Transcription factor networks play a key role in human brain evolution and disorders Von der Fakultät für Mathematik und Informatik der Universität Leipzig eingereichte Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) im Fachgebiet Informatik vorgelegt von Stefano Berto geboren am 24. Juli 1983 in Conegliano (Italien) Die Annahme Der Dissertation wurde empfohlen von: 1. Professor Dr. Erich Bornberg-Bauer (Universität Münster) 2. Professor Dr. Peter F. Stadler (Universität Leipzig) Die Verleihung des akademischen Grades erfolgt mit Bestehen der Verteidigung am 19. Januar 2016 mit dem Gesamtprädikat magna cum laude i ii Acknowledgments I would like to thank my supervisor Katja Nowick for her support, encouragement, and advice throughout my PhD. I am truly thankful to her also for giving me this amazing opportunity to work in such a dynamic and inspiring environment. With her discussions, suggestions, and motivations, I have been able to gain insight into primate and, in particular, brain evolution. I also feel privileged for being part of her group and its multicultural environment. I would like to thank Alvaro Perdomo Sabogal, Maria Beatriz Walter Costa, Sree Rohit Raj Kolora, Sabina Kanton, and all the undergraduate students Lisa, Sandra, Nadine, Anna, and Cladia for all their help during discussions and lab meetings. One particular thank you goes to our lab manager, Katja Jacob, who helped me and followed me during these past years solving problems in the laboratory and teaching me the techniques I am currently working with. I would also like to thank the entire bioinformatic department, with a special thanks to Mario Fasold, Gero Doose, Steve Hoffman, Daniel Gerighausen, Lydia Muller, Christian Arnold, and Lydia Hopp for all the support, help, and suggestions they gave me during my PhD. Finally, I am heavily thankful to my current supervisor, Genevieve Konopka, and her laboratory, the Konopka lab, at UT Southwestern Medical Center in Dallas, for their exceptional support during the last year with suggestions and help to understand better the molecular mechanisms of cognition and brain development. A special thanks also to my friend, Andrea Trevisiol, who has provided me with an amazing Italian coffee machine, an exceptional support during the last PhD year. iii Dedication This thesis is dedicated to my wife Arianna Martin and to my parents. Three amazing and wonderful great apes. Without their help and support, I would not have reached my goals. A special dedication also goes to my little Jackie, a silly dog who has put a smile on my face, even on the most stressed days. “Darwin wasn't just provocative in saying that we descend from the apes - he didn't go far enough. We are apes in every way, from our long arms and tailless bodies to our habits and temperament.” Frans de Waal, Emory University iv v Abstract Although the human brain has been studied over past decades at morphological and histological levels, much remains unknown about its molecular and genetic mechanisms. Furthermore, when compared with our closest relative the chimpanzee, the human brain strikingly shows great morphological changes that have been often associated with our cognitive specializations and skills. Nevertheless, such drastic changes in the human brain may have arisen not only through morphological changes but also through changes in the expression levels of genes and transcripts. Gene regulatory networks are complex and large-scale sets of protein interactions that play a fundamental role at the core of cellular and tissue functions. Among the most important players of such regulatory networks are transcription factors (TFs) and the transcriptional circuitries in which TFs are the central nodes. Over past decades, several studies have focused on the functional characterization of brain-specific TFs, highlighting their pathways, interactions, and target genes implicated in brain development and often disorders. However, one of the main limitations of such studies is the data collection which is generally based on an individual experiment using a single TF. To understand how TFs might contribute to such human-specific cognitive abilities, it is necessary to integrate the TFs into a system level network to emphasize their potential pathways and circuitry. This thesis proceeds with a novel systems biology approach to infer the evolution of these networks. Using human, chimpanzee, and rhesus macaque, we spanned circa 35 million years of evolution to infer ancestral TF networks and the TF-TF interactions that are conserved or shared in important brain regions. Additionally, we developed a novel method to integrate multiple TF networks derived from human frontal lobe next-generation sequencing data into a high confidence consensus network. In this study, we also vi integrated a manually curated list of TFs important for brain function and disorders. Interestingly, such “Brain-TFs” are important hubs of the consensus network, emphasizing their biological role in TF circuitry in the human frontal lobe. This thesis describes two major studies in which DNA microarray and RNA-sequencing (RNA-seq) datasets have been mined, directing the TFs and their potential target genes into co-expression networks in human and non-human primate brain genome-wide expression datasets. In a third study we functionally characterized ZEB2, a TF implicated in brain development and linked with Mowat-Wilson syndrome, using human, chimpanzee, and orangutan cell lines. This work introduces not only an accurate analysis of ZEB2 targets, but also an analysis of the evolution of ZEB2 binding sites and the regulatory network controlled by ZEB2 in great apes, spanning circa 16 million years of evolution. In summary, those studies demonstrated the critical role of TFs on the gene regulatory networks of human frontal lobe evolution and functions, emphasizing the potential relationships between TF circuitries and such cognitive skills that make humans unique. vii viii Contents INTRODUCTION ..................................................................................... 1 1.1 The primate brain: anatomical evolution ..................................... 1 1.2 The primate brain: molecular evolution ....................................... 4 1.3 Transcription factors ..................................................................... 7 1.4 Gene regulation played by transcription factors ........................ 8 1.5 Transcription factors in human .................................................. 10 1.6 Transcription factors and brain development........................... 12 1.7 Transcription factors and networks ........................................... 16 CHAPTERS ............................................................................................ 20 CHAPTER 1 ........................................................................................... 23 Project summary ................................................................................ 23 Introduction ........................................................................................ 24 Results ................................................................................................ 26 Lineage-specific expression pattern changes .......................... 26 TF networks in each species ....................................................... 27 Network evolution ........................................................................ 30 Species differences in the networks of other tissues .............. 33 Expression Changed Sub-Networks ........................................... 35 Discussion .......................................................................................... 38 Methods .............................................................................................. 41 CHAPTER 2 ........................................................................................... 47 Project summary ................................................................................ 47 Introduction ........................................................................................ 48 ix Results ............................................................................................... 51 Transcription factors in cognition, brain development and disorders ................................................................................. 51 Ten different genome-wide TFs co-expression networks .. 53 The consensus network ......................................................... 56 The Brain-TFs and their role in the consensus network .... 58 Discussion ......................................................................................... 62 Methods .............................................................................................. 67 CHAPTER 3 ............................................................................................ 73 Introduction ....................................................................................... 73 Results ............................................................................................... 75 Genomic distribution of ZEB2 binding in three great apes 75 ZEB2 mediated gene expression .......................................... 81 Discussion ......................................................................................... 83 Methods .............................................................................................. 86 Summary, conclusions, and future perspective