Cellular and Structural Studies of Myeloma Mutants

描述（由申请人提供）：在B细胞成熟过程中，Igh基因座的DNA发生了巨大的变化，首先在基因座的5'端通过VDJ连接，然后下游涉及CSR中的Ch序列。这些DNA变化集中在Igh位点，保留了下游相邻序列，并限制了可能导致恶性转化的致突变作用。Igh基因座的主要顺式调控因子是E和一个复杂的3' Igh调控区域，这是本提案的主要焦点。研究表明，在40-150 kb的距离上，高增强子会影响CSR。在B细胞成熟过程中，也会发生复制模式和核亚定位的发育变化，并且Igh复制的一个区域在大多数已知的3'增强子的下游终止。这些观察结果表明，在Igh位点的3'端存在多个水平的调控。染色质免疫沉淀分析将用于识别染色质结构的特征，这些特征标志着Igh复制结构域的限制。Using已经确定了一个5-7 kb的3'调控区域的延伸，该区域在B细胞分化早期与活性染色质标记相关，随后是该区域其他模块的逐步关联。本提案将分析在B细胞分化过程中获得与活性染色质逐步关联的3‘调控区域的分析模块，并寻求3’ Igh序列的其他表观遗传结构域的证据。将分析3'调控区模块与促进染色质变化的转录因子的相互作用。

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.