Histone N-terminal domains are frequent targets of posttranslational modifications. Multiple acetylated lysine residues have been identified in the N-terminal domain of H2B (K6, K11, K16, K17, K21, and K22), but little is known about how these modifications regulate transcription. We systematically mutated the N-terminal domain of histone H2B, both at known sites of lysine acetylation and elsewhere, and characterized the resulting changes in genome-wide expression in each mutant strain. Our results indicate that known sites of lysine acetylation in this domain are required for gene-specific transcriptional activation. However, the entire H2B N-terminal domain is principally required for the transcriptional repression of a large subset of the yeast genome. We find that the histone H2B repression (HBR) domain, comprised of residues 30 to 37, is necessary and sufficient for this repression. Many of the genes repressed by the HBR domain are located adjacent to telomeres or function in vitamin and carbohydrate metabolism. Deletion of the HBR domain also confers an increased sensitivity to DNA damage by UV irradiation. We mapped the critical residues in the HBR domain required for its repression function. Finally, comparisons of these data with previous studies reveal that a surprising number of genes are coregulated by the N-terminal domains of histone H2B, H3, and H4.In eukaryotic cells, DNA is packaged with histones and other proteins into chromatin (5). The principal packaging unit is the nucleosome, which consists of approximately 147 bp of DNA wrapped around a protein core of one histone H3-H4 tetramer and two histone H2A-H2B dimers (21). Because of their close association with DNA in the nucleosome, histones play integral roles in DNA-templated processes, such as DNA transcription, replication, and repair. Each of the four histone proteins is covalently modified at multiple residues, principally in their flexible N-terminal domains (9, 15). Histone modifications, such as lysine acetylation, lysine methylation, and serine phosphorylation, profoundly affect transcription initiation by regulating the association of transcriptional regulatory proteins with DNA (2, 10, 30).While modifications in the N-terminal domains of histone H3 and H4 have been extensively studied, relatively little is known about how N-terminal modifications in histone H2A and H2B regulate transcription. In Saccharomyces cerevisiae, histone H2B is acetylated at six lysine residues in its N-terminal domain (K6, K11, K16, K17, K21, and K22), most likely by the Gcn5 histone acetyltransferase (24, 28). H2B-K16 has been shown to be hypoacetylated in regions of subtelomeric heterochromatin due to the actions of the Hda1 histone deacetylase (23). It is unclear, however, whether there is a functional requirement for histone H2B hypoacetylation in subtelomeric heterochromatin. The histone H2B N-terminal domain does not appear to regulate telomeric silencing (29, 32).Deletion of amino acids 30 to 37 in the H2B N-terminal domain, which is lethal in some s...

Restriction of long-distance movement of tobacco etch virus (TEV) in Arabidopsis ecotype Col-0 plants requires the function of at least three genes: RTM1 (restricted TEV movement 1), RTM2, and RTM3. The mechanism of TEV movement restriction remains poorly understood, although it does not involve a hypersensitive response or systemic acquired resistance. A functional characterization of RTM1 and RTM2 was done. The RTM1 protein was found to be soluble with the potential to form self-interacting complexes. The regulatory regions of both the RTM1 and RTM2 genes were analyzed using reporter constructs. The regulatory sequences from both genes directed expression of ␤-glucuronidase exclusively in phloemassociated cells. Translational fusion proteins containing the green fluorescent protein and RTM1 or RTM2 localized to sieve elements when expressed from their native regulatory sequences. Thus, components of the RTM system may function within phloem, and sieve elements in particular, to restrict TEV long-distance movement.Virus infection of plants is a multiple-step process requiring compatible interactions between host-and virus-encoded factors during genome expression, cell-to-cell movement via plasmodesmata, and longdistance movement through the vascular system (Carrington et al., 1996). Restricted infection may result if cellular factors required by the virus are lacking or incompatible with the virus or if the host responds to the virus and activates a defense response.Arabidopsis ecotypes vary in their ability to support systemic infection by tobacco etch virus (TEV; Mahajan et al., 1998). Some ecotypes (e.g. C24 and Ler) allow long-distance movement of TEV from inoculated rosette leaves to noninoculated inflorescence tissue. Many ecotypes, such as Col-0, support replication and cell-to-cell movement of TEV in inoculated leaves but do not allow systemic movement of the virus. At least three Arabidopsis loci, RTM1 (restricted TEV movement 1), RTM2, and RTM3, are required for restriction of long-distance TEV movement in Col-0 (Mahajan et al., 1998; Whitham et al., 1999; S. Whitham, M. Yamamoto, and J.C. Carrington, unpublished data). Restriction mediated by the RTM system is specific to TEV and does not involve a hypersensitive response or induction of systemic acquired resistance (Mahajan et al., 1998; Whitham et al., 2000). The RTM1 and RTM2 genes were isolated by map-based cloning. The deduced RTM1 protein is similar to the Artocarpus integrifolia lectin, jacalin. Jacalin belongs to a family of related proteins, including at least ten Arabidopsis proteins, that contain one or more copies of a jacalin-like subunit, termed the jacalin repeat (JR; Chisholm et al., 2000). Several JR-containing proteins function in a jasmonate-inducible wound response, the result of which is production of antifungal and insecticidal compounds (Bones and Rossiter, 1996). A JR protein from Maclura pomifera has direct insecticidal activity (Murdock et al., 1990). Thus, proteins with JRs function in plant defense but by mechanisms tha...

No abstract

Histone N-terminal domains play critical roles in regulating chromatin structure and gene transcription. Relatively little is known, however, about the role of the histone H2A N-terminal domain in transcription regulation. We have used DNA microarrays to characterize the changes in genome-wide expression caused by mutations in the N-terminal domain of histone H2A. Our results indicate that the N-terminal domain of histone H2A functions primarily to repress the transcription of a large subset of the Saccharomyces cerevisiae genome and that most of the H2A-repressed genes are also repressed by the histone H2B N-terminal domain. Using the histone H2A microarray data, we selected three reporter genes (BNA1, BNA2, and GCY1), which we subsequently used to map regions in the H2A N-terminal domain responsible for this transcriptional repression. These studies revealed that a small subdomain in the H2A N-terminal tail, comprised of residues 16 to 20, is required for the transcriptional repression of these reporter genes. Deletion of either the entire histone H2A N-terminal domain or just this small subdomain imparts sensitivity to UV irradiation. Finally, we show that two residues in this H2A subdomain, serine-17 and arginine-18, are specifically required for the transcriptional repression of the BNA2 reporter gene.The packaging of DNA into chromatin has profound consequences on the regulation of gene transcription. The principal packaging unit of chromatin is the nucleosome core particle, consisting of two copies each of histones H2A, H2B, H3, and H4 and 147 bp of DNA (16). The arrangement and organization of genomic DNA into nucleosomes are not random processes, but instead appear to play an important regulatory function in transcription (14). Regions of genomic DNA are also distinguished by distinct patterns of posttranslational modifications that decorate the associated histone proteins (11). A large body of scientific literature has shown that histone posttranslational modifications are important regulators of transcription (2).The N-terminal domains of histone proteins play critical roles in both the organization and posttranslational modification of chromatin. Previous studies have shown that the histone N-terminal domains function to control the translational positioning of nucleosomes in vitro (24). Histone N-terminal domains can mediate internucleosome interactions that are required for the formation of higher-order chromatin structures (4, 25). Histone Nterminal domains are also primary sites of posttranslational modifications. These modifications, such as lysine acetylation, lysine and arginine methylation, and serine phosphorylation, can alter the stability of the nucleosome and regulate the association of transcriptional regulatory proteins (2, 6, 21).In Saccharomyces cerevisiae, a single histone H2A N-terminal modification has been verified, i.e., acetylation of H2A K7 (20,22). An additional acetylation site at H2A K4 is suspected (3, 18); however, this modification has yet to be verified in vivo.Both H2A ...

Recent technological advancements have allowed for highly-sophisticated mass spectrometry-based studies of the histone code, which predicts that combinations of post-translational modifications (PTMs) on histone proteins result in defined biological outcomes mediated by effector proteins that recognize such marks. While significant progress has been made in the identification and characterization of histone PTMs, a full appreciation of the complexity of the histone code will require a complete understanding of all the modifications that putatively contribute to it. Here, using the top-down mass spectrometry approach for identifying PTMs on full-length histones, we report that lysine 37 of histone H2B is dimethylated in the budding yeast Saccharomyces cerevisiae. By generating a modification-specific antibody and yeast strains that harbor mutations in the putative site of methylation, we provide evidence that this mark exist in vivo. Importantly, we show that this lysine residue is highly conserved through evolution, and provide evidence that this methylation event also occurs in higher eukaryotes. By identifying a novel site of histone methylation, this study adds to our overall understanding of the complex number of histone modifications that contribute to chromatin function.