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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Dorsal functions as both an activator and repressor of transcription to determine dorsoventral fate in the Drosophila melanogaster embryo. Repression by Dorsal requires the corepressor Groucho (Gro) and is mediated by silencers termed ventral repression regions (VRRs). A VRR in zerknüllt (zen) contains Dorsal binding sites as well as an essential element termed AT2. We have identified and purified an AT2 DNA binding activity in embryos and shown it to consist of cut (ct) and dead ringer (dri) gene products. Studies of loss-of-function mutations in ct and dri demonstrate that both genes are required for the activity of the AT2 site. Dorsal and Dri both bind Gro, acting cooperatively to recruit it to the DNA. Thus, ventral repression may require the formation of a multiprotein complex at the VRR. This complex includes Dorsal, Gro, and additional DNA binding proteins, which appear to convert Dorsal from an activator to a repressor by enabling it to recruit Gro to the template. By showing how binding site context can dramatically alter transcription factor function, these findings help clarify the mechanisms responsible for the regulatory specificity of transcription factors.Establishment of the dorsoventral axis in a Drosophila melanogaster embryo depends upon the maternal morphogen Dorsal. This transcription factor is localized in a monotonic nuclear concentration gradient in early blastoderm embryos, with ventral nuclei containing the highest and dorsal nuclei containing the lowest concentrations of this protein (38,40,44). The dorsoventral fate map of the Drosophila embryo is dictated by the Dorsal nuclear concentration gradient, and mutations that disrupt the gradient also disrupt the fate map. Nuclear localization of Dorsal is dependent upon the activity of 12 maternal gene products, which transduce a signal originating in the ventral perivitelline space to the interior of the embryo, resulting in the release of Dorsal from its cytoplasmic inhibitor, Cactus (for reviews see references 2, 10, and 14). Once Dorsal is free from Cactus, it traverses the nuclear membrane and modifies the transcriptional program of the embryo, generating multiple distinct domains of gene activity along the dorsoventral axis. The ability of Dorsal to subdivide the embryo into multiple domains is critically dependent upon the ability of this factor to function as both an activator and a repressor of transcription. An understanding of what determines whether Dorsal will function as an activator or a repressor of any given target gene is therefore essential to an understanding of pattern formation in the Drosophila embryo.Dorsal functions as a regulator of a number of cellular determinant-encoding genes. For example, it activates the mesoderm-determining genes twist (twi) and snail (sna) and represses the dorsal ectoderm-determining genes zerknüllt (zen) and decapentaplegic (dpp) (37). Dorsal activates twi through ventral activation regions (VARs) upstream of the core promoter. The only elements within the VARs that are essential for ac...

Several biological processes in Trypanosoma brucei are affected by chromatin structure, including gene expression, cell cycle regulation, and life-cycle stage differentiation. In Saccharomyces cerevisiae and other organisms, chromatin structure is dependent upon posttranslational modifications of histones, which have been mapped in detail. The tails of the four core histones of T. brucei are highly diverged from those of mammals and yeasts, so sites of potential modification cannot be reliably inferred, and no cross-species antibodies are available to map the modifications. We therefore undertook an extensive survey to identify posttranslational modifications by Edman degradation and mass spectrometry. Edman analysis showed that the N-terminal alanine of H2A, H2B, and H4 could be monomethylated. We found that the histone H4 N-terminus is heavily modified, while, in contrast to other organisms, the histone H2A and H2B N-termini have relatively few modifications. Histone H3 appears to have a number of modifications at the N-terminus, but we were unable to assign many of these to a specific amino acid. Therefore, we focused our efforts on uncovering modification states of H4. We discuss the potential relevance of these modifications.

To start to understand the role of chromatin structure in regulating transcription in trypanosomes, we analyzed covalent modifications on the four core histones of Trypanosoma brucei. We found unusually few modifications in the N-terminal tails, which are abundantly modified in other organisms and whose sequences, but not composition, are highly divergent in trypanosomes. In contrast, the C-terminal region of H2A appears to be hyper-acetylated. Surprisingly, the N-terminal alanines of H2A, H2B, and H4, were mono-methylated, a modification that has not been described previously for histones. Possible functions and evolutionary explanations for these unusual histone modifications are discussed.