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EPIGENETICS
Cold Spring Harbor Symposium 69 Live:  
Dispatches

On a repressing note, Thomas Jenuwein (IMP, Vienna) described work being done in his lab on histone methylation patterns. Jenuwein’s group has also raised antibodies against modified histones, although these are against mono-, di- and tri-methylated histone residues - either histone H3K9, H3K27 or H4K20. An immunofluorescence analysis in mouse embryonic fibroblasts revealed specific staining patterns for these modifications with a strong association found in the pericentric heterochromatin. Surprisingly, in cells deficient for Suv3-9h1 activity (which is the specific H3K9 methyltransferase), not only are the staining patterns for H3K9 altered as expected, but so are those of trimethylated H4K20. This simple observation led to the identification of two novel histone methyltransferases, Suv4-20h1 and Suv4-20h2. In vitro analysis shows that these novel enzymes have different substrate specificities from the Suv3-9h1 and use octamers and nucleosomes more efficiently as substrates than free histones. Analysis of the fly homolog of these genes shows that Suv4-20 acts as a PEV modifier thus establishing its role as a key epigenetic player in this pathway. This modification was also examined in fission yeast extracts by immunoblotting and was strongly represented implying a global and ancient role for this modification in regulating gene expression. Analysis of the H4K20-trimethyl epitope in mammalian cells reveals its enrichment at repeat elements in the genome and this may very well turn out to have an important function in maintaining genomic integrity. Based on these data, a model was proposed whereby the Suv4-20 enzymes function downstream of Suv3-9, using trimethylated H3K9 histones as substrates to trimethylate H4K20, perhaps in an HP1 dependent manner, to repress these genomic loci.

Moving on from trimethylated H3K9 to trimethylated H3K27, there were three talks that elaborated on recent studies on gene regulation by polycomb complexes. The first was a biochemical analysis presented by Danny Reinberg (UNDMJ, New Jersey) whose group purified tagged PRC complexes using an overexpression system from cultured human cells. In vitro methylation assays revealed that activity of this complex is uniquely modulated by linker H1 histone. Moreover, it was revealed that overexpression of Ezh2 caused changes in endogenous levels of the Eed isoforms implying that polycomb complexes were regulating the expression of their own subunits. In other experiments examining the PRC4 complex, it was demonstrated that the Sirt1 deacetylase was intimately and specifically associated with this complex and this was recapitulated in vitro. Interestingly, this association was dependent on the deacetylase activity of the Sirt1, which modifies the same lysine residue targeted by PRC4 for methylation. Immunoblotting analysis of different cells showed a massive upregulation of PRC subunits in cancerous cells and presently, work is being done to examine the role of PRC using a mouse model of prostate cancer.

Yi Zhang (UNCH) described a series of experiments investigating the regulation of Hox gene expression and argued that aberrant histone methylation by hDot1L resulted in leukemogenesis and discussed experiments done to identify various small molecule inhibitors of this activity. Zhang’s group has also purified tagged human PRC complexes from human cells and reconstituted it in vitro using baculovirus expression vectors. He demonstrated how knockdown of the PRC subunits correlated with a loss of H3K27 methylation with an upregulation of Hox gene expression and it was suggested that this was reminiscent of normal MLL/hDot1L mediated leukemogenesis. To complete the story, he described the identification of hRing2 as the ubiquitin ligase for histone H2B-K119 and presented data that loss of Ring2 function in different species also resulted in upregulation of the Hox gene clusters. A model was presented in which PRC activity co-operated with histone ubiquitination to upregulate Hox gene expression and result in leukemogenesis.

The third talk on this issue was presented by Vincenzo Pirrotta (Univ. of Geneva) whose group is investigating the regulation of Ubx gene expression in flies by the polycomb complex. His results indicate that PcG mediates repression by preventing the initiation of transcription. In addition, data from ChIP experiments revealed that PcG repression occurs in cis via spreading of silencing marks across the entire locus, probably by sequential methylation of nucleosomes. He finally described a novel gene called Esc-like which was probably responsible for maintaining these methylation marks in later development when the normal Esc gene is inactive.

Differential gene expression can be effected not only by histone modifications but also by histone variants and Srinivas Chakravarthy from Karolin Luger’s lab (Colorado State University) presented a crystallographic view of epigenetics with the elucidation of high resolution structures of domains from the macro-H2A histone. This variant is normally found enriched at the inactive X chromosome in female mammals. The analysis reveals that differences in residues constituting the L1-loop in histone H2A confers differential flexibility to these histones, which share otherwise superimposable structures, and this difference may be responsible for protein-protein interactions that determine the fate of gene expression. Domain swapping experiments demonstrated that these residues were sufficient to alter the localization of the major H2A to the inactive X, yet insufficient to disrupt the localization of macro-H2A. suggesting that the unique C terminus of the variant has other localization signals or interaction domains that recruit it to these loci. Further work will elucidate the interacting partners of the L1 loop and the non-histone region.

During this session, Thomas Jenuwein announced the formation of “The Epigenome” initiative to promote Epigenetics research in Europe with funding opportunities for young scientists interested in pursuing research in this field. He also promised an interactive and collegial atmosphere for researchers to exchange ideas and information as well as a website that would be updated with the most recent information in the fields covered in this Symposium.