CELLULAR AND STRUCTURAL STUDIES OF MYELOMA MUTANTS

描述（改编自研究者摘要）：DNA重排，体细胞 发生突变，并表达免疫球蛋白重链（IgH）基因 仅在B细胞中。在过去的几年里，我的实验室 鉴定和表征了候选的顺式和反式元件 VDJ连接、类别转换和IgH基因表达的调节因子。这些 包括鉴定关键B细胞转录因子BSAP， IgH 3'调控区的特征，其包含四个 增强剂。只有最远端的增强子hs 4在整个过程中表现出活性。 B细胞分化，如在培养的细胞系中评估的。我们积极 研究了延伸的3 ′ IgH区在IgH基因复制中的作用， 与罗伊·里布利特和卡尔·希尔德克劳特一起我们发现， 使用DNA复制起点的IgH基因簇在培养的非B细胞 细胞系和代表不同成熟阶段的B细胞系。 增强器激活和起源使用的变化发生在一个单一的 约100 kb的DNA片段。该提案的假设是， 在体内，调节之间存在时间和发育关系， IgH 3'增强子和IgH复制起点的使用， 机械接口我们的第一个目标是确定时间和 作为体内转录增强子的HS 4的细胞类型特异性活性。我们 将产生具有与GFP表达相关的hs 4调节转基因的小鼠， 记者.进行hs 4转录活性的突变分析 在细胞系中。我们的第二个目标是定位和识别IgH的起源， 在非B、B和浆细胞系中复制，并确定是否正常 细胞显示相似的特异性IgH复制起点的利用。第三 目的是确定IgH基因座/染色体结构域的边界是否 位于hs 4处，在MEL中使用的主要下游ori处，或在两者之间。 我们将评估100 kb区域的染色质构型，其中 增强子激活和起源使用的变化都在发生。

DESCRIPTION (adapted from investigator's abstract): DNA rearrangement, somatic mutation, and expression of immunoglobulin heavy chain (IgH) genes occur exclusively in B cells. During the last several years, my laboratory has identified and characterized cis and trans elements that are candidate regulators of VDJ joining, class switching and IgH gene expression. These include identification of a key B cell transcription factor, BSAP, and characterization of the IgH 3' regulatory region, which contains four enhancers. Only the most distal enhancer, hs4, appears to be active throughout B cell differentiation, as assessed in cultured cell lines. We have actively studied the extended 3' IgH region for its role in IgH gene replication, together with Roy Riblet and Carl Schildkraut. We find dramatic differences in usage of origins of DNA replication for the IgH gene cluster in cultured non-B cell lines and in B cell lines representing different stages of maturation. Changes in enhancer activation and origin usage are occurring within a single approximately 100 kb DNA segment. The hypothesis of this proposal is that in vivo, there is a temporal and developmental relationship between regulation of IgH 3' enhancers and usage of IgH origins of replication that involves a mechanistic interface. Our first goal is to determine the temporal and cell-type specific activity of hs4 as a transcriptional enhancer in vivo. We will generate mice with an hs4-regulated transgene linked to a GFP expression reporter. Mutational analysis of hs4 transcriptional activity will be carried out in cell lines. Our second goal is to locate and identify IgH origins of replication in non-B, B and plasma cell lines and to determine whether normal cells show similar utilization of specific IgH replication origins. A third goal is to determine whether a boundary for the IgH locus/ chromosomal domain is located at hs4, at the dominant downstream ori used in MEL, or in between. We will assess the chromatin configuration of the 100 kb region in which changes in enhancer activation and origin usage are both occurring.