Polycomb Complexes & Epigenetics

1. Polycomb Complexes & Epigenetics Adam Baim MBB306 10/2/07

2. Chromatin Structure Adapted from: Alberts, et al., Molecular Biology of the Cell, 4th Ed. (2002)

3. Polycomb (PcG) Complexes • Chromatin-modifying transcriptional repressors with key developmental roles: • Embryonic development • Adult stem cell renewal • Deregulated in cancer • PRC2 • subunits: EZH2, EED, SUZ12, RbAp48 • EZH2 is histone methyltransferase, acts upon H3K27 • PRC1 • subunits: BMI-1, CBX2,4,7,8, other proteins • binds H3K27me3 and represses gene

4. Sparmann, et al (2006) Polycomb silencers control cell fate, development and cancer. Nature Reviews Cancer 6, 846-856

5. What’s known about Polycomb? • Best known as epigenetic regulators of homeotic (HOX) clusters (Ringrose, 2007) • PRC1 has been shown to mediate silencing by ubiquitylation of H2AK119; the protein which reads this modification has yet to be characterized (Schuettengruber, et al., 2007) • The H3K27me3 deposited by PRC2 might directly repress tsc in absence of PRC1 (Schuettengruber, et al., 2007)

6. Trends in Polycomb Research

8. Overview of Paper • In human embryonic fibroblasts: • Examined changes in mRNA expression associated with depletion of PRC1 and PRC2, to find potential PcG target genes • ChIP-on-chip analyses to confirm PcG targets: • Isolated DNA sequences which bind to PRC1,2, and H3K27me3 (ChIP) • Hybridized labeled DNA to tiled array (allows for identification of genes it contains which bind PRC1,2 and H3K27me3) • In human neural progenitor cells: • Evaluated PcG binding at genes involved in neural differentiation

9. siRNA to Deplete Specific Proteins • Create double-stranded RNA copy of mRNA to be blocked, transfect into cells • Internal machinery (e.g., RISC) uses this double-stranded RNA as template for recognizing the gene’s mRNA in vivo • The mRNA is cleaved and degraded, inhibiting protein synthesis Image: http://www.ohsu.edu/research/core/images/sirna_MWG-biotech.gif

10. Effects of PcG depletion on Expression • Used siRNA to block translation of EZH2, EED, and SUZ12 (PRC2 subunits) and BMI-1 (main PRC1 subunit) • Confirmed inhibition (lack of translated protein) via Western blot (A) • Extracted and labeled RNA from PcG-inhibited cells and control cells, hybridized directly to Affymetrix gene expression array • Quantified changes in gene expression (B,C) • Selected 341 of the most significantly changed genes as potential PcG targets

11. Overview of Paper • In human embryonic fibroblasts: • Examined changes in mRNA expression associated with depletion of PRC1 and PRC2, to find potential PcG target genes • ChIP-on-chip analyses to confirm PcG targets: • Isolated DNA sequences which bind to PRC1,2, and H3K27me3…“ChIP” • Hybridized labeled DNA to tiled arrays (allows for identification of genes which bind PRC1,2 and H3K27me3)…“chip” • In human neural progenitor cells: • Evaluated PcG binding at genes involved in neural differentiation

12. Chromatin Immunoprecipitation (ChIP) Protein Antibody to Protein Authors used this technique to make antibodies specific to SUZ12 (PRC2), CBX8 (PRC1), and H3K27me3 DNA that originally bound PRC1,2 and H3K27me3 is now ISOLATED, but it needs to be IDENTIFIED http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/mboc4/ch7f32.gif http://imagecache2.allposters.com/images/ADVG/479.jpg

13. Immunoprecipitated DNA  Gene chip • To validate specificity of ChIP antibodies, authors labeled this DNA and hybridized it to tiled gene chips containing all genes from the HOXA, HOXB, HOXC, and HOXD clusters (known PcG binding sites) Adapted from: Alberts, et al., Molecular Biology of the Cell, 4th Ed. (2002)

14. HOXA Findings • Heavy binding of PRC1,2 and H3K27me3 between HOXA9 and HOXA13 loci • Proteins “blanket” loci rather than binding exclusively at promoters • αHA (-) control

15. Overview of Paper • In human embryonic fibroblasts: • Examined changes in mRNA expression associated with depletion of PRC1 and PRC2, to find potential PcG target genes • ChIP-on-chip analyses to confirm PcG targets: • Isolated DNA sequences which bind to PRC1,2, and H3K27me3…“ChIP” • Hybridized labeled DNA to tiled arrays (allows for identification of genes which bind PRC1,2 and H3K27me3)…“chip” • In human neural progenitor cells: • Evaluated PcG binding at genes involved in neural differentiation

16. Identification of genes from ChIP DNA • Represented the 341 putative PcG target gene loci found in the mRNA expression assay on tiled gene chips as: |------------15kb------------GENE------5kb----| • Hybridized these chips with the same ChIP DNA used in the HOX experiment (i.e., the DNA that binds PRC1,2, and H3K27me3 • Hybridization suggested that PRC1,2 and H3K27me3 bound within the gene loci of 43 individual genes

17. “Bell enrichments” binding is greatest at particular points in the gene locus Depleted Subunit “Blanket enrichments” binding occurs throughout the gene Changes in expression for the 43 genes suggested as PcG/H3K27me3 targets by the ChIP on chip experiment Antibody binding for specific genes

18. “Genome-wide identification of human promoters bound by PcG” • Experiment repeated on chips containing probes for 24,275 promoters, each consisting of: |-------------1300bp----------TSS-----200bp---| =“Transcription Start Site” • PcGs bound promoters for 70% of the genes identified in the previous tiled array • Of the promoters found to bind PcGs, there was a significant overlap between those bound by PRC1, PRC2, and those which were H3K27me3 • The authors believe this is “…consistent with the fact that PRC1 is dependent on PRC2-mediated K27 tri-methylation for its ability to bind chromatin”

19. Suggests there is significant overlap in regions where SUZ12 (PRC2), CBX8 (PRC1), and H3K27me3 are bound

20. Overview of Paper • In human embryonic fibroblasts: • Examined changes in mRNA expression associated with depletion of PRC1 and PRC2 • ChIP-on-chip analyses: • Isolated DNA sequences which bind to PRC1,2, and H3K27me3…“ChIP” • Hybridized labeled DNA to tiled arrays (allows for identification of genes which biund PRC1,2 and H3K27me3)…“chip” • In human neural progenitor cells: • Evaluated PcG binding at genes involved in neural differentiation

21. Many of the PcG target genes suggested in the genome-wide experiment regulate neuronal differentiation

22. PcGs in Neural Differentiation • In human embryonic neural progenitor cells, authors selected four genes with known roles in neuronal differentiation • Experiments showed progressive decrease in PcG binding and H3K27me3 during differentiation (induced by retinoic acid) • Differentiation requires displacement of PcGs which repress differentiation-guiding genes • PcGs replaced when differentiation is finished (e.g., HOX genes)

24. An Unusual Result • Authors then examined a set genes which are highly expressed except during differentiation, when they are strongly silenced • ChIP experiments showed presence of PcGs and H3K27me3 at promoters of these genes in the undifferentiated state, when genes are being expressed • PcGs and H3K27me3 are only slightly increased during differentiation, when the genes are silenced

25. Looking again at HOXA • Authors found binding of EZH2 (PRC2) and CBX8 (PRC1) with H3K27me3 throughout the HOXA locus, both BEFORE and AFTER differentiation

26. Conjecture • The authors postulate that PcG target genes are “pre-programmed to be repressed upon appropriate cell fate signals,” explaining transcription in spite of PRC1,2 binding and H3K27me3 • Support: PcG and trithorax proteins found at polycomb binding sites and HOX promoters before expression levels are established by segmentation factors

27. Key Findings • Only a small number of PcG target genes found in the global-analysis were affected by PcG depletions • Hypothesis: Majority of ~1000 known PcG target genes are silenced in embryonic fibroblasts, perhaps due to EZH2’s ability to methylate H1K26 • Alternative Hypothesis: fibroblasts lack transcriptional activators necessary for expression of tissue-specific genes • ChIP-on-chip revealed 43 novel PcG target genes with “important regulatory roles in mesenchymal differentiation and development.” • Neuronal, bone, and sex differentiation; muscle development, hematopoiesis • PcG binding to target genes “does not strictly correlate with transcriptional silencing.”

28. End. All figures from Bracken, et al., 2006 except where stated otherwise