Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1404 Gene regulation by different proteins of TGFβ superfamily VARUN MATURI ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 ISBN 978-91-513-0172-3 UPPSALA urn:nbn:se:uu:diva-334411 2018 Dissertation presented at Uppsala University to be publicly examined in Room B42, BMC, Husargatan 3, Uppsala, Monday, 29 January 2018 at 09:15 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in English. Faculty examiner: Senior Researcher Anita Göndör (Karolinska Institutet). Abstract Maturi, V. 2018. Gene regulation by different proteins of TGFβ superfamily. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1404. 50 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0172-3. The present thesis discusses how gene regulation by transforming growth factor β (TGFβ) family cytokines is affected by post-translational modifications of different transcription factors. The thesis also focuses on gene regulation by transcription factors involved in TGFβ signaling. The importance of the poly ADP-ribose polymerase (PARP) family in controlling gene expression in response to TGFβ and bone morphogenetic protein (BMP) is analyzed first. PARP2, along with PARP1, ADP-ribosylates Smad2 and Smad3, the signaling mediators of TGFβ. On the other hand, poly ADP-ribose glycohydrolase (PARG) removes the ADP-ribose from Smad2/3 and antagonizes PARP1 and PARP2. ADP-ribosylation of Smads in turn affects their DNA binding capacity. We then illustrate how PARP1 and PARG can regulate gene expression in response to BMP that signals via Smad1, 5. Over-expression of PARP1 suppressed the transcriptional activity of Smad1/5. Knockdown of PARP1 or over-expression of PARG enhanced the transcriptional activity of BMP-Smads on target genes. Hence our data suggest that ADP-ribosylation of Smad proteins controls both TGFβ and BMP signaling. I then focus on elucidating novel genes that are regulated by ZEB1 and Snail1, two key transcriptional factors in TGFβ signaling, known for their ability to induce EMT and cancer metastasis. Chromatin immunoprecipitation-sequencing (ChIP-seq) and targeted whole genome transcriptomics in triple negative breast cancer cells were used, to find binding regions and the functional impact of ZEB1 and Snail1 throughout the genome. ZEB1 binds to the regulatory sequences of a wide range of genes, not only related to cell invasion, pointing to new functions of ZEB1. On the other hand, Snail1 regulated only a few genes, especially related to signal transduction and cellular movement. Further functional analysis revealed that ZEB1 could regulate the anchorage-independent growth of the triple negative breast cancer cells, whereas Snail1 could regulate the expression of BMP6 in these cells. We have therefore elucidated novel functional roles of the two transcription factors, Snail1 and ZEB1 in triple negative breast cancer cells. Keywords: EMT, Snail1, ZEB1, TGFβ, BMP, Gene regulation Varun Maturi, Ludwig Institute for Cancer Research, Box 595, Uppsala University, SE-75124 Uppsala, Sweden. © Varun Maturi 2018 ISSN 1651-6206 ISBN 978-91-513-0172-3 urn:nbn:se:uu:diva-334411 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-334411) To my family and my teachers తృ భవ తృ భవ ఆర భవ In a day when you don’t come across any problems, you can be sure that you are going in a wrong path. - Swamy Vivekananda Main Supervisor Aristidis Moustakas, Professor Department of Medical Biochemistry and Microbiology Uppsala University, Sweden Associate supervisor Carl-Henrik Heldin, Professor Department of Medical Biochemistry and Microbiology, Uppsala Univeristy, Sweden Stefan Enroth, Senior Researcher Department of Immunology Genetics and pathology Uppsala University Sweden Opponent Anita Göndör, Associate Professor Department of Microbiology, Tumor and Cell Biology Karolinska Institute Sweden Committee Members Tanel Punga, Associate Professor Department of Medical Biochemistry and Microbiology Uppsala University Sweden Lars Forsberg, Associate Professor Department of Medical Biochemistry and Microbiology Uppsala University Sweden Lars-Gunnar Larsson, Professor Department of Microbiology, Tumor and Cell Biology Karolinska Institute Sweden List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Markus Dahl*, Maturi V*, Peter Lönn*, Panagiotis Papout- soglou*, Agata Zieba, Michael Vanlandewijck, Lars P. van der Heide, Yukihide Watanabe, Ola Söderberg, Michael O. Hottiger, Carl-Henrik Heldin and Aristidis Moustakas. (2014). Fine tuning of Smad protein function by poly (ADP-ribose) polymerases and poly (ADP-ribose) glycohydrolase during TGFβ signaling PLoS One. Aug 18; 9(8):e103651). II Watanabe, Y.*, Papoutsoglou, P.*, Maturi, V.*, Yutaro Tsu- bakihara, Hottiger, M.O., Heldin, C.-H. and Moustakas, A. (2016). ADP-ribosylating enzymes control bone morphogenetic protein signaling. Journal of Biological chemistry. Apr 21. III Maturi, V. Enroth, S. Heldin, C.-H. and Moustakas, A. (2017) Genome-wide binding of transcription factor ZEB1 in triple neg- ative breast cancer cells. Manuscript. IV Maturi, V. Enroth, S. Heldin, C.-H. and Moustakas, A. (2017) Genome-wide binding of transcription factor Snail1 in triple neg- ative breast cancer cells. Manuscript. *Indicates authors contributed equally to the work Reprints were made with permission from the respective publishers. Other Papers not included in the thesis I. Kahata K, Maturi V, Moustakas A, (2017). TGF-β Family Signaling in Ductal Differentiation and Branching Morphogenesis. Cold Spring Harb Perspect Biol. Mar.13 pii: a031997. II. Lehmann L, Aibara S, Hewitt G, Leitner A, Marklund E, Maturi V, Van der spoel D, Moustakas A, Boulton SJ, and Deindl S. (2017) Mechanistic insights into the auto-inhibition of the oncogenic chromatin remodeler alc1. Molecular Cell. (In Press). Contents Introduction ................................................................................................... 13 Overview .................................................................................................. 13 Gene regulation ........................................................................................ 14 PARP Family............................................................................................ 16 Gene regulation by signaling pathways .................................................... 19 TGFβ family ............................................................................................. 19 TGFβ-dependent Smad signaling ........................................................ 20 Bone Morphogenetic Proteins ............................................................. 21 Negative regulation of TGFβ and BMP pathways ............................... 22 Non-Smad signaling ............................................................................ 23 TGFβ in cancer ......................................................................................... 23 Epithelial-Mesenchymal Transition (EMT) ......................................... 24 Zinc finger E-box binding homeobox1 (ZEB1) .................................. 25 Snail1 ................................................................................................... 26 Methods ........................................................................................................ 28 Proximity ligation assay ........................................................................... 28 Chromatin immunoprecipitation-sequencing ........................................... 29 Ampliseq transcriptomic array ................................................................. 30 CRISPR-Cas9 ........................................................................................... 30 Present Investigation ..................................................................................... 32 Paper I ...................................................................................................... 32 Aim ...................................................................................................... 32 Background .......................................................................................... 32 Methods ............................................................................................... 32 Results ................................................................................................. 32 Conclusions ......................................................................................... 33 Paper II ..................................................................................................... 33 Aim ...................................................................................................... 33 Background .......................................................................................... 33 Methods ............................................................................................... 33 Results ................................................................................................. 34 Conclusions ......................................................................................... 34 Paper III .................................................................................................... 34 Aim ...................................................................................................... 34 Background .........................................................................................