Allosteric Control of ALC1-Catalyzed Nucleosome Remodeling

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2009

Allosteric control of ALC1- catalyzed nucleosome remodeling

LAURA C. LEHMANN

ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-1128-9 UPPSALA urn:nbn:se:uu:diva-429899 2021 Dissertation presented at Uppsala University to be publicly examined in Room A1:111a, Biomedical Center, Husargatan 3, Uppsala, Wednesday, 24 March 2021 at 13:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Felix Müller-Planitz (Institute of Physiological Chemistry, Faculty of Medicine, TU Dresden).

Abstract Lehmann, L. C. 2021. Allosteric control of ALC1-catalyzed nucleosome remodeling. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2009. 45 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1128-9.

The genetic information of eukaryotic cells is packaged inside the nucleus as chromatin. This packaging restricts access to the DNA and therefore represents a barrier for processes such as DNA replication, repair, and gene expression. Specialized enzymes, termed ATP-dependent chromatin remodelers (remodelers), are involved in regulating the chromatin landscape by repositioning, ejecting, and altering the composition of nucleosomes, the smallest building blocks of chromatin. Remodelers are tightly regulated by post-translational modifications, nucleosomal features, as well as by their own domains and subunits. Dysregulation of remodeling activity has been implicated in severe disease states such as various types of cancer. ALC1/CHD1L (Amplified in Liver Cancer 1/Chromodomain-Helicase-DNA-binding protein 1-Like) is an oncogenic remodeler involved in DNA damage repair. This thesis investigates the molecular basis of ALC1 regulation, the role of the nucleosome and its features in ALC1 activation, as well as the role of this remodeler in DNA damage repair. The results presented in this thesis show that, without DNA damage, the catalytic domain of ALC1 adopts an inactive conformation that is stabilized through a conserved electrostatic interface with the macro domain of ALC1. Upon DNA damage, the binding of PAR chains to the macro domain displaces it from the ATPase motor of ALC1, thereby priming the remodeler for activation. Full activation additionally requires the interaction between a regulatory segment in the linker region of ALC1 and the nucleosome acidic patch. This interaction tethers the remodeler to the nucleosome and is required for coupling ATP hydrolysis to nucleosome remodeling. Additionally, investigations of ALC1 in vivo showed that the loss of ALC1 sensitizes cells to PARP inhibitors and is synthetic lethal with homologous recombination deficiency. This makes ALC1 a possible target for therapeutic drugs in combination with PARP inhibitors for cancers that are deficient in homologous recombination.

Keywords: ALC1, CHD1L, ATP-dependent chromatin remodeling, nucleosome, allosteric regulation, autoinhibition, macro domain, PARP, ADP-ribosylation, DNA damage, acidic patch

Laura C. Lehmann, Department of Cell and Molecular Biology, Molecular Systems Biology, Box 596, Uppsala University, SE-751 24 Uppsala, Sweden.

ISSN 1651-6214 ISBN 978-91-513-1128-9 urn:nbn:se:uu:diva-429899 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-429899)

To all my teachers

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I. Lehmann1, L.C., Hewitt1, G., Aibara, S., Leitner, A., Marklund, E., Maslen, S.L., Maturi, V., Chen, Y., van der Spoel, D., Skehel, J.M., et al. (2017). Mechanistic Insights into Autoinhibition of the Onco- genic Chromatin Remodeler ALC1. Molecular Cell, 68(5): 847– 859.e7. II. Lehmann1, L.C., Bacic1, L., Hewitt1, G., Brackmann, K., Sabantsev, A., Gaullier, G., Pytharopoulou, S., Degliesposti, G., Okkenhaug, H., Tan, S., et al. (2020). Mechanistic Insights into Regulation of the ALC1 Remodeler by the Nucleosome Acidic Patch. Cell Reports, 33(12): 108529. III. Hewitt, G., Borel, V., Segura-Bayona, S., Takaki, T., Ruis, P., Bellelli, R., Lehmann, L.C., Sommerova, L., Vancevska, A., Tomas- Loba, A., et al. (2020). Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD. Molecular Cell, https://doi.org/10.1016/j.molcel.2020.12.006.

1These authors contributed equally.

Reprints were made with permission from the respective publishers.

Papers not included in this Thesis

iv. Ballet, C., Correia, M.S.P., Conway, L.P., Locher, T.L., Lehmann, L.C., Garg, N., Vujasinovic, M., Deindl, S., Lohr, J.-M., Globisch, D. (2018). New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites. Chem. Sci., 9(29): 6233–6239. v. Kipper, K., Eremina, N., Marklund, E., Tubasum, S., Mao, G., Leh- mann, L.C., Elf, J., Deindl, D. (2018). Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI. PLoS ONE, 13(6): e0198416. vi. Marklund, E., van Oosten, B., Mao, G., Amselem, E., Kipper, K., Sa- bantsev, A., Emmerich, A., Globisch, D., Zheng, X., Lehmann, L.C., et al. (2020). DNA surface exploration and operator bypassing during target search. Nature, 583: 858–861.

Contents

1. Introduction ...... 9 2. Chromatin and its organization ...... 10 2.1 The nucleosome is a dynamic building block of chromatin ...... 10 2.2 The nucleosome acidic patch ...... 11 3. Chromatin remodeling enzymes: families and function ...... 13 3.1 Remodeler families ...... 13 3.2 Mechanisms of remodeling ...... 15 4. Regulation of remodelers ...... 18 5. The remodeler ALC1 ...... 21 5.1 ALC1 is a PAR-dependent remodeler ...... 21 5.2 The effects of ALC1 on chromatin ...... 22 5.3 ALC1 and cancer ...... 23 6. Paper I: Autoinhibition of ALC1 by its macro domain ...... 24 7. Paper II: ALC1 and the nucleosome acidic patch ...... 27 8. Paper III: Loss of ALC1 confers PARPi sensitivity and synthetic lethality with HRD ...... 30 9. Future Perspectives ...... 33 10. Svensk sammanfattning ...... 35 11. Acknowledgements...... 37 12. References ...... 40

Abbreviations

Å Ångström ADP adenosine diphosphate ALC1 amplified in liver cancer 1 ATP adenosine triphosphate bp base pair CHD chromodomain-helicase-DNA-binding CHD1 chromodomain-helicase-DNA-binding protein 1 CHD1L chromodomain-helicase-DNA-binding protein 1-like cryo-EM cryogenic electron microscopy DNA deoxyribonucleic acid DSB double-strand break HDX-MS hydrogen-deuterium exchange coupled to mass spectrometry HR(D) homologous recombination (deficiency) HSA helicase-SANT HSS HAND-SANT-SLIDE INO80 inositol requiring 80 ISWI imitation switch LANA latency-associated nuclear antigen NAD+ nicotinamide adenine dinucleotide PAR poly(ADP-ribose) PARG poly(ADP-ribose) glycohydrolase PARP poly(ADP-ribose) polymerase PARPi poly(ADP-ribose) polymerase inhibitors PARylation poly(ADP-ribosylation) RNA ribonucleic acid SANT SWI3, ADA2, NCoR, TFIIIB SAXS small-angle x-ray scattering SF2 superfamily 2 SHL superhelical location SLIDE SANT-like ISWI domain SnAC Snf2 ATP coupling Snf2 sucrose nonfermenting 2 SWI/SNF switching defective/sucrose nonfermenting WT wild-type XL-MS crosslinking coupled to mass spectrometry

1. Introduction

Eukaryotic DNA is packaged inside the nucleus as chromatin. The regulation and maintenance of chromatin are vital processes, and dysregulation of these processes or damage to chromatin can lead to a variety of different diseases such as cancer. According to the World Health Organization, cancer is the second leading cause of death, with over nine million deaths worldwide in 2018 (Bray et al., 2018). Although the best way of tackling cancer would be to prevent it, it is equally important to invest in cancer treatment. The identification of new targets for therapeutic drugs requires an in-depth mechanistic understanding of the processes that regulate chromatin. In the following chapters, I will give an introduction into chromatin and its organization, as well as describe a class of enzymes that regulate the chromatin landscape. I will further summarize how these remodelers are regulated in order to ensure their proper function.

9 2. Chromatin and its organization

Eukaryotic genomes are packaged into chromatin, which not only contributes to the condensation and organization of the genome, but also restricts access to the DNA template (Kornberg and Lorch, 2020). This enables the cell to establish transcriptionally repressed as well as more active regions of chromatin. Through precisely controlled dynamic rearrangements of chromatin structure, different patterns of active and repressed chromatin enable diverse transcriptional outcomes and the establishment of various cellular identities from a single genetic blueprint.

2.1 The nucleosome is a dynamic building block of chromatin Chromatin is a complex of DNA and proteins, and the smallest building block of chromatin is the nucleosome. The first high resolution structure of a nucleosome was solved in 1997 (Luger et al., 1997). Nucleosomes consist of 145- 147 base pairs (bp) of DNA wrapped around a protein core, containing two copies of each histone H2A, H2B, H3 and H4 (Figure 1). Histones are mostly positively charged and interact with the negatively charged phosphate back- bone of the DNA. The locations where the major groove of the DNA is facing the histone octamer are referred to as superhelical locations (SHL), numbered from 0 at the dyad to -7 or +7 at each end of the nucleosomal DNA (Luger et al., 1997). The histone core of the nucleosome is an octamer that consists of a central H3-H4 tetramer flanked by H2A-H2B dimers on each side of the tetramer (Arents et al., 1991; Luger et al., 1997).

10 Figure 1. Structure of the nucleosome core particle (PDB: 1AOI, Luger et al., 1997).

Although nucleosomes represent a barrier that restricts access to DNA, they are nonetheless a very dynamic structure. Nucleosomes are in an equilibrium state of rapid unwrapping and rewrapping from their DNA ends, which is thought to facilitate access to buried parts of the nucleosomal DNA (Li and Widom, 2004; Li et al., 2005). Post-translational modifications of histones and the sequence of the nucleosomal DNA have been shown to influence nucleosome dynamics (reviewed in: Zhou et al., 2019). The dynamics as well as the structure of nucleosomes are also regulated by proteins. These proteins can add or remove post-translational modifications, they can exchange histones with variants that have different properties than the canonical histones, they can stabilize nucleosomes, or reposition them (McGinty and Tan, 2016; Zhou et al., 2019). The nucleosome provides a variety of different interaction plat- forms for these proteins. Surfaces for interaction include the nucleosomal DNA (extra-nucleosomal and intra-nucleosomal DNA), histone tails, and the histone core. It is very common that proteins use several of these surfaces to bind to the nucleosome (McGinty and Tan, 2016).

2.2 The nucleosome acidic patch One of the most prominent interaction surfaces on the nucleosome is the acidic patch. It is located on the H2A-H2B dimer and comprises the residues Glu56, Glu61, Glu64, Asp90, Glu91, and Glu92 of H2A and Glu105 and Glu113 of H2B (Figure 2A). Many proteins interact with this negatively charged surface through a so-called ‘arginine anchor’ (McGinty and Tan, 2016). One example

11 for such an interaction is the latency-associated nuclear antigen (LANA) protein from the Kaposi’s sarcoma-associated herpesvirus. The crystal structure of a nucleosome-bound peptide from the N-terminus of LANA revealed that the critical residue that mediates binding to the acidic patch of the nucleosome is an arginine residue (Barbera et al., 2006) (Figure 2B). Comparisons of different structures of proteins binding to the acidic patch did not show much sequence conservation apart from the arginine anchor (McGinty and Tan, 2016). Additional residues that are involved in binding to the nucleosome seem to be individual for each protein.

Figure 2. The nucleosome acidic patch is an interaction platform for many binding factors. (A) Position of the acidic patch on the nucleosome surface (PDB: 1AOI, Lu- ger et al., 1997; adapted from: Lehmann et al., 2020). (B) LANA peptide bound to the acidic patch (PDB: 1ZLA, Barbera et al., 2006).

In addition to mediating binding to other proteins, the nucleosome acidic patch has also been implicated in the formation of higher order chromatin structures through interactions with the H4 tail of neighboring nucleosomes (Davey et al., 2002; Dorigo et al., 2003, 2004; Schalch et al., 2005; also reviewed in: Kalashnikova et al., 2013). In summary, the acidic patch is an important interaction surface for nucleosome-nucleosome contacts as well as for contacts with other proteins.

12 3. Chromatin remodeling enzymes: families and function

The dynamic regulation of chromatin is essential for nuclear processes such as DNA replication, repair and gene expression. Cells have evolved different ways of regulating their chromatin landscape, for example by the action of a class of enzymes called ATP-dependent chromatin remodeling enzymes (remodelers). These enzymes belong to the superfamily 2 (SF2) of helicases, more specifically to the Snf2 (sucrose non-fermenting 2) family of helicase- related proteins (Singleton et al., 2007). Remodelers influence a broad range of nuclear processes by hydrolyzing ATP to move (slide), destabilize, disas- semble, or rearrange nucleosomes. Although single-subunit remodelers can remodel nucleosomes, many remodelers form complexes containing a catalytic subunit and additional non-catalytic subunits (accessory subunits) that give rise to specific remodeling outcomes. Mutations in remodelers and remodeling complexes have been linked to many cancers, which underlines the importance of remodelers in nuclear processes (Mirabella et al., 2016; Narlikar et al., 2013; Pulice and Kadoch, 2016).

3.1 Remodeler families All remodelers share a conserved ATPase motor (also referred to as ATPase domain or motor domain) that consists of two RecA-like lobes (Flaus et al., 2006). Based on the sequence of this ATPase motor, remodelers can be sepa- rated into 24 subfamilies that are conserved between species and generally correlate with biological functions (Flaus et al., 2006; Narlikar et al., 2013) (Figure 3).

Figure 3. Schematic representation of remodeler family organization based on their conserved ATPase domain (adapted from: Flaus et al., 2006).

Apart from the motor domain, remodelers can feature a variety of different accessory domains within the catalytic or accessory subunits that often regulate remodeling activity or mediate interactions with other remodeler subunits or the nucleosome (reviewed in: Bartholomew, 2014; Clapier and Cairns, 2009; Hopfner et al., 2012; Narlikar et al., 2013). Apart from using the conserved ATPase domain as a basis for remodeler families, remodelers can also be grouped into families based on their accessory domains (Figure 4). Some examples of remodeler families and their characteristic accessory domains are described below.

Figure 4. Schematic representation of remodeler family organization based on their accessory domains (adapted from: Clapier and Cairns, 2009; Clapier et al., 2017).

14 Remodelers in the CHD (Chromodomain, Helicase, DNA-binding) subfamily typically slide nucleosomes and feature regulatory chromodomains that can bind methylated histones (Becker and Workman, 2013; Clapier et al., 2017). ISWI (Imitation Switch) is another subfamily of remodelers, members of which are typically small complexes with two to four subunits. ISWI remodelers are involved in chromatin assembly and transcription repression through nucleosome spacing (Clapier and Cairns, 2009; Clapier et al., 2017). The ATPase motor of ISWI remodelers typically contains several regulatory domains, such as the AutoN and NegC domains, or the HAND-SANT-SLIDE (HSS) domain that is required for ISWI regulation and nucleosome spacing (Bartholomew, 2014; Clapier et al., 2017; Narlikar et al., 2013). SWI/SNF (Switching defective/Sucrose non-fermenting) remodelers commonly feature an HSA (helicase-SANT) domain that mediates interactions with other factors and a bromodomain that binds to acetylated lysines (Becker and Workman, 2013). Remodelers in this family form large complexes that slide and eject nucleosomes and are involved in a variety of different nuclear processes (Bartholomew, 2014; Clapier et al., 2017). Remodelers of the INO80 (Inositol requiring 80) subfamily also contain an HSA domain and feature a long insertion between the two RecA-like lobes of the ATPase domain that is needed for interaction with other proteins (Becker and Workman, 2013). Members of this remodeler subfamily also form large complexes that fulfill diverse functions in DNA repair, promoting transcription, and restructuring nucleosomes (Bartholomew, 2014; Clapier and Cairns, 2009; Clapier et al., 2017).

Although the various remodelers are involved in a wide range of different processes, they all share some basic properties: They have an affinity for the nucleosome that goes beyond the affinity for DNA itself; they have accessory domains that can recognize histone modifications and other proteins; they have a DNA-dependent motor domain that uses the energy from ATP hydrolysis to change histone-DNA contacts in the nucleosome; and they have domains that regulate the motor domain (Clapier and Cairns, 2009; Clapier et al., 2017). The following chapters will provide an overview of the core remodeling mechanism and describe examples of remodeler regulation, which can give rise to specific remodeling outcomes.

3.2 Mechanisms of remodeling Remodelers are able to manipulate the nucleosome structure through their ATPase motor, but how precisely they achieve this is a longstanding question in the field. Several models for the remodeler-catalyzed sliding of nucleosomes on DNA have been proposed. This chapter provides a brief overview of two of the main models that have been proposed.

15 During nucleosome remodeling, the two lobes of the remodeler motor domain come together in a specific orientation that allows ATP to bind in the interface between the two lobes. The binding of nucleic acids stabilizes a conformation of the two lobes that stimulates ATP hydrolysis. The motor domain then moves through cycles of ATP binding, hydrolysis, and ADP release, while switching between an open and a closed state, which results in an inchworm-like motion of the motor domain on the DNA (Nodelman and Bowman, 2021). The challenge for a remodeler is to convert this motion into movement of double-stranded DNA around the histone core. Two prominent models have been proposed for the mechanism of nucleosome sliding, the ‘loop propagation’ and the ‘twist diffusion’ model (Figure 5). In the twist diffusion model, a twist defect, which is the gain or loss of a base pair in a helical turn of nucleosomal DNA, can travel to neighboring seg- ments in the DNA through a corkscrew shift (van Holde and Yager, 1985, 2003). According to this model, only very few local DNA-histone contacts are disrupted at any given time. In the loop propagation model, a DNA loop of multiple base pairs is lifted off the nucleosome surface from the entry side, disrupting several DNA-histone contacts at a time, that would then rapidly travel around the nucleosome (Strohner et al., 2005; also reviewed in: Becker and Hörz, 2002; Clapier and Cairns, 2009).

Figure 5. Two models for nucleosome sliding. (A) Loop propagation model and (B) twist diffusion model (adapted from: Nodelman and Bowman, 2021).

16 Nucleosome remodeling implies the breaking of histone-DNA contacts. The hydrolysis of one ATP molecule is likely not enough to break all histone-DNA contacts at the same time. The movement of DNA around the histone octamer is thus discontinuous. Both the loop propagation and twist diffusion models therefore require the nucleosome to accommodate additional base pairs during nucleosome remodeling. Single-molecule experiments demonstrated that entry-side DNA is moved into the nucleosome before exit-side DNA is pushed out while the nucleosome transiently absorbs nucleosomal DNA, which shows that the nucleosome can accommodate additional base pairs (Sabantsev et al., 2019). Several studies have shown that most remodelers engage the nucleosome at the SHL2 position inside the nucleosome rather than at the entry side (McKnight et al., 2011; Saha et al., 2005; Schwanbeck et al., 2004; Zofall et al., 2006). Additionally, crosslinking experiments showed that the binding of Chd1 to the nucleosome was enough to change DNA twists at SHL2 (Winger et al., 2018). These observations are difficult to explain in the context of loop propagation, which led to the following adjustment: If the motor domain engages the nucleosome at SHL2, then DNA binding domains would need to bind the DNA at the entry side of the nucleosome to create tension or to act as a barrier for a loop to form (Clapier and Cairns, 2009; Clapier et al., 2017). Chd1 and ISWI indeed feature DNA-binding domains that bind to DNA at the entry side of the nucleosome. However, studies have shown that these domains are not used for a power stroke, since the addition of a flexible linker between the DNA binding domain and the motor domain does not change the outcome of nucleosome remodeling (Ludwigsen et al., 2013; Nodelman and Bowman, 2013). These findings therefore argue against the loop propagation model. The DNA binding domains in Chd1 and ISWI appear to fulfill a regulatory role rather than a mechanical role in nucleosome sliding as described in the next chapter on the regulation of remodelers. A recent surge of cryo-EM structures of remodelers bound to the nucleosome shed light onto the mechanism of the initial DNA movement before ATP is bound (Chittori et al., 2019; Li et al., 2019; Yan et al., 2019). These important structural insights, combined with recent molecular simulations (Brandani and Takada, 2018; Brandani et al., 2018; Lequieu et al., 2017), strongly support the twist diffusion model for nucleosome sliding.

17 4. Regulation of remodelers

The tight regulation of remodeling activity is important for two reasons: first, it prevents unnecessary ATP hydrolysis in the cells and second, it ensures activation in the right context and when required only. Just as there are many different remodelers, there are many different ways how remodelers are regulated. The subject of remodeler regulation has been extensively reviewed over the years and below are some examples of regulatory mechanisms. The activity of remodelers can be regulated by post-translational modifications (reviewed in: Becker and Workman, 2013; Clapier and Cairns, 2009). The most common post-translational modifications of remodelers are phos- phorylation, acetylation, and poly(ADP-ribosylation) (PARylation). These modifications can activate or inhibit interactions with other proteins and remodeler functions, and are used by the cell to finely tune remodeling activity. The ISWI remodeler, for example, has been shown to be a target for PARyla- tion by PAR polymerase 1 (PARP1), which counteracts ISWI activity (Sala et al., 2008). Another important concept of regulation is the relative position of the two RecA-like lobes in the motor domain (reviewed in: Hauk and Bowman, 2011; Narlikar et al., 2013). Both lobes contain conserved sequence motifs that are needed for ATP binding and hydrolysis as well as for the interaction with DNA (Flaus et al., 2006). The two lobes have to be brought together in the right orientation to form a closed and catalytically competent ATPase motor. Consistent with this, a crystal structure of the zebrafish Rad54 ATPase domain shows a closed conformation of the two lobes (Thoma et al., 2005), while the structure of Sulfolobus solfataricus Rad54 shows that the lobes are flipped 180° relative to the closed conformation (Dürr et al., 2005). A flipped conformation of the two ATPase lobes can also be observed in the structure of Snf2 from Myceliophthora thermophila (Mt), which explains the low basal ATPase activity of this remodeler (Xia et al., 2016). The two lobes are realigned in a structure of yeast Snf2 bound to the nucleosome, indicating the switch to an active conformation upon substrate engagement (Liu et al., 2017). Another example where the ATPase lobes are splayed apart is yeast Chd1. A crystal structure of the catalytic domain shows an autoinhibited conformation where the double chromodomains of Chd1 pack against the two lobes of the motor domain, which stabilizes an open and inactive conformation (Hauk et al., 2010). Consistent with this, in a structure of Chd1 bound to the nucleosome, the chromodomains are shifted towards the nucleosomal DNA,

18 allowing motor domain closure (Farnung et al., 2017). In addition to preventing a closed conformation, the chromodomains also block the DNA binding site, which allows the remodeler to differentiate between naked DNA and nucleosomes (Hauk and Bowman, 2011; Hauk et al., 2010). The example of Chd1 ties in with another form of regulation: regulation of remodeler activity through accessory domains. Accessory domains are conserved within subfamilies of remodelers and can contribute to remodeler specificity and positive or negative regulation of activity (reviewed in: Clapier et al., 2017; Narlikar et al., 2013; Paul and Bar- tholomew, 2018). They can also impact activity through preventing the substrate from binding to the ATPase domain (as discussed above for the chromodomains of Chd1), by stimulating or inhibiting the ATPase hydrolysis rate, or by coupling ATPase activity to nucleosome mobilization. In 2011 Sen et al. discovered a conserved domain in the catalytic subunit of the SWI/SNF remodeling complex that they named SnAC (Snf2 ATP coupling) (Sen et al., 2011). This domain is an example for positive regulation of remodelers, since SnAC enhances ATPase activity (Sen et al., 2011). More importantly, a later study found this domain to be essential for coupling ATP hydrolysis to nucleosome movement by acting as a histone anchor (Sen et al., 2013). Another well-studied example regarding regulation is the remodeler ISWI. Apart from several accessory domains that impact the activity of the catalytic domain, it is also regulated through nucleosomal features. AutoN and NegC, two domains that flank the catalytic domain of ISWI, negatively regulate the ATPase motor. Upon recognition of nucleosomal features, such as the H4 tail and linker DNA, this autoinhibition is relieved (Clapier and Cairns, 2012). Crosslinking studies suggested that the catalytic domain of Drosophila ISWI adopts a more compact structure than Chd1 in the autoinhibited state (Forné et al., 2012). A crystal structure of the MtISWI catalytic core confirms this and shows that the AutoN domain packs against the motor domain, which explains the autoinhibition in the resting state (Yan et al., 2016). Previous studies had shown that a basic patch in the histone H4 tail is needed for ISWI activity (Clapier et al., 2001, 2002). A structure of the MtISWI catalytic core bound to the H4 tail revealed that it relieves the autoinhibition of the motor domain through AutoN by competitive binding to the ATPase motor (Yan et al., 2016). Another domain in ISWI that is involved in regulation is the HSS domain (Grüne et al., 2003). Although the ATPase domain of ISWI has been shown to be an autonomous remodeling enzyme (Mueller-Planitz et al., 2013), the HSS domain is needed for linker length sensing through binding to extra-nucleosomal DNA during ISWI catalyzed nucleosome remodeling (Dang and Bartholomew, 2007; Hota et al., 2013; Yang et al., 2006). The HSS domain samples the extra-nucleosomal DNA and moves faster towards the longer flanking DNA which creates evenly spaced arrays of nucleosomes (Yang et al., 2006). Moreover, for the ISWI-family remodeler ACF, the histone H4 tail

19 was required for linker length sensing (Hwang et al., 2014), illustrating that several layers of regulation can work together.

To summarize the chapters on chromatin remodelers and their regulation, I would like to use the hourglass model of chromatin remodeling suggested by Clapier et al., 2017: A variety of different chromatin remodelers and complexes all use a unifying mechanism for nucleosome mobilization, where the anchoring of an ATPase motor results in directional movement of DNA. Dif- ferent layers of regulation define the outcome of this translocation movement and result in a variety of different processes in the cell.

20 5. The remodeler ALC1

Human ALC1 (Amplified in Liver Cancer 1), also known as CHD1L (Chro- modomain-Helicase-DNA-binding protein 1-Like), is an ATP-dependent chromatin remodeling enzyme. Like all remodelers it contains a motor domain composed of an N-terminal and a C-terminal ATPase lobe (Flaus et al., 2006). But what is unique about ALC1 is that it possesses a macro domain that is connected to the motor domain through a long linker region (Figure 6).

Figure 6. Domain architecture of the human ALC1 protein (adapted from: Lehmann et al., 2017).

The macro domain can bind to poly(ADP-ribose) (PAR) (Karras et al., 2005), which is a polymer that is synthesized by PAR polymerases (PARPs) at sites of DNA damage (Hassa et al., 2006; Lindahl et al., 1995; Satoh and Lindahl, 1992). In the following chapters, I will give an overview of ALC1 and its recruitment to sites of DNA damage, as well as its role in cancer.

5.1 ALC1 is a PAR-dependent remodeler In 2009, two papers identified ALC1 as a PAR-dependent chromatin remodeling enzyme (Ahel et al., 2009; Gottschalk et al., 2009). These papers showed that ALC1 has a macro domain that binds PAR and PARylated PARP1 in vitro and in vivo, whereas the macro domain mutation D723A, which was predicted to substantially decrease the affinity for ADP-ribose (Karras et al., 2005), did not bind PAR efficiently. ALC1 was found to display little ATP hydrolysis activity on its own. However, the hydrolysis rate was elevated in the presence of DNA and even more so in the presence of nucleosomes. ATPase activity was fully stimulated in the presence of PARP1/NAD+ and DNA or nucleosomes. Additionally, Ahel et al. showed that ALC1 is PARylated by PARP1 in vitro and that although ALC1 ATPase activity is most strongly increased in the presence of PARylated PARP1, it does not depend on PARylation of the

21 remodeler itself (Ahel et al., 2009). Both papers further investigated the nucleosome sliding abilities of ALC1. Gottschalk et al. reported that ALC1 remodels nucleosomes in the presence of PARP1/NAD+, whereas nucleosome remodeling does not occur with the ATPase-dead ALC1 E175Q mutant, or in the presence of PARP inhibitors. Ahel et al. found that while wild-type (WT) ALC1 remodels nucleosomes in the presence of ATP, the K77R mutant does not remodel nucleosomes at all, and that PARylated PARP1 is required for full nucleosome sliding activity. Both papers therefore established that PARy- lated PARP1 is needed for full ALC1 activity. Additionally, Ahel et al. also found that the nucleosome H4 tail is needed for stimulation of ATPase activity. Although ALC1 could bind to both WT and H4(16-19)A nucleosomes, only WT nucleosomes were remodeled, which indicates that nucleosomes are the relevant substrate for ALC1 (Ahel et al., 2009). Mass spectrometric analysis revealed PAR-dependent interactions between ALC1 and various proteins involved in DNA repair (Ahel et al., 2009). These observations suggested a role of ALC1 in the DNA damage response. Indeed, both papers showed that ALC1 is rapidly recruited to sites of DNA damage in vivo and that the recruitment is abolished upon PARP inhibitor treatment. Recruitment was dependent on the macro domain binding to PAR at sites of DNA damage, and the release of ALC1 from such sites required ATPase activity of the motor domain (Ahel et al., 2009). Removal or mutation of the macro domain resulted in loss of recruitment (Gottschalk et al., 2009). In summary, these investigations showed that a PARP1-dependent PARy- lation event activates and recruits ALC1 to sites of DNA damage. Gottschalk et al. note that “it will be of interest to determine whether binding of a PARy- lated species, most likely Parp1 itself, to the Alc1 macrodomain results in allosteric activation of the enzyme” (Gottschalk et al., 2009).

5.2 The effects of ALC1 on chromatin During their investigations on ALC1 recruitment to sites of DNA damage, Ahel et al. found that cells that do not express ALC1 are more sensitive to DNA damage (Ahel et al., 2009). This led them to the conclusion that the remodeler ALC1 is localized to sites of DNA damage in a PAR-dependent fashion so as to regulate chromatin during DNA repair. They further investigated the consequences of ALC1 overexpression and found that there was more phleomycin-induced DNA damage in ALC1 overexpressing cells than in cells that do not overexpress ALC1 (Ahel et al., 2009). This phenotype was only observed with phleomycin, but not with ionizing radiation or H2O2. Since a structurally related DNA damaging agent acts on relaxed chromatin, Ahel et al. concluded that ALC1 overexpression is relaxing chromatin, thereby making it more susceptible to DNA damage by phleomycin (Ahel et al., 2009).

22 Indeed, a later study showed that ALC1 contributes to local chromatin relaxation at sites of DNA damage and that a loss of ACL1 leads to impaired chromatin relaxation that can be rescued with catalytically competent ALC1 (Sellou et al., 2016). It was also shown that overexpression of ALC1 leads to an increase in chromatin relaxation (Sellou et al., 2016).

Another study investigated the fate of PARP1 during ALC1-catalyzed nucleosome remodeling (Gottschalk et al., 2012). They found that PARP1 is re- tained longer on DNA/nucleosomes and does not dissociate even at high NAD+ levels in the presence of ALC1. The ALC1-PARP1-DNA/nucleosome complex was found to be stable over a long period of time also in the presence of ATP, suggesting that ATP hydrolysis did not lead to dissociation of PARP1 (Gottschalk et al., 2012). By using a PAR footprinting assay, it was further demonstrated that ALC1 protected PAR chains with 3 to 20 ADP-ribose units from degradation by poly(ADP-ribose) glycohydrolase (PARG). ATPase- dead ALC1 E175Q behaved like the WT protein, whereas the D723A mutation in the macro domain led to a reduction in protection, suggesting that the macro domain of ALC1 is protecting the PAR chains (Gottschalk et al., 2012).

5.3 ALC1 and cancer The ALC1 gene is found in the human chromosome region 1q21 that is fre- quently amplified in hepatocellular carcinoma (Ma et al., 2008; Marchio et al., 1997; Wong et al., 2003). A study in 2008 identified ALC1 as an oncogene, showing that the gene was amplified in 51% of hepatocellular carcinoma cases, and overexpression of the ALC1 protein was detected in 52% of all cases (Ma et al., 2008). The oncogenic role of ALC1 was supported by three observations (Ma et al., 2008): First, ALC1 transfected liver cells formed more colonies in soft agar than non-transfected cells. Additionally, mice developed tumors when transfected with ALC1-overexpressing cells. Second, small in- terfering RNA against ACL1 inhibited tumorigenicity. And third, ALC1 was shown to facilitate DNA synthesis and promote the G1/S phase transition in ALC1-transfected cells, suggesting a role of ALC1 in promoting cell prolifer- ation. Also, ALC1 overexpression inhibited apoptosis and downregulated the expression of apoptosis-associated proteins (Ma et al., 2008). Since then, ALC1 has been linked to many types of solid tumors and has been found to induce spontaneous tumor formation in ALC1-transgenic mice (Chen et al., 2009), to play a role in invasion and metastasis in mice, human hepatocellular carcinoma (Chen et al., 2010), and breast cancer (Mu et al., 2015; also reviewed in: Cheng et al., 2013). A more detailed understanding of this link between ALC1 and cancer requires knowledge of the molecular mechanisms underlying ALC1 regulation and function.

23 6. Paper I: Autoinhibition of ALC1 by its macro domain

The activation and recruitment to sites of DNA damage of the oncogenic remodeler ALC1 was suggested to depend on an intact macro domain and PARylated PARP1 (Ahel et al., 2009; Gottschalk et al., 2009; Ma et al., 2008). This indicated an important role of the macro domain in the regulation of ALC1. However, the underlying regulatory mechanism remained unclear. In order to investigate the role of the macro domain in the regulation of ALC1 activity, we compared the ATPase activities of full-length ALC1 and a version of ALC1 that lacks the macro domain. As previously observed, ALC1 has a low basal ATPase activity that is increased in the presence of DNA and fully stimulated in the presence of PARylated PARP1. However, when remov- ing the macro domain, ALC1 is already strongly stimulated by DNA and there is no further increase in activity in the presence of PARylated PARP1. Despite this elevated ATPase activity, the catalytic domain of ALC1 exhibits very low nucleosome sliding activity. These findings suggest the following: First, macro domain removal abolishes the dependence on PARylated PARP1 for ALC1 activation, i.e., the macro domain has an autoinhibitory activity. Sec- ond, the removal of the macro domain leads to reduced nucleosome sliding activity despite elevated ATPase activity, which suggests a function of the macro domain in efficiently coupling ATPase activity to nucleosome repositioning. But how does the macro domain inhibit ALC1 activity? We applied a variety of structural methods to investigate the autoinhibited state of ALC1. Small-angle x-ray scattering (SAXS) in combination with cross-linking coupled to mass-spectrometry (XL-MS) revealed that ALC1 can exists in two conformational states: One in which the macro domain is juxtaposed to the N- terminal ATPase lobe of the motor domain, and one where the macro domain is juxtaposed to the C-terminal ATPase lobe. This interaction confers catalytic autoinhibition in the absence of PARylated PARP1 by stabilizing an open and inactive conformation of the motor domain (Figure 7).

Figure 7. In the autoinhibited state, the ALC1 motor domain adopts an open and inactive conformation (adapted from: Lehmann et al., 2017).

We further investigated the activation of ALC1 by PARylated PARP1. Sev- eral in vitro and in vivo interaction assays showed that the macro domain sta- bly interacts with the motor domain, and that this interaction is lost in the presence of PARylated PARP1. We compared the structure of ALC1 in the autoinhibited state with that in the presence of PARylated PARP1 by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) and XL- MS. These comparisons revealed that ALC1 is undergoing a major conformational change upon activation, consistent with the release of the macro domain from the motor domain. Upon investigation of a potential interface, we found a number of conserved basic residues in the macro domain of AC1, of which two were sites of somatic mutation in cancer. We hypothesized that these residues might be involved in forming an electrostatic interface between the macro domain and the motor domain. Indeed, mutation of these residues destabilizes the interaction between the macro and the motor domain and results in elevated ATPase and nucleosome sliding activities for ALC1 independent of PARylated PARP1. In addition, live-cell imaging showed that mutations in the interface alter the recruitment kinetics of ALC1 at sites of DNA damage. Based on these findings, we present the following model for regulation of ALC1 in paper I: in the autoinhibited state, the macro domain interacts with the motor domain through a conserved electrostatic interface which holds the motor domain in an open and catalytically inactive conformation. Upon engagement with PARylated PARP1, the macro domain is released from the motor domain, and the motor domain can adopt a closed and active conformation. This mechanism ensures that ALC1 is only activated when needed at sites of DNA damage (Figure 8).

Figure 8. Mechanism of autoinhibition of ALC1 by its macro domain (adapted from: Lehmann et al., 2017).

Another group published their independent findings on the autoinhibition of ALC1 in a complementary, back-to-back paper (Singh et al., 2017). Singh et al. also found that the macro domain of ALC1 binds tightly to the motor domain in vitro and in vivo, leading to an autoinhibited state in the absence of activated PARP1. The interaction is lost and inhibition is relieved in the presence of activated PARP1 or tri-ADP-ribose, which was identified as a macro domain ligand. In addition, the authors of this study also identified cancer- associated mutations that disrupt the macro domain-motor domain interaction and constitutively activate ALC1. These findings are in very good agreement with our own data and provide additional support for the model of ALC1 autoinhibition.

26 7. Paper II: ALC1 and the nucleosome acidic patch

We and others have previously shown that in the autoinhibited state, the macro domain of ALC1 is positioned against the motor domain and that binding of PAR to the macro domain relieves the autoinhibition at sites of DNA damage (Lehmann et al., 2017; Singh et al., 2017). These studies defined the autoinhibited state of ALC1 and showed that it is undergoing major conformational changes upon activation. However, how ALC1 recognizes its substrate, the nucleosome, and how nucleosome binding activates ALC1 remained unclear. Since the acidic patch has previously been shown to be important for many remodelers, we investigated the role of the nucleosome acidic patch in ALC1 activity. Nucleosome sliding assays showed that ALC1 remodels nucleosomes without acidic patches substantially less efficiently than nucleosomes with WT acidic patches. Further investigation with single-molecule sliding assays using asymmetric nucleosomes showed that a WT acidic patch is required on the nucleosomal entry-side for ALC1 to exhibit efficient remodeling. We next investigated which part of ALC1 might interact with the acidic patch. Crosslinking coupled to mass spectrometry analysis suggested that the linker region of ALC1 binds to the vicinity of the acidic patch on the nucleosome. This finding was further strengthened by the discovery of sequence sim- ilarity between the ALC1 linker and the N-terminal portion of the LANA (latency-associated nuclear antigen) protein, which is known to bind to the acidic patch (Barbera et al., 2006) (see chapter 2.2). We termed the region of the ALC1 linker that aligned to LANA ‘ALC1 regulatory linker segment’ (ALC1RLS). Interestingly, the arginine residue of the LANA protein that mediates binding to the acidic patch of the nucleosome (the arginine anchor) aligned with an arginine residue (R611) in the linker region of ALC1. We further showed that an exogenous (synthetic) ALC1RLS peptide binds to WT nucleosomes but not to nucleosomes without acidic patches. In order to characterize the interaction between ALC1RSL and the acidic patch, we determined the cryo-electron microscopy (cryo-EM) structure of a peptide from the linker region of ALC1 crosslinked to the nucleosome at an overall resolution of 2.5 Å (Figure 9).

Figure 9. The ALC1 linker peptide binds to the nucleosome acidic patch via an arginine anchor (adapted from: Lehmann et al., 2020).

This structure shows that the ALC1 peptide engages the nucleosome at the acidic patch through the conserved arginine anchor R611. Mutation of the ALC1RLS region causes a slight reduction in maximal ATPase activity and a stronger reduction in maximal nucleosome sliding rate as well as an increase in the Michaelis constant. These data suggest a role of the ALC1 linker region in binding to the nucleosome and in coupling of ATPase activity to efficient mobilization of nucleosomes. Further single-molecule investigations showed that mutation of the ALC1RLS region increases the pause-phase of nucleosome sliding, suggesting that this phase might be used to probe for an intact acidic patch. Additional in vivo studies showed that mutation of the ALC1RLS region impacts the recruitment of ALC1 to sites of DNA damage. Taken together, these findings show that the interaction of ALC1RLS with the nucleosome acidic patch is needed for full activation of ALC1 nucleosome sliding activity. We therefore propose the following model for the regulation and activation of ALC1: Upon the release of autoinhibition through PARy- lated PARP1 at sites of DNA damage, ALC1 engages the nucleosome. Once fully activated, ALC1RLS engages the acidic patch, thereby tethering the remodeler to the nucleosome and efficiently coupling ATPase activity to nucleosome sliding (Figure 10).

Figure 10. Model for activation of ACL1 through the nucleosome acidic patch (adapted from: Lehmann et al., 2020).

29 8. Paper III: Loss of ALC1 confers PARPi sensitivity and synthetic lethality with HRD

Although it has been suggested that ALC1 remodeling facilitates DNA repair, the precise role of ALC1 in vivo still remains unclear. In paper III, we investigated the role of ALC1 in vivo by creating ALC1 knockout mice. The loss of ALC1 had no impact on viability or lifespan but made the mice less susceptible to mesenchymal and epithelial tumors. Experiments with mouse embry- onic fibroblasts showed that the loss of ALC1 sensitized to the PARP inhibitor (PARPi) olaparib. The same was observed for human eHAP and U2OS cells where the ALC1 gene was knocked out. But how does the loss of ALC1 lead to PARPi sensitization? Further investigation showed that the loss of ALC1 leads to an increase in PARP trapping on chromatin. We reasoned that this might be the cause for the observed increase in PARPi sensitization upon loss of ALC1. A whole-genome CRISPR screen allowed us to screen for genetic vulner- abilities in ALC1 knockout cells. We found a reduced viability of ALC1 knockout cells through the depletion of deoxyuridine triphosphatase (DUT), an enzyme that converts dUTP to dUMP, homologous recombination (HR) factors, and double-strand break (DSB) processing factors. Upon validation of these hits, we confirmed that the loss of ALC1 in combination with either homologous recombination deficiency (HRD) or DSB processing leads to synthetic growth defects and hyper-sensitization to PARP inhibition. During investigation of the genetic interaction between ALC1 and DUT, we hypothesized that the impact of DUT depletion on viability of ALC1 knockout cells might be due to a defect in the DNA processing of misincor- porated uracil. DUT knockout leads to elevated levels of dUTP in the cells, which in turn leads to more uracil misincorporation into DNA. Indeed, our experiments show that ALC1 knockout cells are sensitive to formyl-dU. The depletion of SMUG1, a uracil glycosylase, rescues formyl-dU sensitivity but not PARPi sensitivity, which indicates that SMUG1 creates toxic lesions in the absence of ALC1 and in response to formyl-dU. Further investigation showed that ALC1 is recruited downstream of SMUG1 but upstream of APEX1, an endonuclease in the base excision repair (BER) pathway. Loss of either ALC1 or APEX1 results in an accumulation of toxic lesions.

30 Our initial hypothesis was that ALC1 remodeling is needed for SMUG1 to access sites of uracil misincorporation. In order to test this hypothesis, I conducted in vitro experiments with nucleosomes containing a uracil-guanine (U:G) mismatch protected by the histone octamer. The assembled U:G nucleosomes were remodeled by ALC1 just like nucleosomes without a uracil (Fig- ure 11A). Naked DNA (Figure 11B) or nucleosomes (Figure 11C) were treated with SMUG1, which creates an abasic site where uracil is accessible. NaOH in combination with heat treatment denatures the reaction and creates a single strand break at the abasic site (for methods see: Tarantino et al., 2018). SDS-PAGE analysis of the resulting DNA showed that in non-remodeled nucleosomes where the uracil is protected by the histone octamer, only roughly 20% of the uracil is excised. After remodeling, the fraction of excised uracil increases to roughly 70%, showing that ALC1-catalyzed nucleosome remodeling facilitates uracil excision by SMUG1 (Figure 11D).

Figure 11. (A) ALC1-catalyzed nucleosome remodeling assays of nucleosomes with or without uracil. (B) and (C) Schematic overview of uracil excision assay on DNA (B) and on nucleosomes (C). (D) ALC1-catalyzed nucleosome remodeling facilitates uracil excision by SMUG1.

However, further experiments showed that PARP1/2 and ALC1 recognize the lesion created by SMUG1, and that ALC1 is required for an efficient hand- over from SMUG1 to APEX1 rather than allowing SMUG1 to access the lesion. For this reason, our observation that nucleosome remodeling by ALC1 facilitates uracil excision by SMUG1 may not be physiologically relevant in vivo.

31 Taken together, our investigations show that ALC1-catalyzed nucleosome remodeling is required after lesion excision by glycosylases and before APEX1 action. While a loss of ALC1 does not impact viability, it confers sensitivity to PARPi and synthetic lethality with HRD which makes ALC1 a possible therapeutic target.

32 9. Future Perspectives

In paper I we used a variety of low-resolution structural approaches to describe the autoinhibited state of ALC1 (Lehmann et al., 2017). We found that the macodomain of ALC1 holds the motor domain in an open and inactive conformation, but how exactly the interaction between the macro domain and the motor domain stabilizes this inactive conformation of the motor domain remains unclear. High-resolution structural information is needed to answer this question. A crystal structure of ALC1 in its inactive state might also reveal the precise position of the linker region of ALC1, given that it appears folded and not overly flexible. Moreover, structural information on the full linker of ALC1 may reveal additional layers or regulation. It would also be highly informative to obtain a cryo-EM structure of full- length ALC1 bound to the nucleosome. Such a structure is expected to un- cover interactions between ALC1 and the nucleosome beyond the linker- acidic patch interaction discovered in paper II (Lehmann et al., 2020). Fur- thermore, the structure of an ALC1-nucleosome complex would reveal if the motor domain also binds to SHL2 of the nucleosome, as it has been observed for other remodelers, including Chd1 (Farnung et al., 2017; Sundaramoorthy et al., 2017), Snf2 (Liu et al., 2017), Swr1 (Willhoft et al., 2018), and Isw1 (Yan et al., 2019). It would be interesting to see where the linker region of ALC1 is located in the context of the nucleosome. What is the purpose of such an extended linker region? Lastly, it would also be interesting to see how the histone H4 tail interacts with ALC1, since it has been shown to be important for ALC1 remodeling (Ahel et al., 2009). The histone H4 tail might be needed for stimulation of ALC1 activity, as previously observed for Chd1 (Ferreira et al., 2007) and ISWI-family remodelers (Clapier et al., 2001; Hamiche et al., 2001; Hwang et al., 2014). Apart from obtaining high-resolution structural information, it would also be important to the substrate specificity of ACL1 in vivo. We have shown that the nucleosome acidic patch is important for ALC1 remodeling activity, but how does ALC1 engage nucleosomes with histone variants that alter the acidic patch? The histone variant H2A.Z, for example, has an extended acidic patch (Suto et al., 2000), while H2A.B has no acidic patch at all (Zhou et al., 2007). How would these changes affect nucleosome sliding by ALC1? And are there other substrates for ALC1 altogether? Understanding the precise regulation

33 and function of ALC1 is an important step towards understanding its role in cancer. Our investigations in paper III have shown that loss of ALC1 has no impact on viability on an organismal level, but that it leads to PARPi sensitivity and synthetic lethality with homologous recombination deficiency. These results suggest that ALC1 is an important therapeutic target for inhibition or degradation, especially in HR-deficient cancers, either on its own or in combination with PARPi to enhance sensitivity.

34 10. Svensk sammanfattning

Eukaryot DNA är packat inne i cellkärnan som kromatin. Denna packning förhindrar tillgången till DNA och utgör därför en barriär för processer såsom replikation, reparation av DNA och genuttryck. En cell behöver kunna reglera kromatinets tillgänglighet dynamiskt för att processer i cellkärnan ska kunna ske på rätt plats och vid rätt tidpunkt. Ett sätt för cellen att reglera kromatin- landskapet är med hjälp av en grupp av enzymer, så kallade kromatinremodel- leringsenzymer (remodelers). Dessa enzymer använder energin från ATP- hydrolys för att ändra position (glida), stöta ut och ändra sammansättningen av nukleosomer, kromatinets minsta byggstenar. Remodelers regleras starkt av post-translationella modifieringar, nukleosomernas egenskaper samt av de- ras egna domäner och subenheter. Dessa regleringsätt möjliggör för remodelers att utföra en mängd olika uppgifter. Felaktig reglering av remodelle- ringsaktivitet har kopplats till allvarliga sjukdomstillstånd, såsom olika vari- anter av cancer. ALC1/CHD1L (amplified in liver cancer 1/chromodomain-helicase-DNA- binding-protein 1-like) är en onkogen remodeler som deltar i reparation av skadat DNA. ALC1 har en makrodomän som binder till poly(ADP-ribos) (PAR), en polymer som syntetiseras av poly(ADP-ribos) polymeras 1 (PARP1) vid områden där DNA är skadat. Denna avhandling undersöker den molekylära grunden för reglering av ALC1 och rollen som nukleosomen och dess egenskaper har i aktivering av ALC1, samt dess precisa roll i reparation av skadat DNA.

Resultaten som presenteras i denna avhandling visar att, vid frånvaro av skadat DNA, stabiliserar makrodomänen i ALC1 en inaktiv konformation av den katalytiska domänen genom en konserverad elektrostatisk interaktion. Då den interagerar med PARylerat PARP1, genomgår ALC1 stora konformationsför- ändringar där dess makrodomän förflyttas från dess motordomän. Mutationer i de delar av makrodomänen och motordomänen som är vända mot varandra resulterar i att ALC1 blir konstitutivt aktiv och dess aktivitet blir då oberoende av interaktionen med PARylerat PARP1. Då DNA skadas leder bindningen mellan PAR-kedjor och makrodomänen hos ALC1 till en förflyttning av makrodomänen från ATPas-motordomänen, vilket i sin tur förbereder remodelern för aktivering.

35 Ytterligare experiment, vars mål var att förstå nukleosomens roll i regle- ringen av ALC1, visade att ALC1-aktivitet kräver nukleosomens negativt laddade region för effektiv remodellering. En cryo-EM struktur av ett komplex mellan ett regulatoriskt linker segment hos ALC1 och nukleosomen visade att ALC1 binder till nukleosomens negativt laddade region. Mutationer i de inte- ragerande regionerna påverkar rekryteringen av ALC1 till områden där DNA har skadats och minskar även enzymets aktivitet. Fullständig aktivering av ALC1 kräver därför interaktionen mellan det regulatoriska segmentet i lin- kerregionen hos ALC1 och nukleosomens negativt laddade region. Denna interaktion fäster remodelern på nukleosomen och är nödvändig för att koppla ATP-hydrolys till remodellering av nukleosomer. Undersökning av ALC1 in vivo visade att frånvaro av ALC1 gör celler känsliga mot PARP-inhibitorer och är syntetiskt dödligt i kombination med nedsatt homolog rekombination. Detta gör ALC1 till en potentiell måltavla för läkemedel då de kombineras med PARP-inhibitorer och används mot can- cersarter med nedsatt homolog rekombination.

36 11. Acknowledgements

These past five years have been an amazing journey that would not have been possible without the support of many people. First and foremost I would like to thank my PhD advisor Sebastian Deindl. I am incredibly grateful for the opportunities he has given me and everything he has done for me. As his first PhD student I witnessed the lab grow and develop, and as I grew with it, he was there to support me every step of the way. He taught me everything from ordering lab equipment, using the ÄKTA, data analysis, writing a scientific text, to making Pizza in a lab oven. His passion for science is inspiring and I would not be where I am today without his support. I would also like to thank my co-supervisor Stefan Knight for his support, valuable discussions, and his optimism regarding my crystals. I am grateful to all our collaborators who have worked with us during these years. Special thanks goes to Graeme Hewitt and Simon Boulton for a very fruitful collaboration. I also wish to thank all my teachers on the way, especially Ursula Pfitzner. She was one of my first lab supervisors and has taught me the fundamentals of working in a lab and the importance of self-organization.

Science is a team effort and I could not have asked for a better team to work with than the Deindl Lab. Thank you to every past and present member of the lab who has supported me, challenged me, and made me laugh. Luka, thank you for a lot of interesting discussion about science and other things that I do not wish to further specify. It is always a lot more fun when you are around. I wish you all the best for your PhD and everything that comes afterwards! Klaus, thank you for being “the rock in the waves” for our lab. It is great to know that you can rely on someone when “shit hits the fan”. I am grateful for everything that you have done for me, and your support during paper mode. You also have sparked my interest in many things outside of science, for example dolphins. It is always great to have lunch with you. Anton, thank you for all your help along the way and for being the best personal math teacher one could ask for. I am very grateful for every in-person lesson and every What’s App chat about formulas and calculations. I am astonished by your patience with me. Mao, you are great in the lab and you are great in the kitchen. Thank you for all your help with experiments, especially DNA related ones. I am grateful for your little tips and tricks on how to run pretty gels. And thank you for sharing my passion for Nintendo games. Oanh, thank you for

37 every time you showed me a new assay or walked me through a new experiment. I think you are a great teacher. Thanks for all the girls-talk and every little present you gave me throughout the years. You always know what makes me happy. Guillaume, thank you for all your support and wisdom regarding nucleosomes, proteins, and the ÄKTA. I really enjoy discussing science with you. I also enjoyed each and every one of our crazy lunch discussions, especially the ones initiated by Klaus. I hope that we can soon have a Raclette party again. Sofia, thank you for being an amazing office mate, and for sharing all your candy with me. It is a lot of fun working with you in the lab, especially when we try a new protein purification together (even when we fail). I learned so much from you especially about zodiac signs. Martha, thank you for all the organization in the background. The lab would not be the same without you. You work like a ninja, fast and efficient. I admire your discipline when it comes to working out. It for sure was an experience to join you in the gym. Yang, thank you for everything you taught me about crystallography. It was great working with you and it was great having lunch with you. Brad and Anna, thank you for an incredible fun time, for all the lunches and dinners we had together, and for every game we played. I would also like to thank all past and present members of the Molecular Systems Biology program with special thanks to Jimmy, who is one of those people who can fix everything. Bo, thank you for every coffee break, every fun conversation, and every Pokémon we traded. Javier, you are one of the most positive persons I know and I love your laugh. I am very much looking forward to working with you. Emil, thank you for showing me how to purify and label LacI, and for organizing the Beer Club - I mean the ICM Social Hour. Konrad, thank you for all your support during our time on the ICM board and many thanks for translating my thesis summary to Swedish. A big thank you goes out to every past and present member of the Structural Biology program. You made me feel at home and let me use all your equipment, although I belonged to a different program. A special thanks goes out to Tex. I think you are an incredible person and I admire your organization and planning skills. I have learned so much from you about these things and about crystallography. Thank you for all your help and support and everything you have done for me. Wangshu, thank you for everything, you were like a big PhD sister to me! Sandesh, thank you for all your support and for playing Pokémon Go with Caro and me. Dirk, thank you for sharing your appreciation for southern-German food with me. Ulrich, thank you for all your support during synchrotron trips and for gifting me a lab cat. She has a special place in my office and in my heart. Thank you to everyone at ICM, especially Danna, I love that you love cats so much! Laura R., thank you for being so cheerful. You made Beer Clubs - I mean Social Hour - so much more fun! Jennah, it was great sharing our time as PhD students and our time on the ICM board together. Showgy, it is great having you around. You made my time so much more fun. Soneya, thank you

38 for all your help and support during synchrotron trips. Kristina, thank you for letting us use your equipment. I loved the time in your quiet lab. I would also like to thank Ellie, Silvia, Stefanie, Andrea, and Caro for our time together on the Fikareers organization team.

Apart from people at work, I also received an incredible amount of support from my friends. I am grateful for each and every person that I can call my friend, and I am sorry that I do not have the space to write about everyone. A special thank you goes to Kaj Jansson and Aleksandar Zeljić, my senior- PhD students. You have made my time as a PhD student at Uppsala University so much better by filling it with after-work beers, amazing food, dinner par- ties, and most importantly: board games! A big thank you goes to Andreas Tillberg, Maria Salomonson, and Lennart. You have been my friends since my early days in Sweden and you have done everything to make me feel at home in this county, especially in times when I missed my home in Germany. Thank you for each brunch, each dinner, each walk, and each round of board games! I also want to thank my Girls Gang: Caro, Katrin, Marianne, Anna, Gesa, and Agnes. Every time we are together I forget about all my worries and problems and there is just fun and laughter. I cannot put in words how grateful I am to all of you. An dieser Stelle möchte ich mich von ganzem Herzen bei meiner Familie bedanken. Ohne eure Hilfe und eure Unterstützung wäre all das nicht möglich gewesen. Ihr habt mich immer dazu ermutigt mehr zu wagen und nach den Sternen zu greifen, und das hat mich immerhin bis Schweden gebracht! Mama und Papa, danke, dass ihr euch immer hinter mich gestellt habt. Und danke Carsten, du bist der beste Bruder der Welt! Delyan, thank you for your love and support, and thank you for taking away all the stress of everyday life, so I can dedicate myself fully to my PhD.

39 12. References

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45 Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2009 Editor: The Dean of the Faculty of Science and Technology

A doctoral dissertation from the Faculty of Science and Technology, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology”.)

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