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.

RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription.

We describe a procedure, RNAPol-ChIP, to measure actual transcriptional rate. It consists of the detection, by chromatin immunoprecipitation (ChIP), of RNA polymerase II within the coding region of genes. To do this, the DNA immunoprecipitated with polymerase antibodies is analysed by PCR, using an amplicon well within the coding region of the desired genes to avoid interferences with polymerase paused at the promoter. To validate RNAPol-ChIP, we compare our results to those obtained by classical methods in several genes induced during either liver regeneration or acute pancreatitis. When short half-life mRNA genes are studied (e.g. c-fos and egr1), RNAPol-ChIP gives results similar to those of other procedures. However, in genes whose mRNA is more stable (e.g. the hemopexin, hpx, gene) RNAPol-ChIP informs on real-time transcription with results comparable to those of methods such as nuclear run-on or run-off, which require the isolation of highly purified nuclei. Moreover, RNAPol-ChIP advantageously compares with methods based on the analysis of steady-state mRNA (northern blot or RT-PCR). Additional advantages of RNAPol-ChIP, such as the possibility of combining it with classical ChIP analysis to study transcription-associated changes in chromatin are discussed.