Genome-Wide Chromatin Remodeling Identified at GC-Rich Long Nucleosome-Free Regions

To gain deeper insights into principles of cell biology, it is essential to understand how cells reorganize their genomes by chromatin remodeling. We analyzed chromatin remodeling on next generation sequencing data from resting and activated T cells to determine a whole-genome chromatin remodeling landscape. We consider chromatin remodeling in terms of nucleosome repositioning which can be observed most robustly in long nucleosome-free regions (LNFRs) that are occupied by nucleosomes in another cell state. We found that LNFR sequences are either AT-rich or GC-rich, where nucleosome repositioning was observed much more prominently in GC-rich LNFRs — a considerable proportion of them outside promoter regions. Using support vector machines with string kernels, we identified a GC-rich DNA sequence pattern indicating loci of nucleosome repositioning in resting T cells. This pattern appears to be also typical for CpG islands. We found out that nucleosome repositioning in GC-rich LNFRs is indeed associated with CpG islands and with binding sites of the CpG-island-binding ZF-CXXC proteins KDM2A and CFP1. That this association occurs prominently inside and also prominently outside of promoter regions hints at a mechanism governing nucleosome repositioning that acts on a whole-genome scale.     

Signals from CD28 induce stable epigenetic modification of the IL-2 promoter

CD28 costimulation controls multiple aspects of T cell function, including the expression of proinflammatory cytokine genes. One of these genes encodes IL-2, a growth factor that influences T cell proliferation, survival, and differentiation. Antigenic signaling in the absence of CD28 costimulation leads to anergy, a mechanism of tolerance that renders CD4+ T cells unable to produce IL-2. The molecular mechanisms by which CD28 costimulatory signals induce gene expression are not fully understood. In eukaryotic cells, the expression of many genes is influenced by their physical structure at the level of DNA methylation and local chromatin remodeling. To address whether these epigenetic mechanisms are operative during CD28-dependent gene expression in CD4+ T cells, we compared cytosine methylation and chromatin structure at the IL-2 locus in fully activated CD4+ effector T cells and CD4+ T cells rendered anergic by TCR ligation in the absence of CD28 costimulation. Costimulation through CD28 led to marked, stable histone acetylation and loss of cytosine methylation at the IL-2 promoter/enhancer. This was accompanied by extensive remodeling of the chromatin in this region to a structure highly accessible to DNA binding proteins. Conversely, TCR activation in the absence of CD28 costimulation was not sufficient to promote histone acetylation or cytosine demethylation, and the IL-2 promoter/enhancer in anergic cells remained completely inaccessible. These data suggest that CD28 may function through epigenetic mechanisms to promote CD4+ T cell responses.     

Insulation of the chicken beta-globin chromosomal domain from a chromatin-condensing protein,MENT

Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken beta-globin domain. We observed two sharp transitions of MENT concentration coinciding with the beta-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation.     

Localization of the gene encoding R kappa B (NFRKB), a tissue-specific DNA binding protein, to chromosome 11q24-q25.

Although NF (nuclear factor)-kappa B binds in vitro to several of the kappa B regulatory elements found in cellular and viral genes, another DNA binding protein, R kappa B, also binds to a related variant of the kappa B site that regulates interleukin-2 receptor alpha-chain gene expression, a critical event in T cell activation. Southern blot analysis of a human-mouse somatic cell hybrid panel and in situ hybridization using a fluorescent genomic R kappa B probe have allowed assignment of the R kappa B gene (NFRKB) to 11q24-q25. The NFRKB locus is in close proximity to the chromosomal breakpoint implicated in Ewing sarcoma, but it does not appear to span this region. Nonetheless, NFRKB may be particularly useful as the most telomeric marker thus far assigned to 11q.     

染色质改构因子BRG1降低UV照射引起的细胞凋亡

生命体的遗传物质基础是DNA分子,多种因素可以作用于细胞内的DNA分子,导致多种类型的DNA损伤。若受损的DNA得不到及时和有效的修复,细胞将走向凋亡或发生变异。染色质改构复合物(chromatin remodeling complex)在基因表达调控和DNA复制等方面扮演着重要角色。依赖ATP的染色质改构复合物SWI/SNF的核心亚基Brahma Related Gene1(BRG1)在染色质结构调整和基因转录调控等多个细胞进程中具有重要作用,仅有有限的文献报道BRG1参与到DNA的损伤修复过程。因此,进一步研究与验证BRG1在调控DNA的损伤修复进而挽救细胞凋亡中的作用十分重要。本文通过利用不同强度的UV照射检测细胞凋亡的情况,初步建立了DNA损伤修复的实验体系。将BRG1表达质粒瞬时转染到SW13(BRG1-/-)细胞系中,并利用30J/m2的UV照射,分别在0h、6h和24h检测细胞早期凋亡程度。结果表明,SW13(BRG1-/-)细胞中瞬时表达BRG1可以明显降低由UV照射引起的细胞凋亡,其中UV照射后24h的细胞表现最明显。我们进一步在HeLa细胞中通过瞬时表达BRG1验证了上述结果。由于BRG1通过染色质改构在基因的转录调控、复制和重组等方面起着重要的作用,我们推测BRG1可能通过染色质改构参与了DNA的损伤修复过程,进而影响了细胞凋亡。