The EMBO Meeting 2012

HHMI & Fred Hutchinson Cancer Research Centre

Mapping genome-wide nucleosome dynamics

Eukaryotic gene expression occurs in the context of chromatin, and maintaining a region accessible to DNA-binding proteins for transcriptional regulation requires active processes that mobilize nucleosomes. Our approach to studying these processes has been to map nucleosome dynamics genome-wide, and we have introduced several different strategies to achieve this goal: (1) To measure relative levels of histone replacement across the genome, we have followed incorporation of the replication-independent histone variant, H3.3, which replaces replication-coupled H3 over the course of the cell cycle. (2) To map histone turnover kinetics directly we have developed a novel method based on metabolic labeling of proteins followed by affinity purification of newly synthesized histone core particles. (3) To map classical 'active' chromatin genome-wide we have applied salt fractionation to intact micrococcal nuclease-treated nuclei. (4) To effectively profile chromatin landscapes at single base-pair resolution, we have developed a simple sequencing library preparation protocol and data display method that we have applied to mapping transcription factors, nucleosome remodelers, nucleosomes and kinetochores from a single dataset. (5) To extend genome-wide chromatin profiling to tissues, we have introduced an affinity-based method for purification of nuclei expressing a nuclear envelope protein under control of a cell-type-specific promoter. Application of these methods to model organism genomes suggests that nucleosome turnover is crucial for epigenetic inheritance of gene activity and for maintaining a single centromere on a chromosome.

Biography

Steven Henikoff received a BS degree in Chemistry from the University of Chicago and a Ph.D. degree in Biochemistry and Molecular Biology from Harvard University, and carried out postdoctoral research at the University of Washington. He joined the Fred Hutchinson Cancer Research Center in Seattle in 1981, where he is a Member of the Basic Sciences Division and an Affiliate Professor of Genome Science at the University of Washington. He has been an Investigator of the Howard Hughes Medical Institute since 1990 and a Member of the US National Academy of Sciences since 2005. He is co-Editor-in-Chief of Epigenetics & Chromatin and a member of the Editorial Boards of Trends in Genetics, Current Opinion in Genetics and Development and Genome Biology. His laboratory studies chromatin processes, epigenetic inheritance, centromere structure, function and evolution, and develops tools for epigenomics.

The Wellcome Trust Centre for Cell Biology, University of Edinburgh

The Dinucleotide CG as a Genomic Signalling Module

The DNA sequence 5'CG (CpG) is unusual in several respects. It is self-complementary and can exist in three chemical forms depending on the modification status of its cytosine moiety. To understand the functional significance of the CpG dinucleotide, we study proteins that bind either its methylated or unmethylated form. These proteins are likely mediators of CpG signalling that influence chromatin modification and thereby genome activity. The local density of CpG varies dramatically within genomic DNA. In the bulk genome CpG is rare and highly methylated, but in so-called "CpG islands" (CGIs) it is dense and usually non-methylated. A signature histone mark at non-methylated CGIs and also at transcriptionally active genes is trimethylation of histone H3 lysine 4. We are exploring the mechanisms by which DNA sequence features that are shared by all CGIs influence this and other epigenetic marks. Proteins binding to CpG appear to be key players in setting up "promoter-friendly" chromatin at CGIs. In contrast, proteins that interact with methyl-CpG promote gene silencing by recruiting transcriptional corepressors. In particular mutations in the gene for the methyl-CpG binding protein MeCP2 cause the autism spectrum disorder Rett Syndrome. By studying MeCP2 we are learning about both the biology of DNA methylation and the molecular pathology of this neurological condition.

Biography

Adrian Bird, who holds the Buchanan Chair of Genetics at the University of Edinburgh, studies the basic biology and biomedical significance of DNA methylation. His laboratory identified CpG islands as gene markers in the vertebrate genome and discovered proteins that read the DNA methylation signal to influence chromatin structure. Mutations in one of these proteins, MeCP2, cause the autism spectrum disorder Rett Syndrome. Dr Bird's laboratory established a mouse model of Rett Syndrome and showed that the resulting severe neurological phenotype in mice can be cured. Awards include the Louis-Jeantet Prize for Medicine (1999) and the Gairdner International Award (2011).

German Cancer Research Centre

Non-coding RNA, chromatin remodeling and transcription: Epigenetic control of nucleolar transcription

Epigenetic mechanisms silence a fraction of rRNA genes (rDNA) by establishing heterochromatic features at the rDNA promoter. Silencing of rDNA is mediated by NoRC, a chromatin remodeling complex that interacts with DNA methyltransferase(s), histone deacetylase(s) and histone methyltransferase(s), thereby targeting enzymes to the rDNA promoter that are required for heterochromatin formation and transcriptional repression. Importantly, NoRC function requires the association with 'pRNA' ('promoter-associated RNA'), a 150-250 nt RNA moiety that is complementary in sequence to the rDNA promoter. Antisense-mediated depletion of pRNA leads to displacement of NoRC from nucleoli, decrease in rDNA methylation and activation of Pol I transcription. In contrast, overexpression of pRNA mediates heterochromatin formation, de novo DNA methylation and transcriptional repression. A 20 nt sequence in the 5'-terminal part of pRNA interacts with the target site of the transcription factor TTF-I, forming a triple-stranded structure that is specifically recognized by the DNA methyltransferase DNMT3b. The results reveal a compelling RNA-based strategy for epigenetic programming, implying that ncRNAs can guide DNA methyltransferase to specific genomic sites to methylate DNA and silence transcription. Data will be presented showing that DNA:RNA triplex-mediated targeting of DNMTs is not restricted to rDNA but has the potential to act on other genes that are silenced by DNA methylation.

In addition, rDNA is also transcribed in antisense orientation, yielding a heterogeneous population of long RNA polymerase II-directed transcripts that cover the pre-rRNA coding region and overlap the rDNA promoter. Down-regulation of transcription in response to environmental or developmental cues correlates with increased levels of antisense transcripts and elevated levels of H4K20 trimethylation (H4K20me3) across rDNA. The methyltransferase Suv4-20h2 specifically interacts with antisense RNA, leading to H4K20 trimethylation and compaction of chromatin. An increase in antisense transcripts and enhancement of H4K20me3 in quiescent cells was also observed at promoters of protein-coding genes, suggesting a general RNA-based targeting mechanism that directs Suv4-20h2 to regulatory sequences, thus creating a chromatin environment that impairs transcription. The results link growth-dependent transcriptional regulation to H4K20 trimethylation, suggesting a compelling mechanism of gene regulation that leads to changes of chromatin structure in response to external challenges.

Biography

Ingrid Grummt received her PhD in 1970 at the Humboldt-University in Berlin, worked as a Postdoc at the German Academy of Sciences in Berlin-Buch and the Max-Planck-Institute of Biochemistry in Munich, and has led a research group since 1977. She has been Head of a Research Division at the German Cancer Research Center in Heidelberg since 1990 where she addresses the mechanisms that link transcription to cell proliferation and chromatin structure, focusing on the role of ncRNA in chromatin structure and epigenetic regulation. Her work has uncovered a novel RNA-based strategy for epigenetic programming, demonstrating that DNA:RNA triplexes serve as binding platforms for chromatin modifying enzymes.

She is an elected member of several academic councils and scientific advisory boards and has received numerous awards for her work, ranging from the Gottfried Wilhelm Leibniz Prize for German Scientists in 1990 to the FEBS/EMBO Women in Science Award in 2010.