Research Collection Doctoral Thesis HIF1α dependant transcriptional networks in macrophages and hepatocytes Author(s): Müller, Julius Publication Date: 2009 Permanent Link: https://doi.org/10.3929/ethz-a-005900145 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH Nr. 18594 HIF1 ααα dependant transcriptional networks in macrophages and hepatocytes ABHANDLUNG zur Erlangung des Titels DOKTOR DER WISSENSCHAFTEN der ETH ZÜRICH vorgelegt von Julius Müller Dipl. Biol., Ruprecht-Karls-Universität Heidelberg geboren am 16.09.1977 von Deutschland Angenommen auf Antrag von Prof. Romeo Ricci Prof. Wilhelm Krek Prof. Peter Bühlmann 2009 Index 1. Index 1. Index .......................................................................................................................... 2 2. Acknowledgements .................................................................................................. 4 3. Summary ................................................................................................................... 5 4. Zusammenfassung ................................................................................................... 6 5. Abbreviations ............................................................................................................ 7 6. Introduction ............................................................................................................... 8 6.1. Hypoxia and the Hypoxia Inducible Factor 1 alpha ............................................................... 8 6.2. Transcriptional- and epigenetic regulation ......................................................................... 15 6.3. Methodology to address Genome wide binding patterns................................................... 22 7. Aim of the project ................................................................................................... 24 8. Material and Methods ............................................................................................. 25 8.1. Media and Buffers used for ChIP and ChIP-chip .................................................................. 25 8.2. Cell lines ............................................................................................................................... 28 8.3. ChIP-on-chip......................................................................................................................... 29 8.4. ChIP-Seq ............................................................................................................................... 35 8.5. mRNA Expression Profiling .................................................................................................. 35 8.6. Identification of ChIP-chip Peaks ......................................................................................... 36 8.7. Identification of ChIP-Seq Peaks .......................................................................................... 37 8.8. De novo Motif Analysis ........................................................................................................ 37 8.9. Q-PCR Validation of ChIP Hits .............................................................................................. 38 8.10. Annotation of sequences and association of expression- to binding data .......................... 38 9. Results ..................................................................................................................... 39 9.1. Regulation of HIF1 α and its target genes ............................................................................ 39 9.2. Genome wide expression and binding studies .................................................................... 42 9.3. Genome wide binding study using the murine leukemic monocyte-macrophage cell line (Raw.264) and ChIP-Seq ............................................................................................................................ 54 9.4. Promoters that are occupied by HIF1 α in Raw.264 cells .................................................... 57 9.5. Characterization of the Hypoxia Response Element ........................................................... 63 9.6. Transcription factors interacting with Hif1 α ....................................................................... 68 9.7. Downstream regulatory mechanism regulated by Hif1 α .................................................... 72 Page 2 Index 10. Discussion............................................................................................................... 77 10.1. Binding of HIF1 α is cell type specific ................................................................................... 77 10.2. One out of five genes that are bound by HIF1 α are differentially expressed in PMH and PMM 78 10.3. One out of twenty-five hypoxia responsive genes are bound by HIF1 α in PMH ................ 79 10.4. ChIP-Seq reveals markedly more HIF1 α binding events during hypoxia in Raw.264 cells .. 80 10.5. HIF1 α directly binds to genes associated to glycolysis, angiogenesis and regulation of transcription, depending on the cell type. ............................................................................................... 81 10.6. HIF1 α preferentially binds close to the TSS ......................................................................... 81 10.7. HIF1 α preferentially binds to an HRE consisting of nine base pairs or an tandem core HRE 82 10.8. The TRE consensus motif is overrepresented at enhancer regions targeted by HIF1 α ...... 83 10.9. SP1 is a potential HIF1 α target and might regulate genes in response to hypoxia independent of HIF1 α ............................................................................................................................... 84 10.10. Transcriptional regulation of chromatin modifiers by HIF1 α .............................................. 85 10.11. Comparison to previous genome wide HIF1 α binding studies............................................ 86 11. Outlook .................................................................................................................... 88 12. References .............................................................................................................. 90 13. Supplements ........................................................................................................... 97 13.1. Top 300 up regulated genes in PMH and PMM ................................................................... 97 13.2. Group III genes of PMM, PMH and Raw.264 cells (Top 300) ............................................. 104 Page 3 Acknowledgements 2. Acknowledgements Ich möchte meine Arbeit den folgenden Personen widmen, die alle direkt und indirekt zum erfolgreichen Abschluss meiner Doktorabeit während der letzten vier Jahre beigetragen haben: Meinen Eltern, ohne dessen Unterstützung diese Arbeit niemals möglich gewesen wäre. Romeo Ricci, der mir die Möglichkeit gegeben hat, dieses Projekt bis zum Ende durchzuführen. Meinen Kollegen: Renata Windak, Grzegorz Sumara, Susann Kumpf, Arne Ittner, Helmuth Gehart und Ivan Formentini Meine Semesterstudenten: Yvonne Fink und Andreas Essig Meinem Thesis Committee: Wilhelm Krek und Peter Bühlmann Meinen Kollaboratoren: Andrea Patrignagni (ChIP-chip), Bernard Jost (ChIP-Seq) Meinen Geschwistern: Caroline, Wiebke und Oskar Weitere wichtige Elemente: Susann (Administration und Lehre), Arne und Helmuth (Abend Snacks), Nikolai (Kaffeepausen), Felix (Trainingspartner), Antra (Rauchen), Gerald (Ernährung), Stefan und Strahil (Golf Pros), Iza (General Support), Ivan (Fluchen)… Page 4 Summary 3. Summary HIF1 α is the principal transcription factor that mediates responses to low oxygen levels in eukaryotic cells. By comparing genome-wide promoter binding studies of HIF1 α in primary mouse hepatocytes and primary mouse macrophages, I was able to demonstrate that HIF1 α binding is cell-type specific. Integration of expression data revealed that only a small fraction of genes bound by HIF1 α are differentially expressed. To explore the transcriptional mechanisms, which modulate the differentially expressed genes secondary or independent of HIF1 α, motif analysis of the respective promoters was performed. Among others, transcription factors of the activating protein 2 (AP2) family and SP1 showed a marked overrepresentation suggesting an important function in the regulation of hypoxia responsive genes. To complement these results with genome-wide binding data, an unbiased binding study of HIF1 α using ChIP-Seq in Raw.264 cells, was performed. Although the majority of binding events were localized close to the transcriptional start side (TSS), about 40% of the peaks occurred more than 10kbp away from the TSS. Motif analysis of the Raw.264 binding study revealed that HIF1 α preferably binds to an extended hypoxia response element (HRE) and in 14% of the cases, a tandem core HRE seems to be the consensus site bound by HIF1 α. Moreover, I showed that AP1 might be an important factor cooperating with HIF1 α at enhancer sites to modulate expression levels of developmental genes and genes associated to apoptosis. Another mechanism of transcriptional regulation upon hypoxia may involve the JmjC family of histone demethylases that were found to be direct targets of HIF1 α. Thus, my data refined the HRE motif that appears