Chromosome movements that regulate tissue-specific gene expression

IgH等位基因的3维染色质构型确保1）利用可变（VH）基因区段的多样库和2）类别转换重组（CSR）以在免疫应答期间表达不同的重链同种型。 这些关键过程中的每一个都是由专门的酶启动的，这些酶在这种3D结构的背景下靶向IgH基因座。 RAG重组酶在B细胞发育过程中起作用，活化诱导的脱氨酶（AID）启动CSR。 支架蛋白CTCF和YY 1与IgH等位基因的三维构型的建立有关。 在2020财年，我们实现了以下目标： - 我们产生了在DNA酶I超敏位点缺失的小鼠，该位点位于先前确定的将2 Mb VH结构域折叠成离散的99 kb结构域的区域附近的福尔斯。 远端结构域需要Pax 5来产生，而近端结构域可能由CTCF确定（但目前尚未测试）。 缺失位点位于近端和远端结构域之间的边界处。 正在测试这种缺失对VH库和B细胞发育的影响。 - 我们发起了一项合作，使用多个短寡核苷酸探针对VH结构域的染色质构型进行高分辨率显微镜分析。 - 我们将YY 1融合蛋白募集到TetO/Gal 4取代的Em区，以鉴定Y 1在产生不同VH库中的作用。 - 我们已经发现在来自老年小鼠的pro-B细胞中IgH基因座的3D染色质结构被破坏。 在后续观察中，我们使用来自年轻和老年小鼠的pro-B细胞进行了ChIP-Seq、RNA-Seq、ATAC-Seq和Hi-C。我们发现Em功能由于E2 A蛋白（先前已知）的下调和PU. 1的上调而减弱。 在寻找PU.1上调的机制时，我们发现表观遗传调节因子Ezh 2在旧的pro-B细胞中下调。 因此，双价标记的基因如Sfpi 1（编码PU.1）和Cebpa和B失去了抑制性H3 K27 me 3，并在转录水平上调。重要的是，这些基因对于髓系分化是重要的，这表明Ezh 2随年龄的下调可能有助于老年小鼠造血的髓系偏好。

The 3-dimensional chromatin configuration of IgH alleles ensures 1) utilization of a diverse repertoire of variable (VH) gene segments and 2) class switch recombination (CSR) to express different heavy chain isotypes during immune responses. Each of these critical processes is initiated by specialized enzymes that target the IgH locus in the context of this 3D structure. The RAG recombinase operates during B cell development and activation-induced deaminase (AID) initiates CSR. The scaffolding proteins CTCF and YY1 have been implicated in establishing 3D configuration of IgH alleles. During FY20 we accomplished the following: - we generated mice with a deletion in a DNase I hypersensitive site that falls near a previously determined region that folds the 2Mb VH domain into discrete 99kb domains. The distal domain requires Pax 5 for its generation and the proximal domain is presumable determined by CTCF (but this remains untested for now). The deleted site is at the boundary between proximal and distal domains. The effects of this deletion on VH repertoire and B cell development are being tested. - we initiated a collaboration to carry out high resolution microscopic analysis of the chromatin configuration of the VH domain using multiple short oligonucleotide probes. - we recruited YY1 fusion proteins to the TetO/Gal4 substituted Em region to identify the role of Y1 in generating a diverse VH repertoire. - we had found that the 3D chromatin structure of the IgH locus was disrupted in pro-B cells from old mice. In following up this observation we carried out ChIP-Seq, RNA-Seq, ATAC-Seq and Hi-C using pro-B cells from young and old mice. We found that Em function was attenuated due to down-regulation of E2A proteins (previously known) and up-regulation of PU.1 (our study). In seeking a mechanism for PU.1 up-regulation we found that the epigenetic regulator Ezh2 was down-regulated in old pro-B cells. As a consequence bivalently-marked genes such as Sfpi1 (that encodes PU.1) and Cebpa and b lost repressive H3K27me3 and were up-regulated at the transcriptional level. Importantly, these genes are important for myeloid lineage differentiation, suggesting that Ezh2 down-regulation with age may contribute to the myeloid bias of hematopoeisis in old mice.