50 mM Tris-HCl (pH 8), 50 mM NaCl and 1 mM MgCl for 20 minutes. The reaction contained 0,2 units/µL PARP-1 HSA enzyme (Trevigen), 0.3x activated DNA (from 10x activated DNA, Trevigen) and 200 µM NAD+. PARP1 inhibition was monitored by adding different concentrations of purified macroH2A macrodomains (0 (PARP1 alone), 10, 25 and 50 uM). Reactions were stopped by addition of Laemmli buffer at 95°C for 5min. Samples were analyzed by immunoblotting of PARP1 auto-PARylation. Naphtol blue (Sigma) was used to stain proteins, as a loading control.

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Immunoblot

Cells were collected by scrapping and washed twice with 1X PBS. Cell pellets were directly resuspended with 1X Laemmli and either passed through a syringe or sonicated using Bioruptor (10 min, 30 sec ON-30 sec OFF, Diagenode). After incubation at 95ºC for 5 min, samples were loaded on polyacrylamide gel and run at 36mA (Table 1 & 2). After the sandwich preparation using Whatman paper, proteins from the gel were transferred onto a nitrocellulose membrane (Whatman, GE healthcare) at 220mA for 90 min. Later on, protein profiles were checked by addition of Ponceau staining. Film membranes were then blocked for 20 min using 5% milk (Nestle) prepared in TBST. Membranes were incubated overnight with antibodies of interest. The following day, membranes were washed three times with TBST and incubated for 1 h with the secondary antibody on shaker. Two different secondary antibodies have been used. In one case, secondary-HRP conjugated antibody (DakoCytomation). Membranes were washed three times with TBST and incubated with ThermoScientific chemiluminescent reagent mix 1:1 for 1 min. In a dark room, the dried membranes were overlaid with photographic film (GE Healthcare) for a few minutes. Finally, the membranes were developed using the FujiFilm FPM-100A developer. For Chapter II and III, we also used Odyssey CLX Infrared Imaging System to reveal proteins. In this case, we used IRDye goat anti rabbit antibodies.

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Buffer Composition Concentration 1.5 M Tris Resolving buffer 4X pH 8 1X 0.4%SDS 500 nM Tris Stacking buffer 4X pH 6.8 1X 0.4%SDS 250 mM Tris Running buffer 10X 2 M Glycine 1X 10 g/l SDS 250 mM Tris Transfer buffer 10X 1X 1.92 M Glycine 140 mM NaCl TBST wash buffer 10 X 250 mM Tris 1X 27 mM KCl Blocking 5% milk 5% milk (Nestle) dissolved in TBST 250 m M Tris HCl pH 6.8 50% glycerol Laemmli buffer 5 X 10% SDS 1X 14.3 M b mercaptoethanol 0.05% blue bromphenol Ponceau 0.05% Ponceau S dissolved in 1% acetic acid 10% NaN3 10% sodium-azide 0.05%

Table 1: Buffer composition used to perform immunoblot experiment

Resolving gels 8% 10% 12% 14% Stacking gel 4% Acrylamide 30% (BioRad) 2.6 ml 3.3 ml 3.9 ml 4.6 ml Acrylamide 30% (BioRad) 0.5 WB resolving buffer 4 X pH 8.8 2.5 ml 2.5 ml 2.5 ml 2.5 ml WB resolving buffer 4 X pH 8.8 360ul H2O 5.2 ml 4.5 ml 3.9 ml 3.2 ml H2O 2.1ml APS 10% (Sigma-Aldrich) 80 ul 80 ul 80 ul 80 ul APS 10% (Sigma-Aldrich) 30 ul TEMED (Amresco) 8 ul 8 ul 8 ul 8 ul TEMED (Amresco) 3 ul

Table 2: Gel composition used to perform immunoblot experiment

Immunoprecipitation

For immunoprecipitation, the nuclei were isolated and chromatin was solubilized by sonication. Cells were collected by scraping and lysed in sucrose buffer (0.32M Sucrose, 10mM Tris HCl pH8.0, 3mM CaCl2, 2mM MgOAc, 0.1% Triton buffer, 1:100 PMSF and 1:500 leupeptin). Disruption of cell membranes was facilitated by several passages through a syringe and incubated for 8' on ice. Nuclei were collected by centrifugation for 3 min at 1000g. To maintain their integrity, nuclei were washed with sucrose buffer. Lysis was added to the nuclei (50mM Tris HCl, pH7-8, 135mM NaCl, 0.1% Triton, 1mM EDTA, 1mM DTT, 1:100 PMSF and 1:500 leupeptin). Chromatin was

solubilized by progressive sonication using Bioruptor (10 min, 30 sec ON- 164

30 sec OFF, Diagenode). For the precipitation of PARylated PARP-1, PARG and PARP inhibitors were added to the lysis buffer, 1 µM ADP-HPD (CalBioChem) and 1 µM Olaparib (SelleckChem), respectively. Insoluble fraction was removed by centrifugation and lysates were pre-cleared using sepharose beads (Sigma). During this step, 5% of the total lysate was kept as input material. Lysate was then incubated overnight with antibody-bound beads previously blocked with 1% BSA in lysis buffer. The following day, the beads were washed three times with lysis buffer containing 1% Triton X-100. For immunoblot analysis, 1X Laemmli was added to input and immunoprecipitated protein. Input (1%) and immunoprecipitated material (20%) were warmed at 95ºC for 5 min and loaded. For cell fractionations, nuclei were prepared as described above and the supernatant was kept as a cytosolic fraction. Nuclei were incubated with buffer I (410 mM KCl, 20mM HEPES pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 25% Glycerol, 1% NP-40). Chromatin was separated from the nucleosol by ultracentrifugation (45 000 rpm) for 45 min.

Immunofluorescence

For immunofluorescence, 20,000 C2C12 cells were seeded in 6 well-plates. At day 4 of differentiation, C2C12 cells were fixed in 4% paraformaldehyde (10 min at room temperature). The slides were permeabilized for 10 min with HCl 0,1M, 0.5% Triton-X 100 in PBS, and washed three times with PBST (PBS containing 0,1% Tween 20). Then, slides were pre-blocked with PBST-BSA (5%) for 30 min at room temperature, and incubated with a 1/50 to 1/100 dilution of specific primary antibody for 2 hrs in BSA 5% buffer. After three washes with PBST, the slides were incubated for 1 h at room temperature with a 1/100 dilution of secondary goat anti-rabbit antibody conjugated to Alexa 488 (Thermo Fischer). After successive washes with PBST, slides were mounted with Vectashield® Mounting Medium with DAPI.

165

Fusion Assays and Data analysis

Images of the C2C12 immunofluorescence were obtained using a Leica DMI6000B Advance Fluorescence microscope (Leica) microscope equipped with a 63x/1.4 Plan- Apochromat oil immersion objective. Groups of 10 to 20 images were loaded and analysed in Fiji, a distribution of ImageJ (Schindelin et al., 2012). Myotubes were directly drawn as Regions of Interest (ROIs) on the selected image using Fiji’s manual selection tools on the basis of eMHC staining. The images were then automatically analyzed using a custom ImageJ script with the aim of calculating the number of nuclei inside each myotube, the total number of nuclei in each image and measurements of each myotube. In short, after background removal with a rolling ball algorithm, DAPI signal was automatically thresholded, artifacts removed with binary image functions and subject to the “analyze particles” function to identify and count all nuclei in the image. Then, the centroid of each nucleus was extracted and each region defined as eMHC positive myotube was evaluated to count the number of nuclei it contains. In addition, various measurements (length and perimeter, for example) of the myotubes were obtained using the native “Measure” function in Fiji on the myotube ROIs. The total number of nuclei was normalized on image size. The differentiation index was calculated as the ratio of nuclei in eMHC-positive cells over the total nuclei. the median of the fusion length and perimeter were assessed for the same number of myotubes. Nuclei distribution per myotube was analysed by showing the distribution of nuclei in eMHC-positive cells that contain at least 2 nuclei. The myotube distribution shows different categories of myotubes containing the selected number of nuclei. Total nuclei number and differentiation index were statistically analysed with Student's t-test while we used Wilcoxon test for fusion parameters that were not normally distributed.

Immunochemistry

For immunohistochemistry, 10 µM muscle sections were fixed in 100% cold acetone at

room temperature. Immunostaining was performed by incubating anti-

166 macroH2A1.1 antibody for 1 h in the Ventana UltraView Universal DAB

machine. Nuclei were stained 1 min in Hematoxylin (Sigma-Aldrich). Muscle sections were washed extensively, rinsed in 96% ethanol, dehydrated twice in absolute ethanol and mounted. Frozen biopsy of human muscle was obtained from the Department for Pathologic Anatomy at the Hospital Universitari Germans Trias I Pujol (Badalona, Spain).

RNA isolation and cDNA synthesis

Extraction of total RNA from cell pellets was isolated using the Invitrogen PureLinkTM RNA kit. Briefly, pellets were lysed with lysis buffer from the kit containing 1% β- mercaptoethanol and passed several times through a syringe. One volume of 70% ethanol as added to the lysate and vortexed thoroughly. Lysate was passed and bound to the column provided by the kit by centrifugation at 12000g for 30 sec. Bound RNA was washed in wash buffer I and wash buffer II (containing ethanol) provided by the kit. Between the two washes, DNase-treatment was carried out (Invitrogen, 10ul DNase, 8ul of 10X reaction buffer and 62 ul of water). In order to remove the left-over of ethanol, columns were centrifuged one last time at 12000g for 3 minutes. RNA elution was performed with RNase-free water. For primary myoblasts and myotubes, RNA was extracted using the PureLinkTM RNA Mini kit containing a smaller column, preferentially used for smaller numbers of cells. RNA concentration, and quality, were checked on NanoDrop (Thermoscientific). Complementary DNA (cDNA) was synthesized using First strand cDNA synthesis kit (Fermentas), from 1ug of RNA. To summarize, 1ug of RNA was mixed with 1ug of oligo (dT), nuclease-free water (qsp 11ul), and incubated at 65ºC for 5 min. This allows oligo to anneal to the RNA template. Reaction mix (4ul of 15X reaction buffer, 1ul of RiboLock RNase inhibitor (20u/ul), 2 ul of dNTP mix (10nM), and 2ul of M-MMLC reverse transcriptase (20u/ul)) was added to the previous mix containing RNA. The mixture was incubated 1h at 37ºC for proper enzymatic reaction of the reverse transcriptase. Finally, complementary DNA is terminated by heating mixture at 72ºC for 5min.

167

Semi-quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR)

Relative mRNA levels were quantified by qRT-PCR from the previously prepared cDNA. In 96-well plate, diluted cDNA (1/10) was mixed with RT-reaction (5ul SybrGreen (Roche), 0.5 ul each forward and reverse oligos (stock at 10uM), and 2ul water). Relative expression of the gene of interest was obtained by normalizing their concentration to the average of two housekeeping genes (GAPDH and Rpl7 for mouse samples; and HPRT1 and Rpo for human samples). In order to compare the relative expression macroH2A1.1 and macroH2A1.2, we used a reference sample containing equimolar ratios of macroH2A1.1 and macroH2A1.2 amplicons. This was done only for (Figure 18A) describing the switch of macroH2A1 isoforms. All oligos were purchased from Invitrogen (see list below) and resuspended with water to get a final stock concentration of 100 µM. Oligos quality and amplification capacity was checked by looking at their standard curve and running the final product on 1% agarose gel. The two mitochondrial-encoded (ND3 and CoxI) and nuclear-encoded (Ndufa9, CoxIV) were amplified using TaqMan probes from Applied Biosystems (Life Technologies).

Reaction Temperature(ºC) Time Cycles Preincubation 95 8´ 1 95 10´´ Amplification 60 20´´ 45 72 15´´ 95 15´´ Melting curve 65 1´ 1 96 endless Cooling 40 30´´ 1

Table 3: Reaction condition of qRT-PCR experiment

Restriction Fragment Length Polymorphism (RFLP)

Without antibody availability, we used the RFLP technique to qualitatively assess the

amount of mRNA macroH2A1 isoforms. The cDNA obtained from RNA 168

was used directly to perform PCR reaction. The oligos, used for PCR reaction, specifically targeted exons 5 and exon 8 in mouse or exon 6 and 9 in human of the macroH2A1 transcripts. PCR products (20 µl) were digested with 1ul HpaII/MspI restriction enzyme (ThermoScientific), resuspended in 1X Tango buffer, for 2 h at 37ºC. The digested products were separated by 2% agarose gel electrophoresis for 55 min at 90

V.

Reagent Concentration/Reaction cDNA 25ng Oligo Forward (10uM) 0.1uM Oligo Reverse (10uM) 0.1uM mix of dNTPs (25mM each) 250nM Reaction buffer 10 X (25mM Mg2+) 1X HotMaster Taq DNA Polymerase 5PRIME (5 U/ul) 1.25U

Table 4: Reaction mix used to perform RFLP experiment

Reaction Temperature (ºC) Time Cycles Initial Melting 94 2' 1 Melting 94 20" Oligo Annealing 60 10" 35 Amplification 65 20" Termination 65 5' 1 Hold 16 endless 1

Table 5: PCR condition used for RFLP experiment

Microarray, RNA-sequencing and Data Analysis

The transcriptomic of myotubes at day 4 was analysed. RNA was extracted (as described in the section "RNA isolation and cDNA synthesis") using the PureLinkTM RNA Mini Kit (Ambion, Life Technologies), including on-Column PureLink DNase treatment. RNA concentration was checked using Nanodrop (ThermoScientific). For the microarray, four biological replicates were used (GSE78257). After quantification, RNA quality was checked performing Eukaryote Total RNA Nano assay at the IMPPC Genomics facility. RNA was amplified and loaded onto the microarray

slide Agilent SurePrint G3 Mouse GE 8x60K Microarray. Our laboratory’s

169 bioinformatician, Roberto Malinverni, performed the statistical analysis.

Differentially expressed genes were identified with the LIMMA48 and selected using a q-value cut-off of 0,05 calculated after false discovery rate correction. The Gene Ontology analysis was performed using the ChIPpeakAnno R (Zhu et al., 2010) package using p-value < 0.01 and 30 for minGOterm (minimum count in a genome for a GO term to be included). A Bonferroni multiple hypothesis testing adjustment was finally applied to adjust the enrichment result. The REVIGO online software was used to visualize summaries of the gene ontology analysis 50. Medium was chosen for the SimRel parameter For RNA-seq, RNA was sent for library preparation to the CRG Genomics Unit (CRG Core Facilities). RNA quality control showed RNA integrity number (RIN) was between 9.9 and 10. RNA was amplified using mRNAseq which selects polyA. The sequencing was performed on single read (SR), with an average length of reads of 50 bp, on an Illumina HiSeq2500 sequencer. RNAseq analysis was performed using a pipeline assembled in our laboratory, the quantification of the expression was performed using Salmon software (Patro et al., 2015). The raw reads were “quasi-aligned” on mm10 assembly genome using default option. The statistical analysis was performed using DESeq2 package from Bioconductor. To select statistical differentially expressed genes in each contrast, we applied a cut off of 0.05 on the adjusted p-value, the adjustment was performed by applying a Benjamini- Hochberg false discovery rate procedure. Gene ontology analysis was performed based on the annotated mouse genome from org.Mm.eg.db (Bioconductor). (http://bioconductor.org/packages/release/data/annotation/html/org.Mm.eg.db.html). Pathways analysis was obtained using Paintomics3 web server (http://www.paintomics.org/). For this, the comparison of DEGs with si macroH2A1.1 and si macroH2A1.2 was used as input reference. The lists of opposite deregulated genes (opDEGs) were compared to the input reference. Predicted protein-protein interaction was performed using String web served (https://string-db.org/) with the list of opDEGs.

170

Chromatin Immunoprecipitation, ChIP-sequencing and Data Analysis

Chromatin Immunoprecipitation was performed on proliferating C2C12 myoblasts and terminally differentiated myotubes (day 4). At day 4, myotubes were enriched by a quick PBS and short Trypsinisation (4 mL for 20 secs, Gibco). Trypsine was inhibited by FBS- containing medium. Fresh myoblast and myotube pellets were obtained by centrifugation, 1000 g for 5 min, were resuspended in 10% FBS and cross-linked with 1% Formaldehyde and mix on a rotating wheel for 10 min. Cross-link reaction was quenched with 0,125 M glycine on a rotating wheel for 3 min. Myoblasts and myotubes were gently washed with 1X PBS and stored at -80ºC. Cell pellets were resuspended into Lysis buffer I containing PMSF and Leupeptin (purchased from Sigma-Aldrich) (Table 5) and incubated 10’ on ice. Nuclei extraction was obtained by centrifugation at 5000 x g for 5’ at 4ºC. Nuclei were resuspended in Lysis buffer II containing PMSF and Leupeptin for 10 min. Chromatin was extracted and sonicated 10 min using Bioruptor sonicator (Diagenode). To check that we successfully obtained chromatin fragments of 200 bp we took 10 µl of sonicated chromatin, added 90 µl of water, and incubated the mixture for two hours at 65ºC. DNA extraction was performed with the PCR product kit and chromatin concentration was assessed by Nanodrop (Thermoscientific). 250 ng of chromatin was then run on 1% agarose gel. For the following of ChIP protocol, 35 µg of chromatin were used for each IP reaction and diluted with nine volumes of IP dilution buffer. Samples were precleared with 20 µl of pre-blocked (in 1% BSA for 30’ rotating at 4ºC) slurry Ab binding beads (Diagenode) on a rotating wheel for 2 hrs at 4ºC. At this stage, input samples were taken (10% of the total volume). Chromatin fraction of interest was pull down by specific antibody macroH2A1 (Buschbeck et al., 2009), IgG, and Histone H3, rotating overnight at 4ºC. The following day, chromatin fraction of interest was captured with washed beads on a rotating wheel for 2hs at 4ºC. Beads were spun down and washed quickly twice with each of these buffers in this order: Mixed Micelle wash buffer, LiCl/Detergent wash buffer, Buffer 500. Between all changes of washing buffer, samples were rotating for 5 min on a wheel at RT. Finally, beads were quickly

washed with TE buffer. Chromatin was eluted with elution buffer twice. 171

Chromatin and proteins were reverse cross-linked by addition of NaCl (200 mM), and Proteinase K, followed by an incubation at 65ºC in a shaker overnight. The next day, DNA precipitation was performed by two Phenol/Chlorophorm (Sigma-Aldrich) steps and one chloroform step. Briefly, one volume of Phenol/Chlorophorm mix was added to the sample, vortexed thoroughly, and spun for 5 min at 5000 rpm. The upper aqueous phase containing DNA was recovered, vortexed thoroughly and centrifuge 5 min at 5000 rpm. Phenol/Chlorophorm precipitation was repeated once. Samples were mixed with another volume of Chlorophorm (Sigma-Aldrich). DNA was precipitated by adding three volumes of absolute ethanol and 0,1 volume of 3 M sodium acetate. DNA was kept for 30 min at -80ºC. The DNA pellet (centri) was washed with 70% ethanol, dried at 37ºC for a few minutes and resuspended in 75 µl of water. Final concentration was determined by QubitTM dsDNA HS Assay (Invitrogen). Precipitated DNA was analyzed ChIP-qPCR, normalizing the data on the diluted input (1/50). For Chip-sequencing, 20 ng of enriched DNA was used for library generation and parallel sequencing on Illumina Genome Analyzer at the EMBO facility. After data cleaning and trimming, reads were aligned to the mouse genome (mm9) using Bowtie 2 version 2.0.6, with sensitive pre-setting option (-D 15 -R 2-L 22 -i S,1,1.15) (Langmead & Salzberg, 2012). Enriched genomic regions for multiple overlapping ((peaks) SICER software version 1.1) were used to identify enriched regions using the following settings: redundancy threshold= 2, window size=600, fragment size=250, effective genome fraction = 0.75, gap=1200, FDR=0.05 (Zang et al., 2009). You can find the deposited ChIP GEO: GSE78257.

172

Buffer Composition FA solution 11% FA in 1X PBS 1,25 Glycine dissolved in 1X PBS 5 mM PIPES pH8 85 mM KCl Lysis Buffer I 0.5% NP-40 1mM PMSF 50ug/ml leupeptin 1% SDS 10 mM EDTA pH8 Lysis Buffer II 50 mM Tris pH 8 1mM PMSF 50ug/ml leupeptin 1% Triton X-100 150mM NaCl 2 mM EDTA pH8 IP dilution Buffer 20mM Tris pH 8 1mM PMSF 50ug/ml leupeptin 150 mM 20mM Tris pH 8 5 mM EDTA pH8 Mix Micelle Wash Buffer 5% sucrose (w/v) 0.02% NaN3 1% Triton X-100 0.2% SDS 0.5% deoxycholic acid (w/v) 1mM EDTA 250mM LiCl LiCl/detergent wash 0.5% NP-40 (v/v) 10 mM Tris-HCl pH8 0.2% NaN3 0.1% deoxycholic acid (w/v) 1mM EDTA 50 mM HEPES, pH 7.5 Buffer 500 500mM NaCl 1% Triton X-100 (v/v) 0.2% NaN3 1% SDS Elution buffer 100 mM NaHCO3 10% BSA (Biorad) dissolved in 1X PBS 136 mM NaCl 1X PBS 2.7 mM KCl

Table 5: Buffer composition used to perfrom ChIP experiment

173

Mitochondrial isolation

Hypotonic lysis buffer (10nM NaCl, 1.5mM MgCl2, 10nM Tris-HCl, pH7.5) was used to swell fresh cell pellets on ice from 6 P15 plates. Deacetylase inhibitors (2µM TSA, 10mM NAM) as well as protease inhibitors (1µM leupeptin, 1µM PMSF) were added to the lysis buffer. In order to keep organelle integrity, cell lysate resuspended with homogenization buffer (800nM sucrose, 25mM Tris-HCl, 2.5mM EDTA, 1µM leupeptin, 1µM PMSF, 2µM TSA, 10mM NAM). Mechanical disruption of the cell membranes was performed by 15 strokes with a teflon pestle in a Potter-Elvehjem homogenizer (Signa- Aldrich). Nuclear and cytoplasmic fractions were removed by two successive centrifugations at 1200g for 10 minutes. Mitochondrial isolation was obtained by centrifugation at 16000g for 20 minutes. Mitochondrial pellets were resuspended with homogenization buffer with protease and deacetylase inhibitors. Half of the pellet was used for immunoblot analysis and the other half for metabolites measurement.

Oxygen Consumption and Extracellular Acidification Rate Measurements

Analysis of oxygen consumption (OCR) and extracellular acidification (ECAR) rates was performed using a Seahorse XF96 Flux Analyzer (Seahorse Bioscience), as described (Teperino et al., 2012). Briefly, HepG2 cells, or 24 h post siRNA transfection C2C12 cells were seeded onto XF 96-well cell culture microplates and incubated overnight in incubator. The day after mitochondrial stress test (MST) and Glycolytic Stress Test (GST) were performed. For MST, normal culture medium was changed for minimal medium containing 0.2% FBS, 25 mM glucose, 1mM HEPES, Penicillin/Streptomycin and L- glutamine. Plates were incubated for 2 hrs at 37ºC in incubator. Through the sensor cartridge, mitochondrial stress test was assessed by subsequently adding 1 µM oligomycin, 3µM FCCP and 2 µM Rotenone together with 2 µM Antimycin A. Oxygen

consumption rate (OCR) was recorded as well as extracellular acidification 174

rates (ECAR). At the end of the experiment proteinase K was added to the plate and left over overnight for DNA normalization the day after. GelDye mix (Biotrium, 3ul of GelDye per 100ml of PBS) for DNA quantification. For GST, cells in glucose free medium for 2 hrs. GST was performed by subsequently adding 10 mM glucose and 10 mM 2-deoxyglucose. Results were also normalized on DNA quantity. For fatty acid oxidation, MST cells were previously starved for 24 hours in substrate-limited medium (DMEM containing 0,5 mM glucose, 1 mM glutamax, 0,5 mM carnitine and 1% FBS). Cells were washed with FAO medium (111 mM NaCl, 4.7

mM KCl, 1.25 mM CaCl2, 2 mM MgSO4, 1.2 mM NaH2PO4, 2.5 mM glucose, 0.5 mM carnitine, and 5 mM HEPES at pH 7.4) 45 min prior the assay. Etomoxir (Eto), Palmitate- BSA or BSA alone were added prior MST assay. OCR and ECAR measurements were assessed by adding subsequently MST compounds and as well normalized for DNA quantity.

Lactate and Glucose measurements

To measure lactate, cell culture supernatant was transferred to 5 mm NMR tubes. 1H- NMR spectra were recorded at 300 K on an Avance III 600 spectrometer (Bruker, Germany) operating at a proton frequency of 600.20 MHz using a 5 mm CPTCI triple resonance (1H, 13C, 31P) gradient cryoprobe. One-dimensional 1H pulse experiments were carried out using the nuclear Overhauser effect spectroscopy (NOESY) presaturation sequence to suppress the residual water peak. The acquired spectral width was 12 kHz (20 ppm), and a total of 256 transients were collected into 64 k data points for each 1H spectrum. 1H NMR spectra were referenced to the chemical shift of TSP signal at 0.0 ppm. References of pure compounds from the metabolic profiling AMIX spectra database (Bruker) and Chenomx database were used for lactic acid identification. After baseline correction, the specific NMR region of lactic acid was integrated using the AMIX 3.9 software package. Glucose concentrations were measured using glucose hexokinase method (Siemens, Dimension RHL Max Clinical Chemistry System, Siemens Healthcare Diagnostics) at the clinical biochemistry laboratory of the

Hospital Universitari Germans Trias I Pujol (Spain). 175

Targeted metabolomics

C2C12 cells at day 4 of differentiation were washed 3 times with PBS, immediately collected by scraping and shock frozen. Mitochondria were isolated and shock frozen as well. Metabolites were extracted into the extraction solvent by adding 300µL of cold ACN/H2O (1:1). For protein precipitation, samples were vigorously mixed by vortexing for 30 seconds and stored at –20°C for 1 h to enable protein precipitation. Subsequently, samples were centrifuged 15 minutes at 4°C and 22.600 g and the supernatant was transferred to a LC-MS vial. The samples were analysed using an UHPLC system (1290 Agilent) coupled to a triple quadrupole (QqQ) MS (6490 Agilent Technologies) with iFunnel technology operated in multiple reaction monitoring (MRM), and positive electrospray ionization (ESI+) mode. Metabolites were separated using a C18-RP (ACQUITY UPLC BEH 1.7 µm, Waters) chromatography at a flow rate of 0.3 mL/min. The solvent system was A (20 mM ammonium acetate and 15 mM ammonia in water : acetonitrile [97:3]) and acetonitrile as B. The gradient elution started at 100% A (time 0- 1 min) and finished at 100% B (8-11 min). The injection volume was 5 ml. ESI conditions were: gas temperature, 170 oC; drying gas, 11 L/min; nebulizer, 20 psi; and fragmentor, 380 V. Quality control (QC) consisting of pooled samples were used. Four QC samples were injected repeatedly during the whole analysis. MRM transitions were: NAM (123.06→ 107.10, 80.10), NMN (335.07→ 123.10, 97.00) and NAD+ (664.12→ 428.00, 136.00). The peak areas were manually integrated and the data normalized to total DNA for cells extracts and NDUFA9 protein for isolated mitochondria.

Molecular data mining

MacroH2A sequences were collected from GenBank database by Blast searches using human sequences as query. To get a better representation of species, especially in the transition to the vertebrate lineage, de novo assembly of transcriptomes of jawless fish (hagfish and lamprey; bioproject acc. numbers: PRJDB4902 and PRJNA292033, respectively) and the bowfin, Amia calva (Bioproject acc. number:

PRJNA29203), were carried out using the software Trinity version 2.2.0 in 176

the Galaxy web platform located at the public server usegalaxy.org (Afgan et al., 2016). Briefly, paired-end SRA Fastq files were uploaded from the European Nucleotide Archive (ENA) to the Galaxy platform and their quality analyzed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). All left and right reads were concatenated in two separate files (one for left and one for right reads) and used as input for Trinity leaving all parameters by default. Assembled sequences were used to create local nucleotide databases, and Blast searches were performed as described above. Overall, 159 sequences encompassing 80 metazoan (40 vertebrate and 40 non-vertebrate) and 1 non-metazoan species were retrieved. Three macroH2A variants (mH2A.1.1, mH2A.1.2 and mH2A.2) were collected for all vertebrate species except for the hagfish, Eptatretus atami, and the lamprey, Geotria australis, that lack the variant mH2A.1.2. For all macroH2A proteins, only the globular part of the macrodomain (amino acids 182 to end in human sequences) was used in the analysis. Multiple sequence alignments were performed using MAFFT version 7 (Katoh & Standley, 2013) and Jalview version 2 (Waterhouse et al., 2009), and edited for potential errors in Bioedit version 7 (A. Hall, 1999). Logo plots were generated based on the aligned sequences using WebLogo3 (weblogo.threeplusone.com; (Crooks et al., 2004).

Phylogenetic and molecular evolutionary analyses

Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 7 (Kumar et al., 2016) except where noted. MacroH2A phylogeny was inferred by using the Maximum Likelihood method, with the substitution model that best fit the analyzed protein sequences being LG (Le & Gascuel, 2008), and including gamma-distributed variation among sites. The analysis involved 121 amino acid sequences and a total of 156 positions in the final dataset (positions with less than 95% site coverage were eliminated). The reliability of the reconstructed topology was contrasted by nonparametric bootstrap (BS) method (1,000 replicates). Nucleotide and protein sequence divergence was estimated using uncorrected differences (p-distances, partial deletion 95%) and the rates of evolution of the macro

domains and exon 5 from macroH2A.1.1, macroH2A.1.2, and macroH2A.2 177

were estimated by correlating pairwise protein divergences between pairs of taxa with their corresponding divergence times as defined by the TimeTree database (Hedges, Dudley, & Kumar, 2006).

178

RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies Anti-embryonic Myosin Heavy Chain Gift from dr. Pura Muñoz N/A F1.652 Canoves, UPF, Barcelona Anti-FLAG M2 Sigma F3165 Anti-GFP Santa Cruz sc-9996 Anti-Histone H3 Abcam ab-1791 Anti-macroH2A1 Marcus Buschbeck N/A laboratory Anti-macroH2A1.1 Gift from dr. Andreas N/A Ladurner, LMU, Munich Anti-macroH2A1.2 Gift from dr. Andreas N/A Ladurner, LMU, Munich Anti-macroH2A1.2 Cell Signaling #4827 Anti-MyoD Santa Cruz sc-760 Anti-Myogenin F5D Santa Cruz sc-12732 Anti-NDUFA9 Abcam ab14713 Anti-PAR Ladurner lab. N/A Anti-PAR Trevigen 4336-APC-050 Anti-PARP1 Abcam Ab6079 Anti-PARP1 Trevigen 4338-MC-50 Anti-PARP1 Cell signalling 46D11 #9532 Anti-mouse-HRP DakoCytomation P0447 Anti-rabbit-HRP DakoCytomation P0448 Alexa 488 Thermo Fischer N/A

179

RESOURCES TABLE

Alexa 488 Invitrogen A11001 GFP-trap, nanobodies, magnetic bead ChromoTek gtm-100 coupled Biological Samples Human muscle tissue Pathologic Anatomy N/A Department of the Hospital Universitari Germans Trias i Pujol, Badalona, Spain Chemicals, Peptides, and Recombinant Proteins ADP-HPD inhibitor CalBioChem 118415 Antimycin A Sigma-Aldrich A8674 Basic Fibroblast Growth Factor Invitrogen PHG0264 Benzonase Novagen N/A 2-Deoxy-D-glucose Sigma-Aldrich D6134 DpnI enzyme Fermentas ER1701 Carbonyl cyanide 4- Sigma-Aldrich C2920 (trifluoromethoxy)phenylhydrazone (FCCP) Glucose Sigma-Aldrich G7528 Hematoxylin Sigma-Aldrich HHS80-2,5L HpaII/MspI enzyme Fermentas ER0541 Insulin Sigma-Aldrich 91077C Leupeptin Sigma-Aldrich 62070-10MG Lipofectamine RNAiMAX Transfection Invitrogen 13778150 Reagent Matrigel BDBiosciences 354234 b-Nicotinamide mononucleotide (NMN) Sigma-Aldrich N3501 Nicotinamide (NAM) Sigma-Aldrich 72340

180

RESOURCES TABLE

30-NICK DNA Gift of dr. Gytis N/A Jankevicius, University of Oxford, Oxford NTA-Ni beads Qiagen 30230 Olaparib SelleckChem S1060 Oligomycin Sigma-Aldrich O4876 Paraformaldehyde Sigma-Aldrich 158127-500G PARP1 HAS enzyme Trevigen 4668-02K-01 Polybrene Sigma-Aldrich H9268-5g Phenylmethylsulfonyl Fluoride (PMSF) Sigma-Aldrich 78830-1G Rat-tail collagen I BDBiosciences N/A Rotenone Sigma-Aldrich R8875 Sucrose Merck 1-07651-1000 TSA Sigma-Aldrich T8552

VectaShield Mounting Medium with DAPI Vector Laboratories 53826 Critical Commercial Assays Agilent SurePrint G3 Mouse GE 8x60K Agilent N/A Microarray Total RNA Nano Assay Agilent N/A Deposited Data Microarray and ChIP-seq data This thesis GEO: GSE78257 Experimental Models: Cell Lines C2C12 cells ATCC CRL-1772 HEK293T cells ATCC CRL-3216 GP2-293 Packaging cell line Clontech Laboratories 631458 E.coli Rosetta D3 cells EMD Millipore 70954 Primary mouse myoblasts This thesis N/A

181

RESOURCES TABLE

Primary human myoblasts Gift of Dr. Eduard N/A Gallardo, IR-Sant Pau, Barcelona Recombinant DNA pBabe.puro FLAG-macroH2A1.1 (mouse) This thesis N/A pBabe.puro FLAG-macroH2A1.1 G224E This thesis N/A (mouse) pBabe.puro FLAG-macroH2A1.1 (w/ 4 This thesis N/A silent mutations, mouse) pBabe.puro FLAG-macroH2A1.1 G224E This thesis N/A (w/ 4 silent mutations, mouse) His/GFP-macro1.1 domain (Timinszky et al, 2009) N/A His/GFP-macro1.2 domain (Timinszky et al, 2009) N/A His/GFP-Caps.owz. macro domain (This thesis) N/A His-Caps.owz. N-ter.PARP1 (This thesis) N/A Sequence-Based Reagents siRNA scramble (control) Invitrogen, This thesis N/A cguacgcggaauacuucgatt siRNA against macroH2A1.1-1 Invitrogen, This thesis N/A cgacaaacacugacuucuatt siRNA against macroH2A1.1-2 Invitrogen, This thesis N/A ccgacaaacacugacuucutt siRNA against macroH2A1.2-1 Invitrogen, This thesis N/A gcuuugagguggaggccautt siRNA against macroH2A1.2-2 Invitrogen, This thesis N/A ugacauugaccuuaaagautt siRNA against PARP1-1 Invitrogen, This thesis N/A AGCUGAAGAAAGCGTGUUCTT siRNA against PARP1-2 Invitrogen, This thesis N/A AGGCGUGGCAGGCAAAGGCTT

182

RESOURCES TABLE

siRNA against NMNAT3-1 Invitrogen, This thesis N/A siRNA against NMNAT3-2 Invitrogen, This thesis N/A qPCR Primer for mouse MH2a1 forward Invitrogen, This thesis N/A gacggtgaaaaactgcttgg qPCR Primer for mouse MH2a1 reverse Invitrogen, This thesis N/A ggaggaggacatcgtggag qPCR Primer for mouse MH2a2 forward Invitrogen, This thesis N/A gctggaagagaccatcaaaaa qPCR Primer for mouse MH2a2 reverse Invitrogen, This thesis N/A cgaagtgagccgagatgg qPCR Primer for mouse MH2a1 Exon 5 Invitrogen, This thesis N/A forward cctacagacggcttcactgtc qPCR Primer for mouse MH2a1.2 Exon 6 Invitrogen, This thesis N/A reverse ggtcaatgtcagcattggtagg qPCR Primer for mouse MH2a1.1 Exon 7 Invitrogen, This thesis N/A reverse gtgtagaagtcagtgtttgtcg qPCR Primer for human MH2A1 Exon 9 Invitrogen, This thesis N/A reverse gagttccaggacagcttccac qPCR Primer for human MH2A1.2 Exon Invitrogen, This thesis N/A 6/7 forward tccttggccagaagctgaac qPCR Primer for human MH2A1.1 Exon 8 Invitrogen, This thesis N/A forward ttcacccgacaaacactgac qPCR Primer for human MH2A1 forward Invitrogen, This thesis N/A cctggctgatgataagaagctg qPCR Primer for human MH2A1 reverse Invitrogen, This thesis N/A gacacgaagtaactggagatgg qPCR Primer for human MH2A2 forward Invitrogen, This thesis N/A catggcggcagtcattgag qPCR Primer for human MH2A2 reverse Invitrogen, This thesis N/A attgccggccaattctagaa

183

RESOURCES TABLE

qPCR Primer for mouse Myog forward Invitrogen, This thesis N/A ggtgtgtaagaggaagtctgtg qPCR Primer for mouse Myog reverse Invitrogen, This thesis N/A taggcgctcaatgtactggat qPCR Primer for human MYOG forward Invitrogen, This thesis N/A cagctccctcaaccaggag qPCR Primer for human MYOG reverse Invitrogen, This thesis N/A cactgccccactctggac qPCR Primer for mouse Ckm forward Invitrogen, This thesis N/A acccacagacaagcataagaccga qPCR Primer for mouse Ckm reverse Invitrogen, This thesis N/A aggcagagtgtaacccttgatgct qPCR Primer for human CKM forward Invitrogen, This thesis N/A ctgacaagcacaagactgacc qPCR Primer for human CKM reverse Invitrogen, This thesis N/A ctgctgagcacgtagttaggg qPCR Primer for mouse Rpl7 forward Invitrogen, This thesis N/A gaagctcatctatgagaaggc qPCR Primer for mouse Rpl7 reverse Invitrogen, This thesis N/A aagacgaaggagctgcagaac qPCR Primer for mouse Gapdh forward Invitrogen, This thesis N/A tgcaccaccaactgcttag qPCR Primer for mouse Gapdh reverse Invitrogen, This thesis N/A gatgcagggatgatgttc qPCR Primer for human/mouse RPL0 Invitrogen, This thesis N/A forward ttcattgtgggagcagac qPCR Primer for human/mouse RPL0 Invitrogen, This thesis N/A reverse cagcagtttctccagagc qPCR Primer for mouse Mstn forward Invitrogen, This thesis N/A cgctaccacggaaacaatc

184

RESOURCES TABLE

qPCR Primer for mouse Mstn reverse Invitrogen, This thesis N/A aaagcaacatttgggcttg qPCR Primer for mouse Tmem171 Invitrogen, This thesis N/A forward aaacccaccttcctattccag qPCR Primer for mouse Tmem171 reverse Invitrogen, This thesis N/A atgaaccctgcccagaaatg qPCR Primer for mouse Itga11 forward Invitrogen, This thesis N/A gggaaacctgtggctgac qPCR Primer for mouse Itga11 reverse Invitrogen, This thesis N/A atgaaggggctgtggaac qPCR Primer for mouse Chdr1 forward Invitrogen, This thesis N/A tacagagcgtccaggaaaag qPCR Primer for mouse Chdr1 reverse Invitrogen, This thesis N/A ttgttaggctcctctgcatc qPCR Primer for mouse Coxvb forward Invitrogen, This thesis N/A agcagcacagaagggactg qPCR Primer for mouse Coxvb reverse Invitrogen, This thesis N/A tggacgggactagattaggg qPCR Primer for mouse Cytc forward Invitrogen, This thesis N/A caaatctccacggtctgttc qPCR Primer for mouse Cytc reverse Invitrogen, This thesis N/A tccatcagggtatcctctcc qPCR Primer for mouse Cpt1b forward Invitrogen, This thesis N/A ttgctacaaccctgacgatg qPCR Primer for mouse Cpt1b reverse Invitrogen, This thesis N/A tgcaggagataagggtgaaag qPCR Primer for mouse Acadm forward Invitrogen, This thesis N/A attgtggaagccgacacc qPCR Primer for mouse Acadm reverse Invitrogen, This thesis N/A tttccttaggcactctgacg

185

RESOURCES TABLE

qPCR Primer for mouse Cd36 forward Invitrogen, This thesis N/A gcaaagaacagcagcaaaatc qPCR Primer for mouse Cd36 reverse Invitrogen, This thesis N/A cggggtcctgagttatattttc qPCR Primer for mouse Ucp2 forward Invitrogen, This thesis N/A agttctacaccaagggctcag qPCR Primer for mouse Ucp2 reverse Invitrogen, This thesis N/A aagcggacctttaccacatc qPCR Primer for mouse Cs forward Invitrogen, This thesis N/A ggagccaagaactcatcctg qPCR Primer for mouse Cs reverse Invitrogen, This thesis N/A tctggcctgctccttaggta qPCR Primer for mouse Fabp3 forward Invitrogen, This thesis N/A gacagcagatgaccggaag qPCR Primer for mouse Fabp3 reverse Invitrogen, This thesis N/A gttgtctcctgcccgttc qPCR Primer for mouse Sdha forward Invitrogen, This thesis N/A ctgatggaaaatggggagtg qPCR Primer for mouse Sdha reverse Invitrogen, This thesis N/A tgaagtaggttcgcccgtag qPCR Primer for mouse Ndufa9 forward Invitrogen, This thesis N/A aaggaagctggggttgagag qPCR Primer for mouse Ndufa9 reverse Invitrogen, This thesis N/A tggcttcaggaaacacacttc qPCR Primer for mouse Atp5a1 forward Invitrogen, This thesis N/A ccctcggtaatgctattgatg qPCR Primer for mouse Atp5a1 reverse Invitrogen, This thesis N/A taattccaggggctttcagg qPCR Primer for mouse Cox4i1 forward Invitrogen, This thesis N/A gagcctgattggcaagagag

186

RESOURCES TABLE

qPCR Primer for mouse Cox4i1 reverse Invitrogen, This thesis N/A atcagcgtaagtggggaaag qPCR Primer for mouse Uqcrc2 forward Invitrogen, This thesis N/A gtttcgccgttgggaagtag qPCR Primer for mouse Uqcrc2 reverse Invitrogen, This thesis N/A agccaaggcattcttgtagg qPCR Primer for mouse Slc2a1 forward Invitrogen, This thesis N/A tgcagttcggctataacactg qPCR Primer for mouse Slc2a1 reverse Invitrogen, This thesis N/A gagtgtggtggatgggatg qPCR Primer for mouse Slc2a4 forward Invitrogen, This thesis N/A gcaccctcactacgctctg qPCR Primer for mouse Slc2a4 reverse Invitrogen, This thesis N/A gccagcatagcccttttc qPCR Primer for mouse Hk2 forward Invitrogen, This thesis N/A ggcggatcaaagagaacaag qPCR Primer for mouse Hk2 reverse Invitrogen, This thesis N/A agcctcctcactgccttatg qPCR Primer for mouse Pdhe1a forward Invitrogen, This thesis N/A ggttgtgctaaagggaaagg qPCR Primer for mouse Pdhe1a reverse Invitrogen, This thesis N/A caccatcgccgtataatgtc qPCR Primer for mouse Pdk4 forward Invitrogen, This thesis N/A aaagatgctctgcgaccag qPCR Primer for mouse Pdk4 reverse Invitrogen, This thesis N/A cacaatgtggattggttgg qPCR Primer for mouse Igf1 forward Invitrogen, This thesis N/A tggtggatgctcttcagttc qPCR Primer for mouse Igf1 reverse Invitrogen, This thesis N/A cacaatgcctgtctgaggtg

187

RESOURCES TABLE

qPCR Primer for mouse Nampt forward Invitrogen, Cantó et al., N/A ccgccacagtatctgttcctt 2009, Nature qPCR Primer for mouse Nampt reverse Invitrogen, Cantó et al., N/A agtggccacaaattccagaga 2009, Nature qPCR Primer for mouse Nmnat-1 forward Invitrogen, Cantó et al., N/A aggagtgggtggagactgtg 2009, Nature qPCR Primer for mouse Nmnat-1 reverse Invitrogen, Cantó et al., N/A cagtgcaggtgagctttgtg 2009, Nature qPCR Primer for mouse Nmnat-2 forward Invitrogen, Cantó et al., N/A aaggtgggagaaagcctcag 2009, Nature qPCR Primer for mouse Nmnat-2 reverse Invitrogen, Cantó et al., N/A ctcctcataccgcatcactg 2009, Nature qPCR Primer for mouse Nmnat-3 forward Invitrogen, This thesis N/A cagatcctcagcccagatg qPCR Primer for mouse Nmnat-3 reverse Invitrogen, This thesis N/A gaaggtcttgaggacatcagc qPCR Primer for mouse Parp-1 forward Invitrogen, This thesis N/A cctgaacaacgcagacagc qPCR Primer for mouse Parp-1 reverse Invitrogen, This thesis N/A cgttgtgcgtggtagcatga qPCR Primer for mouse Parp-2 forward Invitrogen, This thesis N/A ggaaggcgagtgctaaatgaa qPCR Primer for mouse Parp-2 reverse Invitrogen, This thesis N/A ggaaggcgagtgctaaatgaa qPCR Primer for mouse Myh1 forward Invitrogen, This thesis N/A ctgaagggcggcaagaag qPCR Primer for mouse Myh1 reverse Invitrogen, This thesis N/A cgcttctgttcattttccac qPCR Primer for mouse Myh4 forward Invitrogen, This thesis N/A gagctactggatgccagtgagcgc

188

RESOURCES TABLE

qPCR Primer for mouse Myh4 reverse Invitrogen, This thesis N/A ctggacgatgtcttccatctctcc qPCR Primer for mouse Myf6 forward Invitrogen, This thesis N/A gcgtggacccctacagctac qPCR Primer for mouse Myf6 reverse Invitrogen, This thesis N/A cgtggaggaggtggtggagaag qPCR Primer for mouse fabp3 forward Invitrogen, This thesis N/A gacagcagatgaccggaag qPCR Primer for mouse fabp3 reverse Invitrogen, This thesis N/A gttgtctcctgcccgttc qPCR Primer for mouse atp9a forward Invitrogen, This thesis N/A tttgggtgttaatcagcatctatc qPCR Primer for mouse atp9a reverse Invitrogen, This thesis N/A ggatgagggatgtgaaggag qPCR Primer for mouse rgcc forward Invitrogen, This thesis N/A TTCAGCGACTCGGAGAGTG qPCR Primer for mouse rgcc reverse Invitrogen, This thesis N/A ATCACTGAAGGTGAAGCTGTC qPCR Primer for mouse camk1g forward Invitrogen, This thesis N/A GGTGAAGCAAAGAGTGACTG qPCR Primer for mouse camk1g reverse Invitrogen, This thesis N/A GGAAGGCTGGTGACTTCTT qPCR Primer for mouse fmod forward Invitrogen, This thesis N/A TCCTCTGGGTCGCTCTACAT qPCR Primer for mouse fmod reverse Invitrogen, This thesis N/A TGCCTCAGCTTGGAGAAGAC qPCR Primer for mouse col1a1 forward Invitrogen, This thesis N/A GCGAGTGCTGTGCTTTCTG qPCR Primer for mouse col1a1 reverse Invitrogen, This thesis N/A GGTCCCTCGACTCCTACATCT

189

RESOURCES TABLE

qPCR Primer for mouse fn1 forward Invitrogen, This thesis N/A GACAACCGAGGAAACCTGCT qPCR Primer for mouse fn1 reverse Invitrogen, This thesis N/A GATCCGGCTGAAGCACTTTG qPCR Primer for mouse cilp forward Invitrogen, This thesis N/A GGCTGTGAAGTCCAAGGTCA qPCR Primer for mouse cilp reverse Invitrogen, This thesis N/A TAGCTCTCTGGGGTTGGGTT qPCR Primer for mouse tmem171 forward Invitrogen, This thesis N/A aaacccaccttcctattccag qPCR Primer for mouse tmem171 reverse Invitrogen, This thesis N/A atgaaccctgcccagaaatg qPCR Primer for human myod1 forward Invitrogen, This thesis N/A CGGCATGATGGACTACAGCG qPCR Primer for human myod1 reverse Invitrogen, This thesis N/A CAGGCAGTCTAGGCTCGAC qPCR Primer for human myog forward Invitrogen, This thesis N/A cagctccctcaaccaggag qPCR Primer for human myog reverse Invitrogen, This thesis N/A cactgccccactctggac qPCR Primer for human ckm forward Invitrogen, This thesis N/A GGGGCAACATGAAGGAGGTT qPCR Primer for human ckm reverse Invitrogen, This thesis N/A TGGTTCCACATGAAGGGGTG qPCR Primer for human myh1 forward Invitrogen, This thesis N/A TGCCATTGAAATTCTGGGCT qPCR Primer for human myh1 reverse Invitrogen, This thesis N/A CATAATGCATCACAGCCCCT qPCR Primer for human myh4 forward Invitrogen, This thesis N/A GCAACAGACACCTCCTTCAA

190

RESOURCES TABLE

qPCR Primer for human myh4 reverse Invitrogen, This thesis N/A CTTGGGCTTCTGGAAGTTGT qPCR Primer for human myf6 forward Invitrogen, This thesis N/A gcgtggacccctacagctac qPCR Primer for human myf6 reverse Invitrogen, This thesis N/A cgtggaggaggtggtggagaag qPCR Primer for human fn1 forward Invitrogen, This thesis N/A TACGGCCACCAAGAAGTGAC qPCR Primer for human fn1 reverse Invitrogen, This thesis N/A AGGGAGTCGTCTCTCCTGTC qPCR Primer for human col1a1 forward Invitrogen, This thesis N/A CCTGCCTGGTGAGAGAGGT qPCR Primer for human col1a1 reverse Invitrogen, This thesis N/A AGTAGCACCATCATTTCCACGA qPCR Primer for human fmod forward Invitrogen, This thesis N/A GGCCTTGTACCTCCAACACA qPCR Primer for human fmod reverse Invitrogen, This thesis N/A CAGCAAGATCAGTGACCGGA qPCR Primer for human rgcc forward Invitrogen, This thesis N/A TAGGAACAGCTTCAGCTTCAGT qPCR Primer for human rgcc reverse Invitrogen, This thesis N/A AGAGCTGGGGTAGAGTCTGT qPCR Primer for human itga11 forward Invitrogen, This thesis N/A GGGTTCACGGACACCTTCAA qPCR Primer for human itga11 reverse Invitrogen, This thesis N/A TGTAGCCAAAGAAGGCGGTC

qPCR Primer for genomic mouse Ndufv1 Invitrogen, This thesis N/A forward cttccccactggcctcaag qPCR Primer for genomic mouse Ndufv1 Invitrogen, This thesis N/A

1 reverse ccaaaacccagtgatccagc 19

RESOURCES TABLE

qPCR Primer for mitochondrial mouse Invitrogen, This thesis N/A Nd2 forward agggatcccactgcacatag qPCR Primer for mitochondrial mouse Invitrogen, This thesis N/A Nd2 reverse ctcctcatgcccctatgaaa qPCR Primer for human HPRT1 forward Invitrogen, This thesis N/A tggacaggactgaacgtcttg qPCR Primer for human HPRT1 reverse Invitrogen, This thesis N/A ccagcaggtcagcaaagaatt

RFLP Primers for human MH2A1 Exon 6 Invitrogen, This thesis N/A forward gcttcacagtcctcctctccacc RFLP Primers for human MH2A1 Exon 9 Invitrogen, This thesis N/A reverse gagttccaggacagcttccac RFLP Primers for mouse Mh2a1 Exon 5 Invitrogen, This thesis N/A forward cctacagacggcttcactgtc RFLP Primers for mouse Mh2a1 Exon 8 Invitrogen, This thesis N/A reverse cgcccttcttctccagtgtg Mutagenesis Primers for mouse mH2A1.1 Invitrogen, This thesis N/A silent_mut_887/890_forward gtgatgctgtcgttcacccgaccaatactgacttctacaccg gtgg Mutagenesis Primers for mouse mH2A1.1 Invitrogen, This thesis N/A silent_mut_887/890_reverse ccaccggtgtagaagtcagtattggtcgggtgaacgacag catcac Mutagenesis Primers for mouse mH2A1.1 Invitrogen, This thesis N/A silent_mut_893/896_forward ctgtcgttcacccgaccaatacggatttctacaccggtggtg aagt Mutagenesis Primers for mouse mH2A1.1 Invitrogen, This thesis N/A silent_mut_893/896_reverse 192

RESOURCES TABLE

acttcaccaccggtgtagaaatccgtattggtcgggtgaac gacag Mutagenesis Primers for mouse mH2A1.1 Invitrogen, This thesis N/A silent_GGT667GAG_forward ccgaccaatacggatttctacaccggtgaggaagtaggaa acacactggagaag Mutagenesis Primers for mouse mH2A1.1 Invitrogen, This thesis N/A silent_GGT667GAG_reverse cttctccagtgtgtttcctacttcctcaccggtgtagaaatcc gtattggtcgg Mutagenesis Primers for Caps. owc. Invitrogen, This thesis N/A mH2A1.1 G224E forward ACCATGTCGTTCGCGGAACAAGTCG GCGGTGCC Mutagenesis Primers for Caps. owc. Invitrogen, This thesis N/A mH2A1.1 G224E reverse GGCACCGCCGACTTGTTCCGCGAAC GACATGGT Mutagenesis Primers for Caps. owc. Invitrogen, This thesis N/A mH2A1.1 G314E forward CCGTCGATCGGCTCTGAAAACAACC ACTTCCCC Mutagenesis Primers for Caps. owc. Invitrogen, This thesis N/A mH2A1.1 G314E reverse GGGGAAGTGGTTGTTTTCAGAGCCG ATCGACGG

Cloning Primers for mouse Invitrogen, This thesis N/A mH2A1.1_aa1_EcoRI_forward gcgaattccatgtcgagccgcggcgggaagaag

193

RESOURCES TABLE

Cloning Primers for mouse Invitrogen, This thesis N/A mH2A1.1_STOP_XhoI_reverse ggctcgag

tcagcctagttggcgtccagcttgg

Software and Algorithms AMIX Bruker version 3.9 Bowtie 2 Langmead and Salzberg, 2012 ChIPpeakAnno R package Zhu el al., 2010 N/A ImageJ http://imagej.net/Contrib version 2.0.0-rc- utors 54/1.51h LIMMA Wettenhall el al., 2004 N/A

Photoshop Adobe Photoshop CS4 version 11.0

REVIGO Supek et al., 2011 N/A

SICER Zhang et al., 2009 Version 1.1

194

Publications

> Posavec Marjanović M.*, Hurtado-Bagès S.*, Lassi M., Valero V., Malinverni R., Delage H., Navarro M., Corujo D., Guberovic I., Douet J., Gama P., Garcia-Roves P.M., Ahel I., Ladurner A.G., Yanes O., Bouvet P., Suelves M., Teperino R., Pospisilik J.A. and Buschbeck M. (2017). MacroH2A1.1 regulates mitochondrial activity by limiting nuclear NAD+ consumption. Nature Structural Molecular Biology. 2017. DOI: 10.1038/nsmb.3481. PMID: 28991266. IF: 13.4. D1. *Shared co-first authorship.

> Hurtado-Bagès S., Guberovic I., Buschbeck M. (2018). The MacroH2A1.1 - PARP1 Axis at the Intersection Between Stress Response and Metabolism. Frontiers in Genetics. DOI: 10.3389/fgene.2018.00417.

> Vieira-Silva T.S., Monteiro-Reis S., Barros-Silva D., Ramalho-Carvalho J., Graça I., Carneiro I., Martins A.T., Oliveira J., Antunes L., Hurtado-Bagès S., Buschbeck M., Henrique R., Jerónimo C. (2019). Histone variant MacroH2A1 is downregulated in prostate cancer and influences malignant cell phenotype. Cancer Cell Int. DOI: 10.1186/s12935-019-0835-9

Student Supervision and Workshop Organisation

> Johanna Albert, Master student, "Role of MacroH2A1 in muscle maturation" (2018). > Organizer of Bioinformatic course ran by EpiChemBio COST Action (March 2019: http://epichembio.eu/bioinformatics-workshop-introduction-to-ngs-data-analysis/)

Scientific communication

International scientific network * Vice Science communication manager of the COST Action CA18127 (International Nucleome Consortium: https://www.cost.eu/actions/CA18127/#tabs|Name:overview)

Oral presentation * EpiChemBio, COST Action. Budapest, Hungary (2015). * Histone variants: molecular functions in health and disease, EMBO. Munich, Germany (2017). * 2nd PhD day: campus Can Ruti, Badalona, Spain. I was awarded the best PhD talk (2019).

Poster * EMBO/FEBS Lecture Course on Chromatin and the Environment, EMBO and FEBS. Spetses, Greece (2016) * SCB Barcelona Chromatin and Epigenetic. Barcelona, Spain (2016) * Epigenetic Mechanisms in Health and Disease, IBMB. Barcelona, Spain (2017) * Advances at the interface between metabolism and epigenetics. Cambridge, UK (2019)

Article revision * Involved in the revision of article published in Nature Communication (2018)

Scientific communication and dissemination to non-scientific audience

* The craft of clear scientific writing (course) organised between Intervals and CÍCLIKS program (PhD in Biomedicine, UPF. Barcelona, Spain (2018) * Rin4 competition, "present your PhD in 4'", PhD in Biomedicine, UPF University. Barcelona, Spain (2016) * Training for 5th BCN Science Slam: organized by the CRG and MCAA in the framework of Barcelona Science Bienal, Barcelona, 2019. * Science Slam competition: organized by the CRG and MCAA in the framework of Barcelona Science Bienal, Barcelona, 2019. "Let me tell you the love story I found in your cells."

Annex

Annex

Annex 1. Deregulated genes by both macroH2A1 isoforms

Subset of genes regulated by both macroH2A1 isoforms pvalue = 0.01 & log2FC [<-0.8 & > 0.8]

DOWN in si mH2A1.1 & UP in si mH2A1.2 (46) DEG_gene_id names si macroH2A1.1 vs si Ctrl (log2FoldChange) si macroH2A1.2 vs si Ctrl (log2FoldChange) si macroH2A1.1 vs si Ctrl (padj) si macroH2A1.2 vs si Ctrl (padj) mgi_description ENSMUSG00000001506 Col1a1 -2,377330465 1,405247912 1,19E-10 0,000958106 collagen type I alpha 1 ENSMUSG00000004791 Pgf -1,256400345 0,869759285 3,67E-12 6,76E-06 placental growth factor ENSMUSG00000004885 Crabp2 -1,169833723 0,92420067 4,15E-06 0,000881273 cellular retinoic acid binding protein II ENSMUSG00000005124 Wisp1 -0,809916751 0,898773842 3,19E-06 3,71E-07 WNT1 inducible signaling pathway protein 1 ENSMUSG00000020053 Igf1 -2,006391632 1,118275354 9,92E-10 0,003473539 insulin-like growth factor 1 ENSMUSG00000020186 Csrp2 -1,009987918 0,830330658 2,37E-07 7,34E-05 cysteine and glycine-rich protein 2 ENSMUSG00000020614 Fam20a -1,196045085 1,724171649 0,003342068 1,24E-05 family with sequence similarity 20 member A ENSMUSG00000021614 Vcan -0,806748849 1,192135877 0,00027519 2,38E-08 versican ENSMUSG00000021803 Cdhr1 -1,604683595 1,982930914 0,000337954 4,73E-06 cadherin-related family member 1 ENSMUSG00000022479 Vdr -0,888975797 0,88472144 0,002131311 0,004252163 vitamin D (125-dihydroxyvitamin D3) receptor ENSMUSG00000022816 Fstl1 -0,962057981 1,27655575 0,001597118 2,11E-05 follistatin-like 1 ENSMUSG00000023411 Nfatc4 -1,394013952 1,142921591 2,00E-05 0,000672738 nuclear factor of activated T cells cytoplasmic calcineurin dependent 4 ENSMUSG00000023886 Smoc2 -1,713985343 1,139261127 5,82E-10 5,31E-05 SPARC related modular calcium binding 2 ENSMUSG00000024544 Ldlrad4 -0,838207311 0,861007035 0,000420975 0,00035848 low density lipoprotein receptor class A domain containing 4 ENSMUSG00000024620 Pdgfrb -1,313002293 1,334196426 1,03E-05 1,53E-05 platelet derived growth factor receptor beta polypeptide ENSMUSG00000026167 Wnt10a -1,233336228 0,931544837 4,35E-07 0,000459361 wingless-type MMTV integration site family member 10A ENSMUSG00000026193 Fn1 -1,059520589 0,892565452 0,000551339 0,009573151 fibronectin 1 ENSMUSG00000026494 Kif26b -0,814525574 1,389358058 0,003399465 7,80E-08 kinesin family member 26B ENSMUSG00000026574 Dpt -1,216508832 1,73192935 0,006590953 5,50E-05 dermatopontin ENSMUSG00000027335 Adra1d -1,346457092 1,554234548 0,003293256 0,000177371 adrenergic receptor alpha 1d ENSMUSG00000027848 Olfml3 -1,769172234 1,558294002 7,14E-10 1,93E-07 olfactomedin-like 3 ENSMUSG00000028226 Mmp16 -0,825948999 0,964411437 0,002110746 0,000208626 matrix metallopeptidase 16 ENSMUSG00000029838 Ptn -1,176869135 1,380292392 0,003359597 0,000693732 pleiotrophin ENSMUSG00000031298 Adgrg2 -0,942848867 1,265539664 0,005132064 8,35E-05 adhesion G protein-coupled receptor G2 ENSMUSG00000031558 Slit2 -0,901391343 1,204007143 0,000832171 4,63E-06 slit guidance ligand 2 ENSMUSG00000031673 Cdh11 -1,934651061 1,713384033 2,22E-12 1,05E-09 cadherin 11 ENSMUSG00000031740 Mmp2 -0,883793352 1,07545122 0,00287744 0,000332809 matrix metallopeptidase 2 ENSMUSG00000032085 Tagln -1,318754225 1,534880772 0,001042337 0,000167068 transgelin ENSMUSG00000032243 Itga11 -3,149978279 1,879377658 3,15E-07 0,00905777 integrin alpha 11 ENSMUSG00000032332 Col12a1 -1,733654286 1,432295523 5,52E-07 0,000139052 collagen type XII alpha 1 ENSMUSG00000037225 Fgf2 -0,837904952 1,21605623 0,003386629 6,24E-06 fibroblast growth factor 2 ENSMUSG00000037379 Spon2 -1,432614162 1,379557827 0,000331844 0,001111903 spondin 2 extracellular matrix protein ENSMUSG00000040152 Thbs1 -1,077328941 0,928377666 9,25E-07 8,28E-05 thrombospondin 1 ENSMUSG00000040270 Bach2 -1,289768709 1,019597249 0,000249597 0,007016235 BTB and CNC homology basic leucine zipper transcription factor 2 ENSMUSG00000041482 Piezo2 -1,096786313 1,215923258 0,000918376 0,00034532 piezo-type mechanosensitive ion channel component 2 ENSMUSG00000041559 Fmod -4,446745388 4,801433124 4,09E-06 6,61E-08 fibromodulin ENSMUSG00000042190 Cmklr1 -1,586106923 1,693917265 5,43E-07 6,77E-08 chemokine-like receptor 1 ENSMUSG00000042254 Cilp -3,121254903 2,270638831 8,40E-17 6,75E-09 cartilage intermediate layer protein nucleotide pyropjosphohydrolase ENSMUSG00000045658 Pid1 -0,918447501 0,979357105 0,006668391 0,004548991 phosphotyrosine interaction domain containing 1 ENSMUSG00000046402 Rbp1 -1,387514035 1,572617472 0,001187498 0,000178049 retinol binding protein 1 cellular ENSMUSG00000048368 Omd -1,714760577 1,228418384 3,80E-06 0,001051485 osteomodulin ENSMUSG00000050212 Eva1b -0,861687404 1,092010651 0,007558927 0,000397723 eva-1 homolog B (C. elegans) ENSMUSG00000052485 Tmem171 -1,910509918 1,308030346 7,14E-10 1,31E-05 transmembrane protein 171 ENSMUSG00000056174 Col8a2 -4,33011077 3,791208294 5,66E-06 2,28E-05 collagen type VII aplha 2 ENSMUSG00000063727 Tnfrsf11b -1,462655337 1,408944824 0,002240068 0,00107613 tumor necrosis factor receptor superfamily member 11b (osteoprotegerin)

DEGs in oppositein (64) direction DEGs ENSMUSG00000070469 Adamtsl3 -2,453245627 1,302798661 1,36E-11 0,00210453 ADAMTS-like 3 UP in si mH2A1.1 & DOWN in si mH2A1.2 (18) ENSMUSG00000001622 Csn3 1,972674574 -1,748696813 2,11E-05 0,006621089 casein kappa ENSMUSG00000002831 Plin4 0,87193693 -0,848637128 1,33E-06 1,37E-05 perilipin 4 ENSMUSG00000005716 Pvalb 1,636233331 -2,899362697 1,27E-06 1,28E-12 parvalbumin ENSMUSG00000020176 Grb10 1,399747795 -1,115211894 4,07E-05 0,004700475 growth factor receptor bound protein 10 ENSMUSG00000026638 Irf6 1,243113062 -1,642476114 0,004292785 0,000486485 interferon regulatory factor 6 ENSMUSG00000027546 Atp9a 3,374752188 -0,967611703 3,47E-43 0,007044066 ATPase class II type 9 A ENSMUSG00000027562 Car2 0,814790514 -0,981620703 0,002113545 0,000378899 carbonic anhydrase 2 ENSMUSG00000027692 Tnik 1,175450299 -1,063398519 2,01E-07 1,87E-05 TRAF2 and NCK interacting kinase ENSMUSG00000029095 Ablim2 1,588140635 -1,301883625 1,16E-06 0,000354017 actin-binding LIM protein 2 ENSMUSG00000030433 Sbk2 0,98337872 -0,814427647 3,15E-07 0,000260396 SH3-binding domain kinase family member 2 ENSMUSG00000033965 Slc16a2 1,193940716 -0,973340903 5,97E-05 0,005494392 solute carrier family 16 (monocarboxylic acid transporters) member 2 ENSMUSG00000034813 Grip1 1,482420036 -1,974719315 0,00362363 0,001705707 glutamate receptor interacting protein 1 ENSMUSG00000034981 Parm1 0,979604956 -1,14619979 0,005123356 0,001881462 prostate androgen-regulated mucin-like protein 1 ENSMUSG00000035923 Myf6 1,905985041 -1,649067327 1,05E-24 6,04E-12 myogenic factor 6 ENSMUSG00000056328 Myh1 1,260536295 -1,761090616 0,001154706 3,33E-06 myosin heavy polypeptide 1 adult ENSMUSG00000062760 1810041L15Rik 0,879450668 -0,883967943 0,000605951 0,001771901 RIKEN cDNA 1810041L15 gene ENSMUSG00000068122 Agtr2 3,022101146 -5,160413158 2,18E-06 0,002694811 angiotensin II receptor type 2 ENSMUSG00000112803 Gm36543 1,825792014 -1,35544017 1,70E-09 0,002515002 predicted gene 36543 UP in si mH2A1.1 & in si mH2A1.2 (16) ENSMUSG00000116461 20,19127078 19,29514526 1,24E-05 7,78E-05 ENSMUSG00000022156 Gzme 4,697279255 4,026378314 3,69E-06 0,000285669 granzyme E ENSMUSG00000047746 Fbxo40 3,313325713 1,188035057 2,93E-30 0,00179792 F-box protein 40 ENSMUSG00000053675 Tgm5 2,481045709 2,031681943 5,50E-07 0,000180984 transglutaminase 5 ENSMUSG00000050272 Dscam 2,134649395 2,24368014 0,003028182 0,003367248 DS cell adhesion molecule ENSMUSG00000015085 Entpd2 2,021761611 2,136858628 0,00054724 0,00044733 ectonucleoside triphosphate diphosphohydrolase 2 ENSMUSG00000031451 Gas6 1,871348075 1,879591378 5,90E-08 1,60E-07 growth arrest specific 6 ENSMUSG00000034555 Tex16 1,865514814 2,301597752 0,003576068 0,000350482 testis expressed gene 16 ENSMUSG00000049303 Syt12 1,795637965 1,09404756 9,71E-13 0,000156552 synaptotagmin XII ENSMUSG00000094707 A830019P07Rik 1,503913502 1,044849153 2,26E-06 0,005605857 RIKEN cDNA A830019P07 gene ENSMUSG00000032511 Scn5a 1,4482707 1,138893243 1,78E-06 0,000691184 sodium channel voltage-gates type V alpha ENSMUSG00000016756 Cmah 1,365344668 1,601108148 4,45E-05 2,18E-06 cytidine monophospho-N-acetylneuraminic acid hydroxylase ENSMUSG00000078945 Naip2 1,301854034 0,846440021 5,50E-08 0,002845813 NLR family apoptosis inhibitory protein 2 ENSMUSG00000078942 Naip6 1,238283824 1,184382877 0,002678396 0,009230676 NLR family apoptosis inhibitory protein 6 ENSMUSG00000035314 Gdpd5 0,992622348 1,051250274 1,94E-05 1,19E-05 glycerophosphodiester phosphodiesterase domain containing 5 ENSMUSG00000034394 Lif 0,909924419 1,002737031 0,000177314 5,50E-05 leukemia inhibitory factor DOWN in si mH2A1.1 & in si mH2A1.2 (18) ENSMUSG00000039976 Tbc1d16 -0,861959862 -0,8375669 0,000119206 0,000413277 TBC1 domain family member 16 ENSMUSG00000058258 Idi1 -0,874866643 -0,809531686 0,001783296 0,008602492 isopentenyl-diphosphate delta isomerase ENSMUSG00000093930 Hmgcs1 -0,878495135 -0,988353746 3,80E-06 3,31E-07 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 ENSMUSG00000015090 Ptgds -0,945128578 -2,402054402 0,001390053 1,21E-16 prostaglandin D2 synthase (brain) ENSMUSG00000038623 Tm6sf1 -1,020422918 -2,082787843 0,003282753 3,92E-11 transmembrane 6 superfamily member 1 ENSMUSG00000083287 Gm13502 -1,047383013 -1,031175956 6,75E-05 0,000197125 predicted gene 13502 ENSMUSG00000023832 Acat2 -1,108040737 -1,154491475 0,000911626 0,000960357 acetyl-Coenzyme A acetyltransferase 2 ENSMUSG00000032942 Ucp3 -1,119903913 -1,300172717 0,001426513 0,000336866 uncoupling protein 3 (mitochondrial proton carrier ENSMUSG00000058454 Dhcr7 -1,135756353 -1,00472707 3,18E-10 1,30E-07 7-dehydrocholesterol reductase ENSMUSG00000047686 Rtl3 -1,138829448 -1,42995334 5,59E-05 5,76E-07 retrotransposon Gag like 3 ENSMUSG00000060716 Plekhh1 -1,236701442 -1,427038063 0,000192688 2,14E-05 pleckstrin homology domain containing family H (with MyTH4 domain) member 1 ENSMUSG00000033105 Lss -1,263150364 -0,997646902 1,77E-13 5,15E-08 lanosterol synthase ENSMUSG00000016356 Col20a1 -1,321620931 -1,546906886 7,29E-06 2,80E-07 collagen type XX alpha 1 ENSMUSG00000006517 Mvd -1,332699625 -1,15760886 2,19E-05 0,000689658 mevalonate (diphospho) decarboxylase DEGs in the same direction (34) direction same the in DEGs ENSMUSG00000036885 Arhgef26 -1,375636149 -0,873185832 1,05E-19 7,94E-08 Rho guanine nucleotide exchange factor (GEF) 26 ENSMUSG00000026676 Ccdc3 -1,570206657 -0,839963516 1,82E-09 0,006267127 coiled-coil domain containing 3 ENSMUSG00000109243 Gm45867 -1,622966789 -2,092413539 0,000143143 1,71E-06 predicted gene 45867 ENSMUSG00000031613 Hpgd -2,124156192 -1,216472976 2,66E-08 0,006372472 hydroxyprostaglandin dehydrogenase 15 (NAD)

Annex 2. Alignment of the analyzed exon 5 from macroH2A1.1

Annex 3. Alignment of the analysed exon 5 from macroH2A2

Annex 4. Alignment of the analyzed exon 5 from macroH2A1.2

Annex 5. MacroH2A amino acid and nucleotide p-distances and selective pressures throughout evolution

Average Numbers of Nucleotide (pNT) and Amino Acid (pAA) differences per 100 Sites in specific regions of macroH2A variants (macro domain and Exon 5). Nucleotide p-distances (pNT ± SE) MacroH2A type Global Mammals Birds Reptiles Amphibians Fish mH2A.1.1 (Macro domain) 0,209 ± 0,010 0,067 ± 0,006 0,047 ± 0,005 0,114 ± 0,010 0,169 ± 0,017 0,299 ± 0,013 mH2A.1.2 (Macro domain) 0,179 ± 0,009 0,064 ± 0,006 0,045 ± 0,005 0,113 ± 0,010 0,150 ± 0,016 0,235 ± 0,012 mH2A.2 (Macro domain) 0,270 ± 0,017 0,079 ± 0,008 0,067 ± 0,009 0,131 ± 0,012 0,191 ± 0,021 0,346 ± 0,025 mH2A.1.1 (Exon 5) 0,160 ± 0,025 0,014 ± 0,007 0,004 ± 0,005 0,030 ± 0,015 0,148 ± 0,045 0,303 ± 0,081 mH2A.1.2 (Exon 5) 0,096 ± 0,028 0,000 ± 0,000 0,000 ± 0,000 0,032 ± 0,015 0,032 ± 0,022 0,193 ± 0,090 mH2A.2 (Exon 5) 0,328 ± 0,069 0,057 ± 0,013 0,029 ± 0,012 0,116 ± 0,029 0,237 ± 0,083 0,456 ± 0,128 Amino acid p-distances (pAA ± SE) MacroH2A type Global Mammals Birds Reptiles Amphibians Fish mH2A.1.1 (Macro domain) 0,100 ± 0,012 0,014 ± 0,004 0,012 ± 0,005 0,045 ± 0,011 0,069 ± 0,017 0,169 ± 0,015 mH2A.1.2 (Macro domain) 0,072 ± 0,009 0,009 ± 0,002 0,010 ± 0,006 0,040 ± 0,010 0,037 ± 0,014 0,113 ± 0,014 mH2A.2 (Macro domain) 0,157 ± 0,014 0,009 ± 0,004 0,012 ± 0,004 0,049 ± 0,012 0,063 ± 0,017 0,218 ± 0,018 mH2A.1.1 (Exon 5) 0,150 ± 0,033 0,028 ± 0,018 0,013 ± 0,014 0,033 ± 0,023 0,200 ± 0,069 0,231 ± 0,043 mH2A.1.2 (Exon 5) 0,082 ± 0,020 0,000 ± 0,000 0,000 ± 0,000 0,000 ± 0,000 0,000 ± 0,000 0,185 ± 0,044 mH2A.2 (Exon 5) 0,205 ± 0,033 0,004 ± 0,004 0,000 ± 0,000 0,096 ± 0,037 0,061 ± 0,042 0,272 ± 0,036 Note 1: SE, standard error. Note 2: The group of mammals encompasses 17 species; birds 5 species; reptiles 4 species; amphibians 2 species; and fish 12 species Note 3: The group of the fish involves 5 different classes which accounts, in part, for the higher divergence showed in p-distance's results

Average Numbers of Synonymous (pS) and Nonsynonymous (pN) nucleotide differences per 100 Sites, z-test for selection, and codon usage bias (ENC) in specific regions of macroH2A variants (macro domain and Exon 5). pS (SE) pN (SE) R Z-test ENC MacroH2A type Global Global Global Global Global mH2A.1.1 (Macro domain) 0,712 ± 0,039 0,067 ± 0,009 2,75 24,669** 53,545 mH2A.1.2 (Macro domain) 0,640 ± 0,037 0,044 ± 0,007 3,07 24,4** 51,932 mH2A.2 (Macro domain) 0,917 ± 0,049 0,109 ± 0,011 2,42 30,243** 49,949 mH2A.1.1 (Exon 5) 0,350 ± 0,071 0,096 ± 0,027 1,24 6,005** 32,076 mH2A.1.2 (Exon 5) 0,262 ± 0,070 0,041 ± 0,011 2,28 4,576** 47,165 mH2A.2 (Exon 5) 1,036 ± 0,070 0,129 ± 0,022 1,48 13,176** 47,375 Note 1: SE, standard error. Note 2: ENC, Effective Number of Codons (codon bias) ranging between 61 (no bias) and 20 (maximum bias). Note 3: R, is the average transition/transversion ratio used in the estimation of pS and pN. **P < 0.001 level in Z-test comparisons (pS > pN).

Annex 6. Protein alignment of Caps. owc. versus mouse (macroH2A1.1 and PARP1)

Protein alignment Caps. ow.c mH2A versus mouse mH2A1.1 (Uniprot analysis)

* Caps. owz. mH2A H2A-fold domain mouse mH2A1.1 linker domain Caps. owz. mH2A macro domain mouse mH2A1.1 Caps. owz. mH2A * The first 9-11 amino acids mouse mH2A1.1 are missing Caps. owz. mH2A D203-G224 (essential amino mouse mH2A1.1 acid for ADP-r binding) Caps. owz. mH2A mouse mH2A1.1 Caps. owz. mH2A mouse mH2A1.1

Protein alignment Caps. ow.c PARP1 versus mouse PARP1 * Caps. owz. PARP1 DNA binding domain mouse PARP1 Automodification domain Caps. owz. PARP1 Catalytic domain mouse PARP1 Caps. owz. PARP1 * The first 3-111 amino acids mouse PARP1 are missing Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1 Caps. owz. PARP1 mouse PARP1

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Author: Sarah Hurtado Proofreaders: Marcus Buschbeck, Iva Guberovic, Michael Maher, David Corujo, François Chataigner, Marguerite-Marie le Pannérer Graphic / editorial design and illustrator: Line Hurtado (linehurtado.wordpress.com / [email protected]). Printed in Barcelona, June 2019. MEDIAative (www.mediaactive.es)