Structural Studies of the Early B-cell Factor (EBF)

描述（由申请方提供）：早期B细胞因子（EBF）是B淋巴细胞中基因的重要调节因子。EBF通过调节编码前B和成熟B细胞受体（前BCR和BCR）的基因，驱动早期祖细胞发育为B细胞。在缺乏EBF的小鼠中，B细胞发育被阻止，并且不产生免疫球蛋白（IG）。在缺乏EBF的情况下，包括mb-1（IG-1）在内的靶基因不能被激活，并驻留在高甲基化的、不可接近的染色质中。最近，Hagman实验室证实，EBF是B淋巴细胞生成过程中mb-1启动子区CpG去甲基化和核小体重塑所必需的，并启动这些过程。需要这些表观遗传修饰来促进mb-1启动子被其他转录因子（包括Pax 5）激活。因此，EBF是一个新的转录因子的“功能层次”的关键组成部分，除了“遗传层次”以前确定的行为。尽管EBF的功能的重要性，很少有人了解其三维结构。EBF及其在果蝇、蠕虫和脊椎动物中的旁系同源物具有高度保守的DNA结合和螺旋-环-螺旋（HLH）结构域。值得注意的是，EBF DNA结合结构域的一级序列表明与其他类型的DNA结合结构域几乎没有关系的新结构。EBF的独特性也由其其他高度保守的结构域表明，这些结构域介导其二聚化和稳定性。因此，我们假设，更好地了解EBF如何在B淋巴细胞和其他靶组织（脂肪细胞和皮质神经元）中发挥作用，将来自其三维结构的知识。在这两个目标中，X射线晶体学和其他方法将用于获得EBF和EBF-DNA复合物的新结构信息。这些信息将在体内验证，并将指导在生物测定中分析突变的EBF蛋白，评估EBF依赖的染色质重塑，蛋白质：蛋白质相互作用和转录激活。总之，这些研究将极大地促进我们对EBF及其对B淋巴细胞基因调控的理解。这些研究将广泛适用于了解先驱转录因子如何启动淋巴细胞以及其他细胞类型的染色质重塑。公共卫生相关性：这项工作与公共卫生有关，因为它将导致更好地了解基因是如何调节的，并可能作为未来治疗的一部分进行操纵。具体而言，许多疾病是不正确的基因表达的结果，并且逆转导致基因失活的表观遗传密码（如DNA甲基化）的能力将为治疗干预提供有希望的方法。然而，为了实现这样的治疗，有必要在分子水平上理解转录因子如EBF如何通过诱导mb-1启动子的DNA去甲基化来启动淋巴基因的转录活性。

DESCRIPTION (provided by applicant): Early B cell Factor (EBF) is an essential regulator of genes in B lymphocytes. EBF drives the development of early progenitors to become B cells by regulating genes encoding the pre-B and mature B cell receptors (pre-BCR and BCR). B cell development is arrested and immunoglobulins (Ig) are not produced in mice lacking EBF. In the absence of EBF, target genes including mb-1 (Ig-1) fail to be activated and reside in hypermethylated, inaccessible chromatin. Recently, the Hagman laboratory demonstrated that EBF is required for and initiates CpG demethylation and the remodeling of nucleosomes at the mb-1 promoter during B lymphopoiesis. These epigenetic modifications are required to facilitate activation of the mb-1 promoter by other transcription factors, including Pax5. Thus, EBF is a key component of a new "functional hierarchy" of transcription factors that acts in addition to the "genetic hierarchy" identified previously. In spite of the functional importance of EBF, very little is understood concerning its three-dimensional structure. EBF and its paralogues in flies, worms and vertebrates possess highly conserved DNA-binding and helix-loop-helix (HLH) domains. Notably, the primary sequence of the EBF DNA binding domain suggests a novel structure with little relation to other types of DNA binding domains. The uniqueness of EBF is also suggested by its other highly conserved domains, which mediate its dimerization and stability. Therefore, we hypothesize that a better understanding of how EBF functions in B lymphocytes and other target tissues (adipocytes and cortical neurons), will come from knowledge of its three-dimensional structure. In the two Aims of this proposal, X-ray crystallography and other methods will be used to obtain new structural information about EBF and EBF-DNA complexes. This information will be validated in vivo and will guide the analysis of mutated EBF proteins in biological assays that assess EBF-dependent chromatin remodeling, protein:protein interactions and transcriptional activation. Together, these studies will greatly facilitate our understanding of EBF and its regulation of genes in B lymphocytes. These studies will be broadly applicable to understanding how pioneer transcription factors initiate chromatin remodeling in lymphoid as well as other cell types. Public Health Relevance: The work is relevant to the public health, because it will lead to a better understanding of how genes are regulated and may be manipulated as part of future therapies. Specifically, many diseases are the result of incorrect gene expression, and the ability to reverse the epigenetic codes that cause gene inactivity, such as DNA methylation, will provide promising approaches for therapeutic intervention. However, to achieve such therapies, it will be necessary to understand at a molecular level how a transcription factor such as EBF initiates transcriptional activity of lymphoid genes through inducing DNA demethylation of the mb-1 promoter.