Global Predictions and Tests of Erythroid Regulation

描述（由申请人提供）：基因表达的适当调控对生物体的正常发育和健康至关重要，而已知异常的基因调控会导致许多遗传疾病，包括一些遗传性贫血，并且被认为是复杂表型（如对常见疾病的易感性）的主要因素。了解基因调控的分子机制可能为治疗干预提供新的候选物。我们的研究旨在更深入地了解红细胞前体细胞成熟成为红细胞这一重要生物学过程中基因调控的全局方面。基于我们使用序列比对模式来预测红系基因的顺式调控模块和解读其进化史的功能相关性的进展，我们建议获取与调控相关的生化特征的全基因组信息，以更全面地了解红系细胞的基因调控。具体来说，我们建议使用高通量生化分析，如染色质免疫沉淀，然后进行微阵列杂交和深度重测序，以获取基因组DNA序列（Aim 1）在体内被关键组织特异性转录因子占据的数据，（Aim 2）与基因激活或抑制相关修饰的组蛋白结合，（Aim 3）在结构改变的染色质中，并且（Aim 4）在恢复关键转录因子GATA-1后成熟的小鼠红细胞模型中转录。然后，我们将（目标5）应用现有软件并开发新的数据处理算法来确定可能代表目标1-4中目标特征位置的信号峰值。Aim 6将挖掘峰值调用结果，以及原始数据、多序列比对和其他信息，研究它们的共变结构，并整合它们来预测顺式调控模块，按功能对模块进行分类，识别与特定蛋白质占用相关的基序，并推断调控模块中关键基序的保存系统发育深度。Aims 7将通过实验测试Aims 6和7中分析产生的生物学假设，确定我们可以验证蛋白质占用和转录本位置的程度，通过功能获得细胞转染测定阳性和阴性顺式调节模块的预测，以及通过定向诱变和体内结合测定与占用相关的基序的作用。我们将测试涉及增强的蛋白质占据DNA片段的基序约束假设是否适用于除GATA-1外的转录因子，我们将进行额外的实验来探索更深层次的生物学问题。这项研究不仅将为红细胞成熟过程中基因调控的机制和作用提供全面的见解，而且这里开发的技术和分析工具可以更好地应用于了解任何组织的发育和分化。

DESCRIPTION (provided by applicant): Proper regulation of gene expression is essential to the normal development and health of organisms, whereas aberrant gene regulation is known to cause many genetic diseases, including some inherited anemias, and it is thought to be a major contributor to complex phenotypes such as susceptibility to common diseases. Understanding the molecular mechanisms of gene regulation may provide novel candidates for therapeutic interventions. Our studies aim for a deeper molecular understanding of global aspects of gene regulation in an important biological process, the maturation of erythroid precursor cells to become red blood cells. Building on our progress using patterns in sequence alignments to predict cis-regulatory modules for erythroid genes and deciphering functional correlations of their evolutionary history, we propose to acquire genome-wide information on biochemical features associated with regulation to reach a more complete understanding of gene regulation in erythroid cells. Specifically, we propose to use high throughput biochemical assays such as chromatin immunoprecipitation followed by hybridization to microarrays and deep re-sequencing to acquire data on genomic DNA sequences (Aim 1) occupied in vivo by critical tissue-specific transcription factors, (Aim 2) bound by histones with modifications associated with gene activation or repression, (Aim 3) in chromatin with an altered structure, and (Aim 4) transcribed in a mouse erythroid cell model that undergoes maturation upon restoration of the critical transcription factor GATA-1. Then we will (Aim 5) apply existing software and develop new data-processing algorithms to determine peaks of signals that are likely to represent the locations of the features targeted in aims 1-4. Aim 6 will mine the peak-calling results, along with raw data, multiple sequence alignments and other information to investigate their covariation structure and integrate them to predict cis-regulatory modules, classify the modules by function, identify motifs associated with specific protein occupancy, and deduce the phylogenetic depth of preservation of critical motifs in the regulatory modules. Aim 7 will experimentally test biological hypotheses that arise from the analyses in Aims 6 and 7, determining the extent to which we can validate the locations of protein occupancy and transcripts, the predictions of both positive and negative cis-regulatory modules by gain-of-function cell transfection assays, and the role of motifs implicated in occupancy by directed mutagenesis and in vivo binding assays. We will test whether the motif- constraint hypothesis for protein-occupied DNA segments involved in enhancement applies to transcription factors in addition to GATA-1, and we will conduct additional experiments probing deeper biological issues. This research will provide not only global insights into mechanisms and effects of gene regulation during erythroid maturation, but the techniques and analytical tools developed here can be applied to better understand the development and differentiation of any tissue. PUBLIC HEALTH RELEVANCE: Proper regulation of gene expression is essential to the normal development and health of organisms, whereas aberrant gene regulation can cause genetic diseases, and it appears to be a major contributor to susceptibility to common diseases. Understanding the molecular mechanisms of gene regulation may provide novel candidates for therapeutic interventions. Our studies collecting genome-wide data on many biochemical features associated with gene regulation, mining the data deeply to predict functional DNA sequences, and experimentally testing those bioinformatic predictions will provide global insights into mechanisms and effects of gene regulation during erythroid maturation and provide techniques and analytical tools to better understand the development and differentiation of any tissue.