Chromatin and epigenetic memory

染色质和表观遗传记忆

• 批准号: 8941510
• 负责人: Keiko Ozato
• 金额: $ 81.85万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8941510
• 关键词: Affect; Binding; Binding Proteins; Bromodomain; Cell Cycle Progression; Cell division; Chromatin; Chromosomes; Complex; Coupled; DNA; DNA biosynthesis; Data; Elongation Factor; Environment; Epigenetic Process; Event; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Goals; Histone Acetylation; Histone H3; Histone H3.3; Histones; Inflammatory; Interferon Type I; Interferons; Memory; Messenger RNA; Mitosis; Molecular Chaperones; Mus; Natural Immunity; Natural Resistance; Nuclear Protein; Nucleosomes; Pathway interactions; Physiological; Play; Positive Transcriptional Elongation Factor B; Process; Property; Recruitment Activity; Regulation; Reporting; Role; Shapes; Stimulus; Testing; Transcription Initiation Site; Transcriptional Activation; Variant; Virus; base; cell growth; cell type; daughter cell; factor EF-P; inhibitor/antagonist; interest; macrophage; negative elongation factor; pathogen; small hairpin RNA; small molecule

BRD4 regulates genes involved in cell cycle progression from G0, G1 through S, thus impacting on cell growth. Recently, it has been shown that BRD4 is required for induction of inflammatory genes in macrophages, suggesting its role in innate immunity. We asked whether BRD4 plays a role in transcription of interferon (IFN) stimulated genes (ISGs). Type I IFN activates a large number of genes through the JAK/STAT pathway and establishes natural resistance against viruses and other pathogens. We found that IFN stimulation triggers recruitment of BRD4 to the transcription start site (TSS) of a number of ISGs. Recruitment of BRD4 to ISGs coincided with increased histone acetylation at the TSS and induction of ISG mRNAs. Further, BRD4 recruitment coincided with the binding of the elongation factor, P-TEFb, which is known to interact with BRD4. We also show that the pausing complex, NELF and DSIF are recruited to the ISGs after IFN stimulation. IFN-induced NELF/DSIF recruitment was unexpected, since the pausing complex was reported to be present prior to stimulation and dissociate after activation as studed for some inducible genes. Furthermore, another elongation factor, SPT6 was recruited to ISGs after IFN stimulation, a factor known to function as a histone H3 chaperon. These results gave a picture where a number of elongation factors are induced to assemble on the ISGs. To delineate a hierarchical order of the assembly of these factors, we tested the effect of small molecule inhibitor that inhibits binding of acetylated histones to bromodomains. Results showed that this inhibitor not only inhibits BRD4 recruitment, but recruitment of P-TEFb, NELF/DSIF and SPT6. In addition, the inhibitor markedly reduced ISG transcription. Similarly shRNA-based BRD4 knockdown led to reduced recruitment all of above factors. These data support a view that BRD4 recruitment is the primary event that initiates a cascade of factor binding that shapes overall ISG transcription. Additional knockdown studies showed that NELF and SPT6 have opposing activities in elongation, the former repressing ISG transcription, while the latter promoting transcription. Together, our results indicate that BRD4 plays a central role in SG transcription for its ability to sequentially recruit various elongation factors. These studies are extended to analysis of Brd4f/f mice expressing cell type specific Cre.

BRD4调节来自G0，G1至S的细胞周期进程的基因，从而影响细胞生长。最近，已经表明，BRD4是诱导巨噬细胞中炎症基因所必需的，这表明其在先天免疫中的作用。我们询问BRD4是否在干扰素（IFN）刺激基因（ISGS）的转录中起作用。 I型IFN通过JAK/STAT途径激活了大量基因，并建立了对病毒和其他病原体的天然抗性。 我们发现，IFN刺激会触发BRD4募集到许多ISG的转录起始位点（TSS）。 BRD4对ISG的募集与TSS时组蛋白乙酰化的增加并诱导ISG mRNA。 此外，BRD4募集与伸长因子P-TEFB的结合相吻合，P-TEFB已知与BRD4相互作用。 我们还表明，在IFN刺激后，将暂停的复合物，NELF和DSIF募集到ISGS。 IFN诱导的NELF/DSIF募集是出乎意料的，因为据报道暂停复合物在刺激前存在，并在激活后作为某些诱导基因的激活而解离。此外，在IFN刺激后，将SPT6的另一个伸长因子募集到ISG，这是一种被称为组蛋白H3伴侣的因子。这些结果给出了一个图片，其中诱导许多伸长因子在ISG上组装。 为了描绘这些因素组装的分层顺序，我们测试了小分子抑制剂的作用，该抑制剂抑制了乙酰化组蛋白与溴结构域的结合。结果表明，该抑制剂不仅抑制了BRD4募集，还抑制了P-TEFB，NELF/DSIF和SPT6的募集。另外，抑制剂显着降低了ISG转录。同样，基于SHRNA的BRD4敲低导致募集减少了上述所有因素。这些数据支持一种观点，即BRD4募集是启动一系列因素结合的主要事件，该因素结合构成了整体ISG转录。额外的敲低研究表明，NELF和SPT6在伸长率中具有相反的活动，前者是抑制ISG转录，而后者则促进转录。总之，我们的结果表明，BRD4在SG转录中起着核心作用，其能够依次募集各种伸长因子。这些研究扩展到表达细胞类型特异性CRE的BRD4F/F小鼠的分析。