Cellular and Structural Studies of Myeloma Mutants

描述（由申请人提供）：Igh基因座在B细胞成熟过程中其DNA发生了巨大变化，首先在基因座的5'端通过VDJ连接，然后在下游涉及CSR中的Ch序列。这些DNA变化集中在Igh基因座上，保留下游相邻序列，并限制可能导致恶性转化的诱变效应。Igh基因座的主要顺式调节子是E和复杂的3' Igh调节区，这是该提议的主要焦点。已显示3' Igh增强子在40-150 kb的距离影响CSR。在B细胞成熟过程中，Igh基因座的复制模式和核亚定位也发生发育变化，Igh复制的结构域终止于最3'端已知增强子的下游。这些观察结果表明在Igh基因座的3'端存在多个水平的调节。染色质免疫沉淀分析将用于鉴定标记Igh复制结构域限制的染色质结构特征。使用已经鉴定了3'调控区的5-7 kb延伸，其在B细胞分化早期与活性染色质的标记物相关，随后逐步与该区域的其它模块相关。该建议将分析在B细胞分化过程中获得与活性染色质逐步缔合的3'调控区的分析模块，并将寻找3' Igh序列的其他表观遗传结构域的证据。将分析3'调控区的模块与促进染色质变化的转录因子的相互作用。 该提议集中于分析hs 4下游的5-7 kb区域，hs 4是最已知的3'增强子，其与pro-B细胞中的开放染色质相关。在B细胞分化过程中，3'调控区的其他模块似乎逐步与活性染色质结合。 在B细胞分化的早期，DNA重排活性首先发生在Igh基因座的5'末端以产生可变区。随着进入B细胞阶段，DNA重排通过类别转换转移到下游恒定区基因。只有两个主要的顺式调控元件是已知的，即内含子增强子r和含有四个已知增强子的复合物w'调控区-一个主要靶。

DESCRIPTION (provided by applicant): The Igh locus undergoes massive changes in its DNA during B cell maturation, first at the 5' end of the locus via VDJ joining, and then downstream to involve Ch sequences in CSR. These DNA changes are focused on the Igh locus, sparing downstream adjacent sequences, and limiting mutagenic effects that could, otherwise, result in malignant transformation. The prime cis-regulators of the Igh locus are E and a complex 3' Igh regulatory region that is the major focus of this proposal. 3' Igh enhancers have been shown to influence CSR at distances of 40-150 kb. Developmental changes in replication patterns and nuclear sublocalization of the Igh locus also occur during B cell maturation and a domain of Igh replication terminates considerably downstream of the most 3' known enhancer. These observations suggest multiple levels of regulation at the 3' end of the Igh locus. Chromatin immunoprecipitation analysis will be used to identify features of chromatin architecture that mark the limits of the Igh replicative domain. Using has identified a 5-7 kb extension of the 3' regulatory region that becomes associated with markers of active chromatin early in B cell differentiation, followed by step-wise association of other modules of this region. This proposal will assay the analymodules of the 3' regulatory region that acquire a step-wise association with active chromatin during B cell differentiation, and evidence for other epigenetic domains of 3' Igh sequences will be sought. The interaction of modules of the 3' regulatory region with transcription factors that foster changes in chromatin will be analyzed. This proposal is focused on analysis of a 5-7 kb region downstream of hs4, the most 3' known enhancer, which is associated with open chromatin in pro-B cells. Other modules of the 3' regulatory region appear to gain step-wise association with active chromatin during B cell differentiation. Early in B cell differentiation, DNA rearrangement activity first occurs at the 5' end of the Igh locus to generate the variable region. With commitment to the B cell stage, DNA rearrangements shift to downstream constant region genes, via class switching. Only two major cis regulatory elements are known, i.e. the intronic enhance r and a complex w' regulatory region containing four known enhancers-a major target.