Bioinformatics of Transcriptional Regulation

Transcription

1 Bioinformatics of Transcriptional Regulation Carl Herrmann IPMB & DKFZ

2 Wechselwirkung von Maßnahmen und Auswirkungen Einflussmöglichkeiten in einem Dialog

3 From genes to active compounds transcription factor A transcription factor B cellular output target gene cis-regulatory module erna lincrna (RIP-seq) ncrna chromatin structure mirna mrna PMID

4 Program of these lectures Introduction ; representation of TFBS specificities ; experimental approaches ; clustering motifs Predicting TFBS in sequences using matrices (pattern matching) ; using additional information to improve TFBS predictions Motif discovery in sequences (word counting, EM,...) ; including phylogenetic information Predicting cis-regulatory modules (supervised and unsupervised) Applications to ChIP datasets Analysis of RNA-seq data : statistical approaches, measuring differential expression

5 First example of a regulatory system glucose is the prefered source of carbon for E. coli lac operon needs to be turned on if ONLY lactose available 2 mechanisms [Jacob & Monod, J.Mol.Bio. (1961)] camp favors binding of RNAP ; [camp] inversely related to [glucose] repressor protein (lac I gene) : constitutively active ; cannot bind the DNA element in presence of lactose

6 Organismal complexity [D.Potier, PhD thesis, 2011]

7 «Junk DNA?»

8 Organismal complexity ~ regulatory complexity [Ahnert, Fink, Zinovyev, 2008] Proportion of non-coding DNA correlates with organismal complexity Proportion of transcription factors correlates with organismal complexity

9 Transcriptional regulation in «higher» eukaryotes 2. Chromatin structure epigenetic factors chromatin wrapped around nucleosomes 3. 3D chromatin conformation 1. Binding of transcription factors 4. Readout : gene expression [ Wasserman & Sandelin, Nat.Rev.Gen (2004) ]

10 What experimental data? Genetic factors (epigenetic) chromatin structure: ChIP-seq post-translational modifications of histons DNA methylation assays DNA methylation DNAse I hypersensitive regions, FAIRE-seq open chromatin regions Chromatin 3D conformation ChIP-seq, ChIP-exo binding sites for transcription factors 3C/4C/Hi-C interaction points Readout : gene expression microarray : cdna, oligoarrays RNA sequencing (RNA-seq)

11 Chromatin Immunoprecipitation (ChIP) Chromatin immunoprecipitation (ChIP) yields DNA fragments, that are bound by the protein of interest. Identification of the fragments : hybridisation on microarrays (mostly tilling arrays = ChIP-chip) : genome-wide PCR/qPCR targeted expriment Important aspect sequencing (ChIP-seq) genome-wide Quality/Specificity of the antibody? DNA fragment (~ bp) binding site (~10 bp)?

12 ChIP-sequencing [Wilbanks & Faccioti PLoS One (2010)] Perfect world Real life Nkx2.5 ChIP-seq in mouse cell lines (GSE21529)

13 Experimental identification of binding sites ChIP-exo [Rhee & Pugh, Cell 2011] ChIP fragments are digested with exo-nuclease much higher resolution ("single-base") than ChIP-chip/ChIP-seq

14 Histone modifications histones are subject to post-translational modifications at their N-terminal tail Lysine methylation Lysine/arginine acetylation Serine phosphorylation ubiquitylation they modify the physical properties of the DNA-nucleosome interactions

15 Chromatin binding proteins Histone modifying enzymes [Wikipedia]

16 Histone modification histone modifications are a good proxy of gene expression and presence of regulatory elements [Barth et al. Trends in Biochemical Sciences, 2010]

17 Example 3 Datasets EZH2 Methyltransferase H3K27me3 H3K27me3 HNF4A transcription factor Distal binding site (away from gene) 2 cell lines (ENCODE) Strong correlation between enzyme binding and corresponding histone modification

18 Long range chromatin interactions Chromosome conformation capture (3C) allows to verify physical interaction of 2 loci («one-to-one») Circularized 3C (4C) allows to identify all interaction points of a given locus («one-to-all») [Wikipedia]

19 Long range chromatin interactions Yet another C : Hi-C («all-to-all») [E Lieberman-Aiden et al. Science 2009] Hi-C can determine the global structure of the DNA molecule (low resolution ~ 1 Mb)

20 Long range chromatin interactions X-ray diffraction patterns (R. Franklin)

21 Long range chromatin interactions Chromatin interaction analysis by paired-end tag sequencing (ChIA-pet) : identify interactions mediated through a particular protein of interest (polymerase, transcription factor, CTCF,...) [MJ Fullwood et al. Nature 462, (2009)]

22 Long range chromatin interactions ChIA-pet shows numerous enhancer-promoter interactions but also many promoter-promoter interactions Hypothesis : Promoters kann act in trans and be used as enhancers for distal genes [Li G et al. Cell (2012)]

23 Do we still understand anything? promoters can act as enhancers enhancers can act as promoters coding exons can serve as enhancers enhancers are transcribed as lincrna

24 transcription factor promoter transcript

25 bi-directionnally transcribed promoter long-range enhancer acting as promoter coding exon enhancer enhancer RNA (erna) polycomb associated gene silencer

26 Operon : born-again? "Intriguingly, the multigene complexes illustrated in this study are, in principle, akin to the bacterial operon as a mechanism for coordinated transcriptional regulation of related genes, suggesting the possibility of a chromatin-based operon mechanism (chro-operon or chroperon) for spatiotemporal regulation of gene transcription in eukaryotic nuclei."