A comprehensive approach to the study of epigenetic regulation of gene expression during development by the C. elegans HP1 proteins – EpiDev

The balance between repressive and activating chromatin is maintained by the dynamic activity of specific proteins which mediate histone variant incorporation, histone modifications, nucleosome remodeling and DNA methylation. The heritability of the epigenetic profiles established by these proteins insures the maintenance of developmental and differentiation programs, and alterations in these profiles can result in profound and diverse effects on normal development. The study of the various chromatin remodeling factors in a developmental context is therefore of fundamental importance to understanding how epigenetic mechanisms influence metazoan development. - The aim of the proposed research project is to improve our understanding of epigenetic mechanisms of gene regulation in a developmental context using the nematode C. elegans. This nematode offers great potential for genetic analysis because of its rapid life cycle, small size, and ease of laboratory cultivation. The simple anatomy of C. elegans make it also an excellent model organism to study development at the systems level using functional genomics, and there are exceptionally good genomic resources available. The complete genome sequence of C. elegans has been determined, DNA microarrays and an RNAi library containing nearly every gene in the genome are readily available, and protein interactions have been studied on a large scale. - We have been studying the nematode homologues of the highly conserved HP1 (Heterochromatin Protein 1) family. HP1 proteins are key player in the dynamic organization of nuclear architecture, chromatin remodelling and transcriptional silencing, and directly contribute to a repressive chromatin structure by binding to histone H3 methylated on the lysine 9 residue (H3-K9Me). Evidence from mammals suggests than in additional to playing an essential role in heterochromatin formation, HP1 family proteins have a broader role in the epigenetic regulation of gene expression in euchromatic regions. The primordial importance of HP1 in the epigenetic regulation of gene expression is underlined by the fact that it is associated with diverse tumorigenic processes in mammals. Nonetheless, the function of these proteins in specific developmental pathways remains largely unknown. - In 2002, we identified for the first time two HP1 homologues in the C. elegans genome, which we named HPL-1 and HPL-2. We have shown that HPL-2 acts in a transcriptional repressor pathway which includes homologues of the human Rb complex. HPL-2 forms a complex with the LIN-13 Zinc finger protein, another member of the Rb related pathway which includes HPL-2. LIN-13 is required for the recruitment of HPL-2 to chromatin and we have identified potential targets for both genes. Given that both HPL-2 and LIN-13 contain Rb binding motifs, we believe that both proteins may act via Rb to bring about the repression of specific genes. Deletion of hpl-2 results in sterility and growth defects. In contrast, hpl-1 is dispensable for both germline and somatic development. However, HPL-1 and HPL-2 are redundantly required for post-embryonic development, as hpl-1;hpl-2 double mutants do not develop past the larval stage. Our data provides the first direct evidence for both redundant and unique functions of HP1 family proteins in metazoan development . In an RNAi based screen, we have isolated SET-2, the homolog of the human SET1 and mixed lineage leukemia (Mll) genes, as a suppressor of hpl-2 mutant phenotypes. Mammalian SET1/MLL proteins are found in a histone H3K4 specific methyltransferase complex and we have found that the nematode counterparts of other complex subunits also suppress hpl-2 mutant phenotypes. Furthermore, the H3K4 HMT activity of these proteins appears to be conserved in C. elegans. These results suggest that a C. elegans SET1/MLL complex may antagonize HPL-2 repressor function in development. - We plan to pursue the study of HPL-2 by using complementary approaches, i.