Global Predictions and Tests of Erythroid Regulation

描述(由申请人提供)：基因表达的适当调节对生物体的正常发育和健康至关重要，而基因调节的异常被认为会导致许多遗传性疾病，包括一些遗传性贫血，它被认为是导致复杂表型的主要因素，例如对常见疾病的易感性。了解基因调控的分子机制可能为治疗干预提供新的候选者。我们的研究旨在更深入地从分子水平理解红系前体细胞成熟为红血球这一重要生物学过程中基因调控的全球方面。在我们使用序列比对模式预测红系基因顺式调控模块和破译其进化历史的功能相关性的进展的基础上，我们建议获取与调控相关的全基因组信息，以更全面地了解红系细胞的基因调控。具体地说，我们建议使用高通量生化分析，如染色质免疫沉淀，然后杂交到微阵列和深度重新测序，以获得关于基因组DNA序列(AIM 1)的数据，这些序列在体内由关键组织特异性转录因子(AIM 1)占据，(AIM 2)与与基因激活或抑制相关的修饰的组蛋白结合，(AIM 3)在结构改变的染色质中转录，以及(AIM 4)在关键转录因子GATA-1恢复后经历成熟的小鼠红系细胞模型中转录。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，以确定可能代表目标1-4中目标特征位置的信号峰值。AIM 6将挖掘峰值呼叫结果，以及原始数据、多个序列比对和其他信息，以调查它们的协变结构并将它们整合到预测顺式调控模块，根据功能对模块进行分类，识别与特定蛋白质占据相关的基序，并推断关键基序在调控模块中保存的系统发育深度。AIM 7将通过实验测试从AIMS 6和7中的分析中产生的生物学假说，确定我们可以在多大程度上验证蛋白质占据和转录本的位置，通过功能获得细胞转染分析预测正和负顺式调节模块，以及通过定向突变和体内结合分析涉及占据的基序的作用。我们将测试参与增强的蛋白质占据的DNA片段的基序约束假说是否适用于GATA-1以外的转录因子，我们还将进行额外的实验，探索更深层次的生物学问题。这项研究不仅将提供对红系成熟过程中基因调控机制和影响的全球见解，而且这里开发的技术和分析工具可以应用于更好地理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.