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Type 1 diabetes is an autoimmune disease in which insulin-producing pancreatic isletβcells are the target of self-reactive B and T cells. T cells reactive with epitopes derived from insulin and/or IGRP are critical for the initiation and maintenance of disease, but T cells reactive with other islet antigens likely have an essential role in disease progression. We sought to identify candidate CD8+T cell epitopes that are pathogenic in type 1 diabetes. Proteins that elicit autoantibodies in human type 1 diabetes were analyzed by predictive algorithms for candidate epitopes. Using several different tolerizing regimes using synthetic peptides, two new predicted tolerogenic CD8+T cell epitopes were identified in the murine homolog of the major human islet autoantigen zinc transporter ZnT8 (aa 158–166 and 282–290) and one in a non-βcell protein, dopamineβ-hydroxylase (aa 233–241). Tolerizing vaccination of NOD mice with a cDNA plasmid expressing full-length proinsulin prevented diabetes, whereas plasmids encoding ZnT8 and DβH did not. However, tolerizing vaccination of NOD mice with the proinsulin plasmid in combination with plasmids expressing ZnT8 and DβH decreased insulitis and enhanced prevention of disease compared to vaccination with the plasmid encoding proinsulin alone.

The histone H3 amino terminus, but not that of H4, is required to prevent the constitutively bound activator Cha4 from remodeling chromatin and activating transcription at the CHA1 gene in Saccharomyces cerevisiae . Here we show that neither the modifiable lysine residues nor any specific region of the H3 tail is required for repression of CHA1 . We then screened for histone H3 mutations that cause derepression of the uninduced CHA1 promoter and identified six mutants, three of which are also temperature-sensitive mutants and four of which exhibit a sin − phenotype. Histone mutant levels were similar to that of wild-type H3, and the mutations did not cause gross alterations in nucleosome structure. One specific and strongly derepressing mutation, H3 A111G, was examined in depth and found to cause a constitutively active chromatin configuration at the uninduced CHA1 promoter as well as at the ADH2 promoter. Transcriptional derepression and altered chromatin structure of the CHA1 promoter depend on the activator Cha4. These results indicate that modest perturbations in distinct regions of the nucleosome can substantially affect the repressive function of chromatin, allowing activation in the absence of a normal inducing signal (at CHA1 ) or of Swi/Snf (resulting in a sin − phenotype).

Histone amino termini are post-translationally modified by both transcriptional coactivators and corepressors, but the extent to which the relevant histone modifications contribute to gene expression, and the mechanisms by which they do so, are incompletely understood. To address this issue, we have examined the contributions of the histone H3 and H4 amino termini, and of the coactivator and histone acetyltransferase Gcn5p, to activation of a small group of Gcn4p-activated genes. The histone H3 tail exerts a modest (about 2-fold) but significant effect on activation that correlates with a requirement for Gcn5p and is distributed over multiple lysine residues. The H4 tail also plays a positive role in activation of some of those genes tested, but this does not correlate as closely with Gcn5p coactivation. Microarray experiments did not reveal a close correspondence between those genes activated by Gcn4p and genes requiring the H3 or H4 tail, and analysis of published microarray data indicates that Gcn4p-regulated genes are not in general strongly dependent on Gcn5p. However, a large fraction of genes activated by Gcn4p were found to be repressed by the H3 and H4 amino termini under non-inducing conditions, indicating that one role for Gcn4p is to overcome repression mediated by the histone tails.

DNA in eukaryotes is packaged into chromatin by the histone proteins. The four core histones, H2A, H2B, H3, and H4, have amino-terminal “tails” that extend from the structured interior and that are subject to numerous posttranslational modifications (7, 54). Acetylation and deacetylation of the histone H3 and H4 amino termini are particularly important for gene regulation, but basic questions about the mechanism by which they affect transcription remain unanswered. For instance, a variety of enzymes can acetylate or deacetylate the H3 and/or H4 tails with different specificities, but it is not known whether modifications of specific lysine residues vary in importance at particular promoters or whether acetylation activates transcription by removing a repressive influence (unacetylated histone tails) or by providing a required positive stimulus (e.g., providing a binding surface for general transcription factors) (1). Furthermore, histone-modifying enzymes may also posttranslationally target other, nonhistone targets, complicating analysis of their functions (6, 22, 43). In an effort to understand how histone modifications affect gene regulation, we have focused in this study on histone deacetylase Rpd3p from the budding yeast Saccharomyces cerevisiae. This protein was first identified as a histone deacetylase in a screen for rat proteins binding to trapoxin, a histone deacetylase inhibitor; sequence analysis then showed it to be a homolog of the known yeast corepressor Rpd3p (46). Subsequent work demonstrated that the histone deacetylase activity of Rpd3p is needed for it to function as a corepressor and that promoter-bound histones show decreased acetylation upon recruitment of Rpd3p to nearby sites (18, 20, 35). The loss of Rpd3p results in a global increase in the acetylation of lysine residues 5, 8, 12, and 16 of histone H4 and lysine residues 9 or 18 and 14 of histone H3 (32, 34, 50), whereas promoter-bound nucleosomes near sites of Rpd3p recruitment have been reported in various studies to show the deacetylation of principally K5 of histone H4 (35); K5 and K12 of H4 and undetermined sites of histone H3 (20); and K5, K8, and K12 of H4, K9, K14, K18, K23, and K27 of H3, K27 of histone H2A and, to somewhat lesser extent, K11 and K16 of histone H2B (45). Rpd3p affects the regulation of a substantial number of genes in S. cerevisiae (3). It can be recruited to gene promoters by the repressor Ume6p but evidently by other mechanisms as well (19, 23, 32), and it is also responsible for a lower level of untargeted histone deacetylation throughout the genome in S. cerevisiae (50). Chromatin immunoprecipitation experiments have shown that repression by Rpd3p is associated with reduced levels of promoter-bound TATA binding protein, SWI/SNF, and SAGA components, and it has been suggested that reduced acetylation of histone amino termini could weaken the interactions of components of SWI/SNF and/or SAGA with promoter-bound nucleosomes, resulting in decreased levels of transcription (9, 41). However, other histone-modifying enzymes have been shown to target nonhistone proteins with functional consequences (22, 43), and it is possible that Rpd3p does so as well. A prediction that has not yet been tested is that if the histone amino termini are the principal functionally important targets of Rpd3p, then the deletion of Rpd3p should have a reduced (or no) effect in their absence. A similar approach was used previously to test the importance of the histone H3 and H4 amino termini for regulation by Gcn5p in S. cerevisiae (53). Here we report experiments testing this prediction at the gene-specific and genome-wide levels. We also compare, by microarray analysis, the roles of the H3 and H4 amino termini in gene regulation at the genome-wide level and examine the extent to which the loss of the H3 and H4 tails and the mutation of lysines to glutamines results in overlapping phenotypes with respect to transcriptional regulation. Our results provide a global view of the roles of the H3 and H4 amino termini in gene regulation in S. cerevisiae and their relationship to regulation by Rpd3p.

DNA in eukaryotes is packaged into chromatin by association with the four histone proteins, H2A, H2B, H3, and H4, to form nucleosomes. Transcription factors must recognize particular DNA sequences to activate genes and so must compete with the histones for occupancy of their cognate binding sites. Beyond this repressive role, the histones have also adopted regulatory roles, both positive and negative, which are largely mediated by amino terminal “tails” that do not contribute to the basic structure of the nucleosome. Here, we describe some of our recent progress in understanding how transcription factors compete with histones for binding site occupancy and also discuss recent results on the global role of the histone H3 amino terminus in regulating gene expression in the budding yeast Saccharomyces cerevisiae.