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

描述（由申请人提供）：早期B细胞因子（EBF）是B淋巴细胞中必不可少的基因调节因子。EBF通过调节编码前B细胞和成熟B细胞受体（pre-BCR和BCR）的基因，推动早期祖细胞发育为B细胞。缺乏EBF的小鼠B细胞发育受阻，免疫球蛋白（Ig）不能产生。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实验室证明，在B淋巴生成过程中，EBF是启动CpG去甲基化和mb-1启动子核小体重构所必需的。这些表观遗传修饰是促进mb-1启动子被其他转录因子激活所必需的，包括Pax5。因此，EBF是一个新的转录因子“功能层次”的关键组成部分，除了先前确定的“遗传层次”之外，它还起作用。尽管EBF具有重要的功能，但对其三维结构的了解甚少。EBF及其在果蝇、蠕虫和脊椎动物中的类似物具有高度保守的dna结合和螺旋-环-螺旋（HLH）结构域。值得注意的是，EBF DNA结合域的一级序列表明了一种新的结构，与其他类型的DNA结合域几乎没有关系。EBF的其他高度保守结构域也表明了它的独特性，这些结构域介导了它的二聚化和稳定性。因此，我们假设更好地理解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，将来自对其三维结构的了解。在本文的两个目标中，将使用x射线晶体学和其他方法来获得EBF和EBF- dna复合物的新结构信息。这些信息将在体内得到验证，并将指导突变EBF蛋白的生物分析，以评估EBF依赖性染色质重塑、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理解先锋转录因子如何在淋巴细胞以及其他细胞类型中启动染色质重塑。公共卫生相关性：这项工作与公共卫生相关，因为它将使人们更好地了解基因是如何被调节的，并可能作为未来治疗的一部分被操纵。具体来说，许多疾病是基因表达错误的结果，而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了解EBF等转录因子如何通过诱导mb-1启动子的DNA去甲基化来启动淋巴样基因的转录活性。

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.