Nucleosome-specific, time-dependent changes in histone modifications during activation of the early growth response 1 (Egr1) gene.

Histone post-translational modifications and nucleosome remodeling are coordinate events involved in eukaryotic transcriptional regulation. There are relatively few data on the time course with which these events occur in individual nucleosomes. As a contribution to fill this gap, we first describe the nature and time course of structural changes in the nucleosomes -2, -1, and +1 of the murine Egr1 gene upon induction. To initiate the transient activation of the gene, we used the stimulation of MLP29 cells with phorbol esters and the in vivo activation after partial hepatectomy. In both models, nucleosomes -1 and +1 are partially evicted, whereas nucleosomes +1 and -2 slide downstream during transcription. The sliding of the latter nucleosome allows the EGR1 protein to bind its site, resulting in the repression of the gene. To decide whether EGR1 is involved in the sliding of nucleosome -2, Egr1 was knocked down. In the absence of detectable EGR1, the nucleosome still slides and remains downstream longer than in control cells, suggesting that the product of the gene may be rather involved in the returning of the nucleosome to the basal position. Moreover, the presence of eight epigenetic histone marks has been determined at a mononucleosomal level in that chromatin region. H3S10phK14ac, H3K4me3, H3K9me3, and H3K27me3 are characteristic of nucleosome +1, and H3K9ac and H4K16ac are mainly found in nucleosome -1, and H3K27ac predominates in nucleosomes -2 and -1. The temporal changes in these marks suggest distinct functions for some of them, although changes in H3K4me3 may result from histone turnover.

Factor binding and chromatin modification in the promoter of murine Egr1 gene upon induction.

The influence of chromatin on immediate-early gene expression has been studied in a model of Egr1 induction in intact mouse cells. ChIP analysis of factor and RNA polymerase binding reveals that the gene is constitutively poised for transcription in nonstimulated cells, but a repressing chromatin structure hampers productive transcription. Stimulation with phorbol esters results in a transient activation, which starts at 5 min and peaks at 30 min. Quantitative mapping of promoter occupancy by the different factors shows for the first time that no direct competition between SP1 and EGR1 occurs. The phosphorylation of ELK1 and CREB, which involves both the cascades of MEK1/2 and p38 kinases, is required for gene expression, which ceases following the binding of NAB1 and NAB2 to the promoter. The changes in histone acetylation and the differential recruitment of histone-modifying complexes further show the role of chromatin in the activation of this immediate-early gene.

Alterations in p53, BRCA1, ATM, PIK3CA, and HER2 genes and their effect in modifying clinicopathological characteristics and overall survival of Bulgarian patients with breast cancer.

PURPOSE: Though p53, BRCA1, ATM, PIK3CA, and HER2 genes are shown to be involved in various aspects of breast carcinogenesis, their functional relationship and clinical value are still disputable. We investigated the genetic status or expression profile of these genes to further elucidate their clinical significance. METHODS: PCR-SSCP-Sequencing of p53, BRCA1, ATM, and PIK3CA was performed in 145 Bulgarian patients with sporadic breast cancer. Expression profiles of HER2 were determined by ICH and CISH. Relationship between mutations and clinicopathological characteristics was evaluated by Chi-squared and Fisher's exact tests. Multivariate Cox proportional hazard test and Kaplan-Meier analysis were used to evaluate differences in overall survival between groups. RESULTS: The frequency of p53 (22.07%), BRCA1 (0.69%), ATM (7.59%), and PIK3CA (31.25%) alterations and HER2 (21.21%) overexpression was estimated. Mutated p53 was associated with tumor size (P = 0.033) and grade of malignancy (P = 0.001), ATM--with grade of malignancy (P = 0.032), and PIK3CA--with PR-positive tumors (P = 0.047). HER2 overexpression correlated with age of diagnosis (P = 0.009), tumor size (P = 0.0004), and ER expression (P = 0.011). Univariate survival analysis showed that mutated p53 is an indicator for worse outcome (P = 0.041). Combination of two genetic abnormalities did not correlate with more aggressive carcinogenesis and worse overall survival. CONCLUSIONS: Our data indicated that p53, BRCA1, ATM, PIK3CA, and HER2 alterations specifically correlate with clinicopathological characteristics of Bulgarian patients with breast cancer. Of these genes, only mutated p53 showed significant, though not independent, negative effect on overall survival.

Transcription of the MAT2A gene, coding for methionine adenosyltransferase, is up-regulated by E2F and Sp1 at a chromatin level during proliferation of liver cells.

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine, the main methyl donor. Two MAT-encoding genes (MAT1A, MAT2A) are found in mammals. The latter is expressed in proliferating liver, dedifferentiation and cancer, whereas MAT1A is expressed in adult quiescent hepatocytes. Here, we report studies on the molecular mechanisms controlling the induction of MAT2A in regenerating rat liver and in proliferating hepatocytes. The MAT2A is up-regulated at two discrete moments during liver regeneration, as confirmed by RNApol-ChIP analysis. The first one coincides with hepatocyte priming (i.e. G0-G1 transition), while the second one takes place at the G1-S interface. Electrophoretic mobility shift assays showed that a putative E2F sequence present in MAT2A promoter binds this factor and ChIP assays confirmed that E2F1, E2F3 and E2F4, as well as the pocket protein p130, are bound to the promoter in quiescent liver. MAT2A activation is accompanied by changes in the binding of histone-modifying enzymes to the promoter. Interestingly, p130 is not displaced from MAT2A promoter during hepatocyte priming, but it is in the late expression of the gene at the G1-S transition. Finally, the transcription factor Sp1 seems to play a decisive role in MAT2A induction, as it binds the promoter when the gene is being actively transcribed. In summary, the present work shows that the molecular mechanism of MAT2A expression is different during G0-G1 or G1-S transition and this may be related to the distinct requirements of S-adenosylmethionine during liver regeneration.

A short-range gradient of histone H3 acetylation and Tup1p redistribution at the promoter of the Saccharomyces cerevisiae SUC2 gene.

Chromatin immunoprecipitation assays are used to map H3 and H4 acetylation over the promoter nucleosomes and the coding region of the Saccharomyces cerevisiae SUC2 gene, under repressed and derepressed conditions, using wild type and mutant strains. In wild type cells, a high level of H3 acetylation at the distal end of the promoter drops sharply toward the proximal nucleosome that covers the TATA box, a gradient that become even steeper on derepression. In contrast, substantial H4 acetylation shows no such gradient and extends into the coding region. Overall levels of both H3 and H4 acetylation rise on derepression. Mutation of GCN5 or SNF2 lead to substantially reduced SUC2 expression; in gnc5 there is no reduction in basal H3 acetylation, but large reductions occur on derepression. SNF2 mutation has little effect on H3 acetylation, so SAGA and SWI/SNF recruitment seem to be independent events. H4 acetylation is little affected by either GCN5 or SNF2 mutation. In a double snf2/gcn5 mutant (very low SUC2 expression), H3 acetylation is at the minimal level, but H4 acetylation remains largely unaffected. Transcription is thus linked to H3 but not H4 acetylation. Chromatin immunoprecipitation assays show that Tup1p is evenly distributed over the four promoter nucleosomes in repressed wild type cells but redistributes upstream on derepression, a movement probably linked to its conversion from a repressor to an activator.