The Chromatin State The scientific quest to decipher the histone code Lior Zimmerman

Histones Discovered in 1884 by Albrecht Kossel Considered an inert packaging material for the DNA Nobel prize in 1910

The Nucleosome Core Histones o H2A H2AF H2A1 H2A2 o H2B H2BF H2B1 H2B2 o H3 H3A1 H3A2 H3A3 o H4 H41 H44 Image from: wikipedia.org

Histone Modifications Image from: Covalent modifications of histones during development and disease pathogenesis, Bhaumik et al, Nature Structural & Molecular Biology

Histone Modifications In the 60’s Allfrey et al showed in a series of studies that acetylation of histone tails is strongly correlated with transcription Acetylation neutralizes the positive charge on the histone tail, reducing the affinity to DNA. Image taken from wikipedia.org

Histone Modifications (cont) Mutation in H3 Core = Lethality Mutation in H3 tail = 1.Disruption to cell cycle 2.Repression of genes Findings suggests that Histones have a role in the cell’s regulatory mechanism. Grunstein et al, Cell, 1988

Histone Modifications (cont) Brian Strahl et al, PNAS, 1999

Histone modifications and gene transcription Gene transcriptionHistone modifications High throughput Tech. Image taken from wikipedia.org Image taken from illumina.com Barski, Zhao et al, Cell, 2007 ChIP-chip ChIP-Seq

ChIP on chip Image taken from wikipedia.org

ChIP-Seq overview Ligating short adapters to sequences

ChIP-Seq overview Attaching DNA to surface

ChIP-Seq overview Cluster creation

ChIP-Seq overview Fluorescent Nucleotides Determine sequence

Histone Methylation near Transcription Start Sites Single nucleosome resolution of 20 histone lysine and arginine methylations ~12,000 human genes, CD4+ T-Cells, divided to 4 groups based on expression levels The genes in each group were aligned relative to the TSS Barski, Zhao et al, Cell, 2007

RNA Pol II at TSS First showing RNS Pol II as control Large number of counts at highly transcribed genes

Histone Methylation near Transcription Start Sites

Gene Activity and Histone Methylations in Transcribed Regions Inspecting the correlation between transcription and several types of modifications. Genes were separated into groups of 100 genes based on their expression levels from high to low (left to right on the x axis) Right y axis = expression level of each group Left y axis = histone methylation level

Histone Methylations at Enhancers By inspecting the Hyper Sensitivity to DNAse removed the HS sites that overlapped with CTCF and Pol II binding sites to remove bias

Conclusions The histone code is highly complex, multiple sites of modifications, multiple types of modifications (ac,me1-3 + more) H3K4me3 – largely correlated with activation H3K27me3 – correlated with repression

Same DNA – Different Expression profiles What could account for the difference among cells?

What about enhancers? Heintzman, Ren et al, 2009, Nature Histone modifications on enhancers in different cell types Image taken from Robert Tjian, "Molecular Machines that Control Genes," Scientific American

Histone modifications at human enhancers Heintzman, Ren et al performed ChIP-chip analysis to determine binding of o CTCF (binds to insulators) o p300 (known enhancer binder) o patterns of histone modifications In five human cell lines o HeLa o immortalized lymphoblast GM06690 o Leukaemia K562 o embryonic stem cells (ES) o BMP4-induced ES cells

Cell type specific Histone code First, took 1% of human genome selected by the ENCODE consortium Examined H3K4me1, H3K4me3, H3K27ac at well- annotated promoters Will the state of these histone modifications vary in a cell-type-specific manner?

Cell type specific Histone code Answer: NO. *logR is the log ratio of enrichment of each marker as determined by ChIP-chip

Prediction of enhancers Performed ChIP-chip on HeLa, K562 and GM cells. Prediction of new enhancers based on H3K4me1 and H3K4me3 On the ENCODE regions (1% of genome)

Training classifier P-300 is a transcriptional coactivator protein that is known to bind enhancers On the ENCODE regions (1% of genome)

Prediction of enhancers Highly cell type specific histone modification patterns

Prediction of enhancers Genome-wide ChIP-chip for H3K4me1 and H3K4me3 on HeLa and K562 cells

Histone Code and Gene Expression HeLa Cell IFN-γ STAT1 DNA ChIP-Seq

Histone Code and Gene Expression H3K4me1 chromatin signature is present before induction at STAT1 binding site No signature at STAT1 binding site Before induction Genes are much more likely to be upregulated if the H3K4me1 chromatin sig. is present!

Chromatin state dynamics in 9 human cell types 9 cell types were probed for genome wide histone modifications 90 whole genome chromatin maps (9 cell types * (9 types of modifications + RNA Pol)) Multivariate hidden Markov model that uses combinatorial patterns of chromatin marks to distinguish chromatin states. Jason Ernst et al, Nature, 2011

Chromatin state dynamics in 9 human cell types Notice: 1.CTCF in insulators 2.H3K4me3 in active promotors 3.H3K4me1 in Strong enhancers

Validating predictions Validating with Luciferase gene activity

Epigenomic map

Cell specific functional enrichments Clustering of genomic locations assigned to active promotors (level 1) and active enhancers For each cluster, enriched gene ontology terms are shown with hyper-geometric P value and fold enrichment (based on the nearest TSS).

Enhancer activity profiles The authors Investigated functional interconnections among enhancers Factors that activate or repress enhancers And the genes whose expression they regulate All that, by defining ‘activity profiles’ for each across the cell types.

Epigenetics and diseases Fold Enrichment Erythrocyte phenotypes at Leukemia cells Colorectal cancer at Hepatocellular Carcinoma

Zooming on Erythrocytes phenotypes

Enhancer activity profiles

Predicted causal regulators

Epigenomic Landscapes of Pluripotent and Lineage Committed Human Cells All share the same genetic code, yet are completely different!

Epigenomic Landscapes of Pluripotent and Lineage Committed Human Cells Hawkins et al studied epigenomic landscapes in pluripotent and differentiated cells and the links between histone modifications and DNA methylation

Histone modification architecture 83% of H3K27me3-marked promoters are H3K4me3/H3K27me3 bivalent promoters in hESCs compared to 50% in IMR90 cells

Histone modification architecture Bivalent mark Constitutive repression

Summary Epigenetics along with DNA elements play a central role in gene transcription regulation Histone modifications could explain the existance of mysterious DNA sequences Could epigenetic ‘malfunction’ be a cause for some types of malignancies? Inheritance of epigenetic traits?

Questions?

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