The Drosophila gene groucho (gro) encodes a transcriptional corepressor that has critical roles in many development processes. In an effort to illuminate the mechanism of Gro-mediated repression, we have employed Gro as an affinity reagent to purify Gro-binding proteins from embryonic nuclear extracts. One of these proteins was found to be the histone deacetylase Rpd3. Protein-protein interaction assays suggest that Gro and Rpd3 form a complex in vivo and that they interact directly via the glycine/proline rich (GP) domain in Gro. Cell culture assays demonstrate that Rpd3 potentiates repression by the GP domain. Furthermore, experiments employing a histone deacetylase inhibitor, as well as a catalytically inactive form of Rpd3, imply that histone deacetylase activity is required for efficient Gro-mediated repression. Finally, mutations in gro and rpd3 have synergistic effects on embryonic lethality and pattern formation. These findings support the view that Gro mediates repression, at least in part, by the direct recruitment of the histone deacetylase Rpd3 to the template, where it can modulate local chromatin structure. They also provide evidence for a specific role of Rpd3 in early development.

SUMMARY The H3K4me3 mark in chromatin is closely correlated with actively transcribed genes, although the mechanisms involved in its generation and function are not fully understood. In vitro studies with recombinant chromatin and purified human factors demonstrate a robust SET1 complex (SET1C)-mediated H3K4 trimethylation that is dependent upon p53- and p300-mediated H3 acetylation, a corresponding SET1C-mediated enhancement of p53- and p300-dependent transcription that reflects a primary effect of SET1C through H3K4 trimethylation, and direct SET1C-p53 and SET1C-p300 interactions indicative of a targeted recruitment mechanism. Complementary cell-based assays demonstrate a DNA-damage-induced p53-SET1C interaction, a corresponding enrichment of SET1C and H3K4me3 on a p53 target gene (p21/WAF1), and a corresponding codependency of H3K4 trimethylation and transcription upon p300 and SET1C. These results establish a mechanism in which SET1C and p300 act cooperatively, through direct interactions and coupled histone modifications, to facilitate the function of p53.

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As the resource laboratory for Rockefeller University our emphasis continues to be on methodology development for the routine analysis of low abundance proteins isolated from native sources. In the past ten years, gel electrophoresis of proteins has become the method of choice for the preparation of microgram and submicrogram quantities of protein for primary structural characterization, and over 95% of the samples submitted for protein identification are either in a gel or bound to polyvinyl difluoride membranes (PVDF). As such, we employ multiple microanalytical approaches encompassing Edman sequence degradation, amino acid and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometric analysis to provide an integrated protein characterization of such samples. Here we describe the two major services we employ when providing protein identification from in-gel or PVDF-bound proteins.

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