In vivo function of BRD4

BRD4的体内功能

• 批准号: 10702619
• 负责人: Dinah S. Singer
• 金额: $ 19.56万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/10702619
• 关键词: Acetylation; Acetyltransferase; Affect; Binding; Binding Proteins; Biological Assay; Blood Cells; Bone Marrow Cells; CD8B1 gene; CRISPR/Cas technology; Cell Cycle; Cell Line; Cell Proliferation; Cell physiology; Cell surface; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Coculture Techniques; DNA; DNA Polymerase II; DNA Repair; Defect; Dependence; Development; Developmental Process; Disease; Embryo; Embryonic Development; Enhancers; Epithelial; Frequencies; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Hematologic Neoplasms; Hematopoietic stem cells; Histones; Human; Immune; Immune system; Inflammation; Inflammatory; Intestines; Knockout Mice; Link; Lysine; Maintenance; Mesenchymal; Metabolic; Metabolic Pathway; Metabolism; Mitotic; Modeling; Monitor; Mus; NF-kappa B; Nuclear; Nucleosomes; Organ; Pathway interactions; Pattern; Peripheral; Phenotype; Phosphorylation; Phosphotransferases; Play; Population; Positioning Attribute; Process; Proliferating; Proteins; RNA Sequence Analysis; Regulation; Role; Signal Transduction; Solid; Surface; T-Cell Receptor; T-Lymphocyte; TAF7 gene; Tail; Thymocyte Development; Thymus Gland; Tissues; Transcription Elongation; Transcriptional Regulation; Transferase; Transgenic Mice; aurora B kinase; base; c-myc Genes; cdc Genes; cell type; chromatin remodeling; differential expression; epigenetic regulation; falls; genome-wide; hematopoietic differentiation; in vivo; mutant; promoter; reconstitution; recruit; scaffold; skin hyperplasia; stem cells; therapeutic target; thymocyte; transcription factor; vector

BRD4 is being widely investigated as a therapeutic target in inflammatory diseases, as well as a variety of solid and hematological cancers (). It is a chromatin-binding protein with both kinase and acetyl transferase activities that links chromatin structure and transcription and plays an active role in regulating early embryonic development. BRD4 contributes to gene expression in multiple ways, as a scaffold that delivers transcription factors and nucleates superenhancers, as a chromatin remodeler, as a transcription factor that phosphorylates the Pol II CTD. Because deletion of BRD4 is embryonic lethal, the role of BRD4 in normal differentiation has not been extensively investigated in vivo. Using conditional BRD4 knock-out mice, we have shown that consistent with its role as a chromatin remodeler, BRD4-/- thymocytes have significantly reduced levels of acetylated histones necessary for chromatin decompaction and transcription, suggesting the BRD4 plays an active role in thymocyte development. An examination of the in vivo requirement for BRD4 during thymocyte development showed that the ISP stage, which represents a discrete subpopulation with a gene expression profile distinct from either the DN precursors or the DP successors, is selectively inhibited by BRD4 deletion, although BRD4 is expressed throughout all stages of thymocyte differentiation. The maturation of thymocytes is accompanied by large changes in gene expression profiles which are reflected in the patterns of cell surface markers and cell cycle genes. Whereas the DN thymocytes express neither CD4 nor CD8 and are highly proliferative, the ISPs express CD8 and undergo only a single round of proliferation. Accordingly, the transition from DN to ISP is accompanied by large changes in both immune and cell cycle pathways. Interestingly, despite the differences in proliferation, both cell populations express c-Myc at approximately equal levels. Thus, Myc expression alone does not determine the extent or frequency of proliferation. Although the maturation from ISP to DP is accomplished during a single round of cell cycle, it is accompanied by the differential expression of a large number of genes in immune and metabolic pathways. Unlike either the DN or ISP populations, the DP thymocytes do not express Myc, consistent with their quiescent phenotype. Of particular note, the ISP expression profile is distinct from both the DN and the DP. Thus, it is not a transitional population, midway between the DN and DP. Rather, the ISPs are a distinct thymocyte subpopulation. The ISP subpopulation is also distinct in its dependence on BRD4. Although BRD4 is expressed throughout thymocyte differentiation - and at equivalent levels - BRD4 depletion selectively affects the ISP subpopulation. In the absence of BRD4, DN thymocytes differentiate relatively normally, although BRD4-/- DN4 thymocytes are smaller than WT and accumulate in somewhat larger numbers. However, BRD4-/- DN4 thymocytes proliferate and undergo TcRbeta rearrangement normally, reflecting the finding that the expression of only 100 genes is affected. Among the pathways affected are those involved in inflammation and jak-stat signaling. Similarly, BRD4 plays a relatively small role in DP or SP thymocytes which are phenotypically normally and regulates expression of only between 300-400 genes in each cell type which fall into immune system and metabolic processes. In sharp contrast to the modest effects of BRD4 deletion in the DN, DP and SP thymocytes, the ISP are dramatically affected. The expression of over 1100 genes - most in cell cycle and metabolic pathways - is affected. This results in BRD4-/- ISP that are smaller than the WT, unable to undergo cell cycle and deficient in metabolic activity. Importantly, a large fraction of the genes uniquely expressed in ISPs are regulated by BRD4. This leads to the unexpected conclusion that BRD4 is a determining factor in ISP differentiation. BRD4 deletion profoundly affects ISP maturation and progression to the DP stage. Since that progression requires a round of cell cycle, this finding is consistent with the observed loss of the cell cycle regulator c-Myc in the absence of BRD4. Based on our findings, we have proposed a model in which the transition from the highly proliferative DN stage to the quiescent DP stage requires a reprogramming through the ISP stage that is regulated by BRD4. The complete conditional deletion of BRD4 did not allow us to distinguish the roles of its two enzymatic activities in thymocyte differentiation. BRD4 has intrinsic kinase and HAT activities, which have been shown to contribute to the regulation of transcription and chromatin organization, respectively. Replacement of wild type BRD4 with mutants of either kinase or HAT activities results in reduced cell cycle and proliferation of cell lines. Indeed, RNA sequence analysis shows dramatic changes in gene expression with either the BRD4 kinase or HAT mutant. Therefore, we have generated transgenic mice that express either BRD4 kinase-deficient or HAT-deficient mutant forms in a vector with a Dox-inducible promoter. These mice have been bred to the BRD4 conditional deletion to allow selective deletion of the endogenous wild type BRD4 and allow us to determine the respective roles of each of these activities in differentiation in vivo and in different tissues. Unfortunately, although induction in cell lines by Dox was vigorous, all attempts to induce expression in transgenic mice failed. Therefore, we have now generated mice in which the endogenous BRD4 gene has been deleted of either the HAT domain or the B domain using CRISPR technology. These mice are currently being analyzed.

BRD4被广泛研究为炎症性疾病中的治疗靶标，以及多种固体和血液学癌（）。它是一种具有激酶和乙酰转移酶活性的染色质结合蛋白，它将染色质结构和转录联系起来，并在调节早期胚胎发育中起积极作用。 BRD4以多种方式有助于基因表达，作为传递转录因子并成核作为染色质重塑剂的脚手架，作为磷酸化Pol II CTD的转录因子。由于BRD4的缺失是胚胎致死的，因此BRD4在正常分化中的作用尚未在体内进行广泛研究。使用条件BRD4敲除小鼠，我们已经表明，与其作为染色质重塑剂的作用一致，BRD4 - / - 胸腺细胞的乙酰化组蛋白水平显着降低了染色质分解和转录所需的水平，这表明BRD4在胸腺细胞发育中起积极作用。对胸腺细胞发育过程中BRD4的体内需求的检查表明，ISP阶段代表具有与DN前体或DP接班人不同的基因表达谱的分散亚群，尽管BRD4缺失选择性地抑制了BRD4，但BRD4在胸腺细胞分化的所有阶段都表达。胸腺细胞的成熟伴随着基因表达谱的巨大变化，这些谱反映在细胞表面标记和细胞周期基因的模式中。 DN胸腺细胞既不表达CD4也不表达CD8，并且具有高度增殖，而ISPS表达CD8，仅经历一轮增殖。因此，从DN到ISP的过渡伴随着免疫周期和细胞周期途径的巨大变化。 有趣的是，尽管增殖存在差异，但两个细胞群体在大约相等的水平上表达C-MYC。因此，单独的MYC表达不能确定增殖的程度或频率。尽管从ISP到DP的成熟是在单轮周期中完成的，但它伴随着免疫和代谢途径中大量基因的差异表达。与DN或ISP种群不同，DP胸腺细胞不表达MYC，这与它们的静态表型一致。特别要注意的是，ISP表达式曲线与DN和DP都不同。因此，它不是DN和DP之间中途的过渡人群。相反，ISP是一个独特的胸腺细胞亚群。 ISP亚群在其对BRD4的依赖性方面也很明显。 尽管BRD4在整个胸腺细胞分化（在等效水平上）都表达，但BRD4耗竭对ISP亚群有选择地影响。在不存在BRD4的情况下，DN胸腺细胞相对正常，尽管BRD4 - / - DN4胸腺细胞小于WT，并且积聚的数量较大。然而，BRD4 - / - DN4胸腺细胞正常地增殖并进行TCRBETA重排，反映出仅影响100个基因的表达受到影响。在受影响的途径中，有炎症和JAK-STAT信号传导。同样，BRD4在表型上正常的DP或SP胸腺细胞中起着相对较小的作用，并且在每个细胞类型中仅调节300-400个基因的表达，这些基因属于免疫系统和代谢过程。与DN，DP和SP胸腺细胞中BRD4缺失的适度作用形成鲜明对比，ISP受到了巨大影响。 超过1100个基因的表达（在细胞周期和代谢途径中最多）受到影响。 这导致BRD4 - / - ISP小于WT，无法经历细胞周期且缺乏代谢活性。重要的是，在ISP中唯一表达的基因的很大一部分受BRD4调节。这导致了意外的结论，即BRD4是ISP分化的决定因素。 BRD4删除深刻影响ISP成熟和发展到DP阶段。由于该进展需要一轮细胞周期，因此该发现与在没有BRD4的情况下观察到的细胞周期调节剂C-MYC的丢失是一致的。根据我们的发现，我们提出了一个模型，在这种模型中，从高度增殖的DN阶段到静态DP阶段的过渡需要通过ISP阶段进行重编程，而ISP阶段则由BRD4调节。 BRD4的完整条件缺失不允许我们区分其两种酶促活性在胸腺细胞分化中的作用。 BRD4具有内在的激酶和HAT活性，已显示出分别有助于转录和染色质组织的调节。用激酶或HAT活性的突变体代替野生型BRD4会导致细胞周期和细胞系的增殖减少。实际上，RNA序列分析显示了用BRD4激酶或HAT突变体的基因表达发生巨大变化。因此，我们产生的转基因小鼠在具有DOX诱导启动子的载体中表达BRD4激酶缺陷或缺陷的突变体形式。这些小鼠已被繁殖到BRD4条件缺失，以允许选择性删除内源性野生型BRD4，并允许我们确定每个活动在体内和不同组织中分化中的各自作用。 不幸的是，尽管DOX诱导细胞系的诱导是剧烈的，但所有诱导转基因小鼠表达的尝试都失败了。因此，我们现在已经生成了使用CRISPR技术删除内源性BRD4基因或B域的小鼠。这些小鼠目前正在分析中。