T细胞受体(T cell receptor, TCR)的高度多样性是其识别抗原的基础，在适应性免疫中发挥重要作用。TCR多样性来自胸腺T细胞发育过程中的V(D)J重排。在4个TCR基因中， Tcra和 Tcrd基因位于同一个基因座，共用特定的V基因，因此其重排和调节具有一定的特殊性。抗原受体基因的调控研究主要集中于顺式和反式调控作用。然而，近期的研究表明染色质空间组织在抗原受体基因重排中发挥重要功能。其中染色质组织蛋白质CTCF-Cohesin通过环挤出方式影响重排的效率和TCR组库多样性，而重组酶RAG也可以扫描染色质。本综述描述 Tcra和 Tcrd基因的重排及其调节机制研究的新进展，特别是染色质空间组织对重排的影响。

The three-dimensional organization of the genome plays a crucial role in regulating gene expression patterns in metazoans (Ong and Corces,2014).The nuclear architectural proteins are known to facilitate the formation of topological domains within the genome through mediating inter-and intra-chromosomal interactions.In vertebrate,CCCTC-binding factor (CTCF) is the main architectural protein that mediates long-range chromosomal interactions among its DNA binding sites through a process that is stabilized by cohesin (Parelho et al.,2008;Wendt et al.,2008).

尿苷二磷酸葡萄糖醛酸转移酶(UDP-glucuronosyltransferase,UGT)是一类重要的Ⅱ相药物代谢酶,通过葡萄糖醛酸接合反应代谢大量内外源小分子化合物,对机体维持内部动态平衡具有重要意义.UGT基因突变或表达异常会造成高胆红素血症等多种疾病、影响药物疗效或减弱代谢药物能力,因此探索UGT表达调控机制将会为人类疾病的预防和个体化医疗以及精准医学提供科学依据.脊椎动物UGT分为UGT1和UGT2两个亚家族,UGT1基因簇结构与原钙粘蛋白(protocadherin,Pcdh)、免疫球蛋白或B细胞受体(immunoglobulin or B-cell receptor)、T细胞受体(T-cell receptor)基因簇类似,但与UGT2结构不同,分为可变区和恒定区,可变区包含成串排列的外显子,任意一个外显子都可以被可变剪接到下游同一套恒定区外显子上,形成9种UGT1信使RNA并翻译成不同UGT1葡醛酸转移酶亚型.本实验室前期工作发现,染色质架构蛋白CTCF结合DNA的方向性在染色质三维结构构建中发挥重要作用.基于此,为了进一步解析UGT1复杂基因簇的三维转录调控机制,本研究分析和比较了人和小鼠UGT1基因簇的CTCF结合位点(CTCF binding site,CBS)的方向性分布,发现人和小鼠的UGT1基因簇中CBS分布差异很大.以人肺癌细胞系A549为模型,通过RNAi敲低细胞中CTCF和SMC3(cohesin亚基),证明了CTCF和cohesin蛋白参与调控人UGT1基因簇的转录表达.进一步采用CRISPR介导的DNA片段编辑技术对hCBS1进行了原位反转(in situ inversion)和删除,并通过RNA-seq分析技术发现hCBS1删除能够显著降低UGT1A6、UGT1A7和UGT1A9的表达水平,然而hCBS1反转仅仅显著降低UGT1A7的表达水平.上述研究表明hCBS1参与UGT1A6、UGT1A7和UGT1A9的转录调节,是人UGT1基因簇的潜在转录调控元件.本研究为未来进一步探索UGT1基因簇的三维基因转录调控机制提供了实验基础.

CCCTC结合因子(CCCTC-binding factor,CTCF)是一种高度保守的多功能转录因子,含有11-锌指结构,可与DNA、蛋白质和RNA相互作用,参与基因的表达调控.CTCF可通过基因DNA甲基化水平的改变、基因突变、CTCF/Cohesin复合体、BORIS/CTCF系统调控CTCF的功能,参与肿瘤的发生和发展.该文就转录因子CTCF在肿瘤发展中的调控机制作一综述.

Dear Editor,Similar to higher-order folding of polypeptide chains into functional proteins,linear DNA molecules are spatially folded in a hieramhical and dynamic manner into three-dimensional (3D) functional chromatin structures in eukaryotic nuclei (Huang and Wu,2016;Rowley and Comes,2018).This dynamic folding is closely related to many nuclear processes such as DNA replication and repair,chromosomal translo cation,recombination,and segregation,as well as RNAtranscription,splicing,and transport.In particular,dynamic long-distance chromatin looping interactions,which result in close spatial contacts between distal enhancers and target promoters,are thought to play a role in controlling precise spatiotemporal as well as cell-type specific gene expression during animal development (Rowley and Comes,2018).

基因的表达调控与基因组在细胞核内的三维空间架构相辅相成,原钙粘蛋白(protocadherin,Pcdh)基因簇在大脑发育中起到关键作用,可以作为研究基因表达调控机制的模式基因.转录因子RFX5(regulatory factor x 5)是翼螺旋家族(winged HLH family)的成员,其蛋白由寡聚化结构域、DNA结合域、螺旋结构域和激活域组成,在调控免疫系统的主要组织相容性复合物II类(major histocompatibility complex class II,MHC II)的表达中起着至关重要的作用.本研究发现RFX5与CTCF在全基因组上结合的位点有部分重叠,利用CRISPR/Cas9 DNA大片段编辑技术,构建了RFX5基因缺失的HEC-1-B细胞系.通过RNA-seq实验,发现RFX5敲除能够显著升高Pcdhα6、Pcdhα12、Pcdhαc2的表达水平.通过ChIP-nexus实验,发现敲除RFX5导致染色质架构蛋白CTCF和cohesin在原钙粘蛋白 α 基因簇处的结合增加.最后,染色质构象捕获QHR-4C实验发现Pcdhα6、Pcdhα12启动子与远端增强子HS5-1的染色质远距离相互作用增强.上述研究表明RFX5蛋白可能通过调控染色质高级结构影响原钙粘蛋白α基因簇的表达,为未来进一步探索RFX5的功能提供了参考.

CCCTC-binding factor(CTCF)is a multifunctional zinc finger protein that is conserved in metazoan species.CTCF is consistently found to play an important role in many diverse biological processes.CTCF/cohesin-mediated active chromatin'loop extrusion'architects three-dimensional(3D)genome folding.The 3D architectural role of CTCF underlies its multifarious functions,including developmental regula-tion of gene expression,protocadherin(Pcdh)promoter choice in the nervous system,immunoglobulin(Ig)and T-cell receptor(Tcr)V(D)J recombination in the immune system,homeobox(Hox)gene control during limb development,as well as many other aspects of biology.Here,we review the pleiotropic functions of CTCF from the perspective of its essential role in 3D genome architecture and topological promoter/enhancer selection.We envision the 3D genome as an enormous complex architecture,with tens of thousands of CTCF sites as connecting nodes and CTCF proteins as mysterious bonds that glue together genomic building parts with distinct articulation joints.In particular,we focus on the internal mechanisms by which CTCF controls higher order chromatin structures that manifest its many fa?ades of physiological and pathological functions.We also discuss the dichotomic role of CTCF sites as intriguing 3D genome nodes for seemingly contradictory'looping bridges'and'topological insulators'to frame a beautiful magnificent house for a cell's nuclear home.

CTCF(CCCTC-binding factor)是一种重要的染色质架构蛋白,其与绝缘子的方向性结合在哺乳动物基因组三维空间结构形成和维持中起着至关重要的作用.正向–反向相对方向的CTCF结合位点(简称CTCF位点)可以在染色质黏连蛋白(cohesin)的协助下,形成染色质环,介导远距离DNA元件之间的相互作用;而在染色质拓扑结构域边界区域的CTCF位点呈现反向–正向相背方向分布,发挥绝缘子的功能.为进一步研究CTCF介导染色质环的形成与其绝缘功能之间的关系,本研究采用DNA片段编辑方法通过设计成对sgRNA(dual sgRNA)构建了一系列HOXD基因簇区域CTCF位点反转的单细胞克隆.定量高分辨率染色质构象捕获实验显示边界区域CTCF位点反转会改变原有的染色质环方向,通过环挤出模型(loop extrusion)形成新的染色质环,引起染色质拓扑结构域边界漂移至新形成的一对反向–正向CTCF位点处.此外,串联排列的CTCF位点可以通过阻碍反方向渗透的黏连蛋白继续滑动发挥绝缘子的功能.RNA-seq实验发现CTCF位点反转引起的局部基因组空间结构变化会进一步影响基因的表达.上述研究表明相邻两个染色质拓扑结构域边界区域的反向-正向CTCF位点可以通过与各自所在拓扑结构域内相向的CTCF位点形成染色质环,阻碍黏连蛋白滑动,该发现为进一步研究CTCF的绝缘功能和其对基因组拓扑结构的影响提供了参考.