A network of bHLHZip transcription factors in melanoma: Interactions of MITF, TFEB and TFE3 Josué A. Ballesteros Álvarez Thesis for the degree of Philosophiae Doctor January 2019 Net bHLHZip umritunarþátta í sortuæxlum: Samstarf milli MITF, TFEB og TFE3 Josué A. Ballesteros Álvarez Ritgerð til doktorsgráðu Leiðbeinandi/leiðbeinendur: Eiríkur Steingrímsson Doktorsnefnd: Margrét H. Ögmundsdóttir Þórarinn Guðjónsson Jórunn E. Eyfjörð Lars Rönnstrand Janúar 2019 Thesis for a doctoral degree at tHe University of Iceland. All rigHts reserved. No Part of tHis Publication may be reProduced in any form witHout tHe Prior permission of the copyright holder. © Josue A. Ballesteros Álvarez. 2019 ISBN 978-9935-9421-4-2 Printing by HáskólaPrent Reykjavik, Iceland 2019 Ágrip StjórnPróteinin MITF , TFEB, TFE3 og TFEC (stundum nefnd MiT-TFE þættirnir) tilheyra bHLHZip fjölskyldu umritunarþátta sem bindast DNA og stjórna tjáningu gena. MITF er mikilvægt fyrir myndun og starfsemi litfruma en ættingjar þess, TFEB og TFE3, stjórna myndun og starfsemi lysósóma og sjálfsáti. Sjálfsát er líffræðilegt ferli sem gegnir mikilvægu hlutverki í starfsemi fruma en getur einnig haft áHrif á myndun og meðHöndlun sjúkdóma. Í verkefni þessu var samstarf MITF, TFE3 og TFEB Próteinanna skoðað í sortuæxlisfrumum og hvaða áhrif þau Hafa á tjáningu hvers annars. Eins og MITF eru TFEB og TFE3 genin tjáð í sortuæxlisfrumum og sortuæxlum; TFEC er ekki tjáð í þessum frumum og var því ekki skoðað í þessu verkefni. Með notkun sérvirkra hindra var sýnt að boðleiðir hafa áhrif á staðsetningu próteinanna þriggja í sortuæxlisfrumum. Umritunarþættir þessir geta bundist skyldum DNA-bindisetum og haft áhrif á tjáningu gena sem eru nauðsynleg fyrir myndun bæði lýsósóma og melanósóma. Áhugavert er að hver umritunarþáttanna þriggja stjórnar tjáningu gena sem hinir hafa engin áhrif á en ekki er ljóst hvernig þessari takmörkun er stjórnað. Þessi þættir stjórna tjáningu Hvers annars, bæði beint og óbeint. Með mótefnisfellingu kom í ljós að MITF, TFEB og TFE3 geta myndað mistvenndir í æxlisfrumum en geta ekki myndað mistvenndir með öðrum bHLHZiP Próteinum eins og MAX. Þriggja amínósýru-svæði, sem er til staðar í HLH-ZiP svæðinu, er nauðsynlegt fyrir þessa sértæku tvenndarmyndun. Hlutverk þessara amínósýra var greint frekar og, með tilfærslu þessara þriggja amínósýra, var reynt að útbúa MITF prótein sem einungis geta myndað einstvenndir. Það tókst ekki en áhugaverðar upplýsingar fengust um hvernig tvenndarmyndun MITF fer fram. Niðurstaðan er að sambandið milli MITF, TFEB og TFE3 í sortuæxlisfrumum er flókið og felst í áhrifum á stjórnun genatjáningar, samstarfi próteina með myndun mistvennda og áHrifum boðleiða á staðsetningu í frumulíffærum. Mikilvægt er að greina og skilja þetta samband í sortuæxlum til að öðlast betri skilning á því hvernig unnt er að hafa áhrif á þau ferli sem umritunarþættir þessir stjórna. Lykilorð: Sortuæxli, MITF, TFEB, TFE3, sjálfsát 5 Abstract The MITF, TFEB, TFE3 and TFEC (MiT-TFE) Proteins belong to a larger family of basic helix-looP-helix leucine zipper transcription factors that are able to bind DNA and regulate gene exPression. MITF is crucial for melanocyte development and has been called a lineage specific oncogene in melanoma whereas its relatives, TFEB and TFE3 are involved in the biogenesis and function of lysosomes and autoPHagosomes, regulating cellular clearance pathways. Autophagy is a biological process that plays a prominent role in human disease and can also have an impact on the outcome of therapeutic approaches. WhetHer MITF, TFEB and TFE3 regulate eacH otHers expression and whether they cooperate in the regulation of gene expression is presently unknown. I have investigated tHe interactions and cross-regulatory relationsHiP of MITF, TFE3 and TFEB in melanoma cells. Like MITF, tHe TFEB and TFE3 genes are expressed in melanoma cells and tissues. I show that these factors regulate eacH other’s exPression. Also, using co-immunoPreciPitation studies, I demonstrate that MITF, TFEB and TFE3 are able to heterodimerize in melanoma cells. However, tHey fail to interact witH otHer members of tHe bHLH family sucH as MAX. FurtHermore, I show that a three amino acid region present in the HLH-ZiP region is resPonsible for dimerization sPecificity. In addition, several signaling pathways can post-translationally modify these factors, affecting their nuclear localization, activity and stability. RePorter gene and ChIP assays show that tHe three factors are able to bind and activate similar DNA regulatory motifs and drive the expression of genes involved in autophagy and pigmentation. Interestingly, some genes are exclusively regulated by one of the factors. The relationsHiP between MITF, TFEB and TFE3 is comPlex and involves regulation of gene exPression, Protein-protein interactions resulting in complementary functions. It is important to further unravel this relationship in melanoma to gain a better understanding of tHe cross-regulatory mecHanisms involved and How tHese factors regulate lysosomal function. Keywords: Melanoma, MITF, TFEB, TFE3, autoPHagy 7 8 Acknowledgements All tHe work Presented in tHis dissertation has been performed in the laboratory of Prof. Eiríkur Steingrímsson, the supervisor of this PhD project. Eiríkur’s lab is Part of tHe DePartment of BiocHemistry and Molecular Biology at tHe BioMedical Center of tHe University of Iceland in Reykjavík, Iceland. This work could not have been carried out without the financial support of the Icelandic ResearcH Fund (Rannís). Thank you so mucH Eiríkur for making tHis Possible. WHen I first visited your lab back in 2013 asking for a job I could not imagine how much I would learn througHout these years, and How this entire exPerience would sHaPe me, not only professionally as a scientist, but also the way I think and how I understand the reality around me. Thank you Margrét for all tHe scientific discussions that took place on a regular basis and were crucial to the develoPment of my entire PHD Project. Also, you conveyed to me your deeP interest in autoPHagy, and I really tHink I would like to continue tHis subject in tHe future. I wisH you success in your growing “autophagy lab”. Thank you to tHe remaining members of my doctoral committee, Prof. Þórarinn Guðjónsson, Prof. Jórunn Erla Eyfjörð and Prof. Lars Rönnstrand. They Provided critical assessment of my Project during tHe regular PhD committee meetings held to discuss the project. There is no way to build a solid piece of research without the constructive criticism that allows you to be your own biggest critic. Also, a big thank you to Drs. Vivian Pogenberg and Mathias Wilmanns at tHe EMBL in Hamburg. It was (still is) an Honor to collaborate with such experts in understanding the structure of MITF. And how much more there is yet to discover. Thank you to Ramile DilsHat, Ludwig Karl, Ingibjörg Sigvaldadóttir, Katrín Möller, Ásgeir Örn Árnþórsson and Kimberley Anderson for your direct contribution to this thesis with your hard work. You are greatly talented people. My gratitude to Lionel Larue for contributing to tHis tHesis witH tHe microarray expression data. Also, thank you Prof. Colin Goding and Dr. Erna Magnusdóttir for the Plasmid constructs thorougHly used in this work. THank you Dr. Sævar Ingþórsson for all your assistance with the confocal microscoPe at the imaging core of the BioMedical Center. 9 To all tHe lab members not yet mentioned, Valerie Fock, Sara Sigurbjörnsdóttir, DiaHann AtacHo, Sigurður Rúnar Guðmundsson, Kristín Bergsteinsdóttir, Hilmar Gunnlaugsson, Lára Stefansson, Linda Sooman, Freyja Imsland and Berglind Einarsdóttir. I learned a lot from eacH one of you during your lab meetings and day to day interactions. I am looking forward to the next cHaPter in my life but I will definitely miss you. I wisH you all the best! A Heartfelt tHank you to Prof. Guðmundur Hrafn Guðmundsson for introducing me to Iceland and Planting tHe first seed of my scientific career. Thank you so mucH Borys for your suPPort and for tHe fun times tHat made easier to go through the slightly stressful final stages of my PhD. Thank you Alba for our everyday conversations about my researcH and your researcH in NaPles. We never stoP laugHing and it is great to Have a friend for so long that exactly understands you. Finally, but most imPortantly, tHank you to my loving Parents CHelo and Luis and my siblings Alberto, Elda and Zoila. It is the biggest blessing to Have a family that loves you and suPPorts you unconditionally and without reservations. Mum and dad, you Have always encouraged me since I was a kid to study and to pursue my career of choice. For this, you gave me all the emotional and material support I would need, and I could never thank you enough for this. I love you. 10 Contents Ágrip .............................................................................................................. 5 Abstract ......................................................................................................... 7 Acknowledgements ...................................................................................... 9 Contents ...................................................................................................... 11 List of abbreviations ................................................................................... 15 List of figures .............................................................................................. 23 List of tables ...............................................................................................