Declaration of conflict of interest. No conflict of interest

Transcription

1 Declaration of conflict of interest No conflict of interest

2 Crosstalk between chromatin remodelling, transcription factors and histone modifications Silke R. Sperling Cardiovascular Genetics Experimental and Clinical Research Center Charite University Medicine Berlin Max Delbrück Center for moleculare Medicine Max Planck Institute for molecular genetics Department Biology, Chemistry, Pharmacy at Freie Universität Berlin

3 Stochastic Biological networks driving cardiac development Histone mod & DNA met Genes & RNA a E b I J A B C D F G H? Biological networks Environment CHD-1 CHD-4 CHD-5 CHD-3 CHD-6 CHD-7 Congenital heart disease Normal heart

4 Chromatin regulation by Baf60c and Dpf3b/Baf45c Chromatin remodeling Bruneau, 2010 Curr Opin Genet Dev Zeng et al. 2010, Nature Lange et al. 2008, Genes Dev

5 Gene expression profiling as an approach to discover genes and pathways involved in cardiac (dys)development Kaynak et al. 2003, Circulation Tönjes at al. 2008, MolBioSystems

6 DPF3 / BAF45c - upregulated in hypertrophic human hearts Kaynak et al. Circulation 2003 Tetralogy of Fallot Overriding aorta Pulmonary stenosis Ventricular septal defect Right ventricular hypertrophy Sample Lange et al. 2008, Genes Dev

7 BAF45c - cardiac and somite expression in mouse

8 DPF3a and b associate with BAF chromatin remodeling complexes Isolation of nuclear binding partner of DPF3a and DPF3b in HEK293T cells Tandem affinity purification (TAP) and mass spectrometry

9 DPF3b interacts with methylated and acetylated lysine residues of histones 3 and 4 Analysis of a broad panel of histone 3 and 4 peptides harboring specific modifications such as methylations, acetylations or phosphorylations PHD1 PHD2 W311E C360R C363R

10 DPF3 expression in chicken and zebrafish

11 Morpholino knockdown of BAF45c in zebrafish - Loss of DPF3 impacts highly on cardiac and skeletal muscle development and function (e.g. dysregulation of cmya1, hand2, troponin, actin) wt MO

12 Chromatin regulation by Dpf3b/Baf45c Is there a functional link between histone modifications and DNA-binding transcription factors?

13 Modulation of the cardiac transcription network by histone modifications and DNA-binding transcription factors HL-1 cells Comparison of HL-1 cardiomyocytes with mouse/human hearts

14 Cooperative function of Gata4, Mef2a, Nkx2.5, Srf 84 94% of TF binding events harbor respective TRANSFAC motifs or in case of Srf the CArG-box Binding site conservation: - 10 % complete conservation between human and mouse - 27% are localized in conserved regions (PhasCons) Gata4 447 Mef2a 999 Nkx Srf 1,335 Overrepresented GO terms: e.g. Mef2a and Nkx2.5 - muscle cell differentiation - heart looping Schlesinger et al. 2011, PLoS Genetics

15 Combinatorial regulation by multiple transcription factors

16 Functional consequences of transcription factor binding Genes bound by multiple factors are significantly less likely differentially expressed in sirna knockdown of respected factors than expected (X 2 -test p<0.001) Knockdown efficiency (Western Blot)

17 Gata4, Mef2a, Nkx2.5 and Srf buffer each other

18 Gata4, Mef2a, Nkx2.5 and Srf buffer each other TF binding TF knockdown effect pairwise co-binding to target genes shared differentially expressed transcripts Negative correlation between pairwise binding and pairwise differential expression Genes bound by multiple TFs are less likely differentially expressed in sirna knockdown (p<0.001) Combinatorial regulation of downstream targets by multiple transcription factors stabilizes transcription networks Schlesinger et al. 2011, PLoS Genetics

19 H3ac modulates effect of transcription factors 60% of histone 3 acetylations overlap with any of the TFs studied, in random situation only 23% would be expected Histone 3 acetylation enhances activating effect of Gata4 and Srf p300 acetylates lysine residues of histone 3 as well as GATA4 Srf-cofactor Myocardin recruits p300 to Srf binding sites - acetylating histone 3

20 sirna SRF knockdown reduces H3ac marks of co- and down-regulated genes Schlesinger et al. 2011, PLoS Genetics

21 Srf leaves expression memory epigenetic mark Global scale using ChIP-seq and mrna-seq in HL1-cardiomyocytes H3ac co-occurence buffers Srf knockdown

22 Model of SRF driven gene expression in smooth muscle cells Srf-cofactor Myocardin recruits p300 to Srf binding site - acetylating histone 4 modified from McDonald and Owens, Circ Res 2007 Zhang et al. Mol BioSys 2012

23 ANOVA models predicting changes in H3ac in correlation of Srf and p300 changes over time (E18.5, E0.5, E4.5) down up unaltered Srf/p300

24 Representative examples of variable gene regulation in the time series E18.5, P0.5 and P4.5

25 Model of SRF driven gene expression in cardiomyocytes Zhang et al. Mol BioSys 2012

26 Model of cooperative function of histone modifications and DNA-binding transcription factors Brg1 H3K4me H3ac H4ac T-box Baf60c Gata4 Gata Tbx5

27 Acknowledgements Sperling Lab Martin Lange Bogac Kaynak Martje Tönjes Jenny Schlesinger Jenny J. Fischer Dominik Seelow Ilona Dunkel Qin Zhang Cornelia Dorn Tammo Krueger Markus Schueler Marcel Grunert Huan Cui Free University Berlin New York University Anne-Katrin Emde Haito Li Knut Reinert Dinshaw Patel MPI for Molecular Genetics German Heart Center Bernd Timmermann Felix Berger Martin Vingron Roland Hetzer Max Delbrück Center Salim Abdelilah-Seyfried Funding FP6 Heart Repair; FP7 CardioGeNet, FP7 CardioNet Studienstiftung des Deutschen Volkes (MT), DAAD (QZ, HC) German Research Foundation (DFG)