Stimulating Conversations between HP1a and Histone Demethylase dKDM4A

In eukaryotic cells, transcription involves highly regulated interactions between RNA polymerase, transcription factors, and the DNA template. Access to DNA is largely governed by chromatin packaging. The bulk of the genome is packaged into nucleosomes, which are generally repressive for transcription. Accessibility for transcriptional activation can be achieved by nucleosome loss or remodeling, using histone variants and/or specific post-translational modifications. Histone modifications are spatially partitioned into domains within the genome and along genes. For example, histone H3 lysine 9 di- and trimethylation (H3K9me2 and H3K9me3) is enriched near centric regions of chromosomes. These epigenetic marks serve as a docking site for Hetero-chromatin Protein 1 (HP1, aka HP1a), a conserved protein initially discovered in Drosophila and named for its predominant localization to centric heterochromatin (James and Elgin, 1986). HP1a, when resident near centromeres, plays a role in the gene silencing associated with heterochromatin packaging; indeed, HP1a is sufficient to nucleate repressive chromatin when targeted to noncentric sites within the genome. The mechanism of silencing involves altered nucleosome packaging and the occlusion of transcription factors from their binding sites (Danzer and Wallrath, 2004). However, there are several cases in which Drosophila HP1a is found associated with active genes. In particular, HP1a has been shown to be required for full expression of genes that naturally reside within heterochromatin (Yasuhara and Wakimoto, 2006), suggesting that such genes require this specific chromatin architecture for optimal expression. Perhaps more surprising, HP1a is found in association with some active euchromatic genes, studied in detail in cytological region 31 (Cryderman et al., 2005). In addition, HP1a is observed at heat shock and developmentally regulated puffs in polytene chromosomes (Piacentini et al., 2003). Expression of these genes decreases in larvae deficient for HP1a. These puzzling cases of HP1a localization and positive function in gene activity may be explained by results reported by Jerry Workman and colleagues in this issue of Molecular Cell (Lin et al., 2008). Here Lin et al. have shown that the D. melanogaster JmjC domain-containing protein dKDM4A is a histone H3K36 demethylase; this has also been reported by Lloret-Linares et al. (2008). The proteomic approach used by Lin et al. has revealed that HP1a is an interacting partner of dKDM4A. This interaction is specific to HP1a, as family members HP1b and HP1c do not interact with dKDM4A. A direct interaction between dKDM4A and HP1a is supported by immunoprecipitation and mutagenesis experiments with tagged proteins. HP1a and dKDM4A appear to associate through the PxVxL binding motif generated by dimerization of the HP1a chromo shadow domain (CSD), as the interaction is lost with single amino acid substitutions in the HP1a CSD that disrupt dimerization (I191E) or PxVxL partner interactions (W200A). Binding is also lost upon alanine substitution for the central valine in a PVVKL peptide of dKDM4a. While structural studies have not yet been undertaken, direct binding through the PxVxL platform is supported by the impact of these reciprocal mutations. Lin et al. further show that HP1a binding stimulates demethylation of H3K36me2 and H3K36me3 by dKDM4a. Oddly enough, this activity is lost with an amino acid substitution (V26M) in the HP1a chromodomain (CD), the key site for H3K9me2 binding. This suggests that either the CD is used here in a critical way, perhaps for a recognition event, or that perturbation of the CD structure prevents function generally. Loss of HP1a leads to increased levels of H3K36me3. Thus HP1a appears to play a critical role in regulation of this modification (Lin et al., 2008). Histone H3 methylation has proven to be one of the consistent marks distinguishing alternative chromatin packaging states: methylation at H3K4, H3K36, and H3K79 is associated with active transcription, while methylation at H3K9 and H3K27 is associated with gene silencing. Methylation of H3K36 is present along the coding regions of genes, as opposed to H3K4me3, which is enriched just downstream of the transcription start site. In S. cerevisiae, H3K36 methylation is carried out by Set2, which associates with elongating RNA Pol II (Li et al., 2003) (Figure 1). The homolog in Drosophila, dSet2, catalyzes H3K36me3, while dMes-4 catalyzes H3K36me2 (Bell et al., 2007). Methylated H3K36 is recognized by the histone deacetylase Rpd3S. Loss of acetylation is repressive and helps to prevent cryptic transcription initiation throughout the body of the gene, behind the passing RNA Pol II (Carrozza et al., 2005). By catalyzing H3K36 demethylation, dKDM4A might inhibit recruitment of deacetylases and consequently promote transcription. In this model, HP1a would play a positive role in transcription by stimulating the demethylase activity. The model suggests that HP1a-stimulated H3K36 demethylation might be an important step in returning the chromatin to its ground state for subsequent refiring, particularly at genes being transcribed at a high rate. The ability of the demethylase to redirect deacetylases might be necessary for acetyltransferases to prime the gene for subsequent rounds of transcription. Further investigation of the balance between histone acetylation and the methylation status of H3K36 should provide insights. Figure 1 Model for dKDM4A Re-establishing the “Ground State” of Chromatin Following Transcription This model is complicated by the many interactions known to occur between HP1a and other chromosomal proteins; how HP1a selects partners is a critical question about which little is known. Tests of this model might include determining whether the transcription stimulation by HP1a reported in cases described above is lost or reduced in the presence of mutations in dKDM4A. Another unanswered question concerns the mechanisms of targeting HP1a to different chromosomal sites. HP1a localization at centric regions is significantly dependent on an interaction with H3K9me (Schotta et al., 2002), most likely enhanced by additional factors. In contrast, HP1a localization at euchromatic sites is not H3K9me dependent. Association of HP1a at puffs is sensitive to RNase treatment, whereas binding to other chromosomal sites is insensitive (Piacentini et al., 2003). Clearly HP1a must use multiple mechanisms for localization, which are likely to be dictated by partner selection. Further studies directed at determining the genomic distribution of dKDM4A in relation to HP1a and H3K9me, and of genetic interactions among the histone modification machinery mutants, are needed. This new and unanticipated partnership between HP1a and dKDM4A both provides an explanation for prior observations and stimulates new questions about the relationships among the epigenetic marks that rule transcription.