Comprehensive Analysis of Estrogen Receptor Genomic Action

说明(申请人提供)：雌激素在正常生理和乳腺癌的病理过程中起着多重作用。人类基因组测序的完成使得对蛋白质编码基因的表达区域的鉴定近乎完整，然而，关于它们的顺式调控元件的组织方式却知之甚少。在最近发表的研究中，我们通过将染色质免疫沉淀(CHIP)与高密度平铺微阵列(CHIP-CHIP)相结合，完成了雌激素受体(ER)与人类染色体21和22的整个非重复序列之间的染色体规模图谱。在这些研究中，我们发现ER选择性地结合到这两条染色体上的有限数量的位置。有趣的是，这些基因中的大多数与雌激素调节基因的转录起始点有很大的距离(10kb)。对这些真正的ER结合位点的无偏向序列查询表明，ER结合通常既涉及雌激素反应元件(ERE)，也涉及近距离存在的叉头因子结合。此外，下调叉头因子FoxA1的表达既减少了ER与这些位点的关联，也减少了雌激素诱导的基因表达。这些研究虽然仅限于两条染色体，仅确定了57个结合位点，但证实了在整个人类基因组中定义全套预测的~4000个ER结合位点的可行性。我们假设，一旦所有的ER结合位点都被确定，根据ER结合区内或附近存在的转录因子基序，将有不同类别的ER靶点。此外，将这些研究扩展到包括整个基因组，应该可以研究雌激素诱导和抑制的基因。这些假设将在目标1中解决。我们的发现表明，FoxA1对于定义乳腺癌细胞中重要的ER靶点是必要的假设。此外，我们假设在目标1中定义的不同类别的内质网靶基因将代表不同功能类别的靶基因。《目标2》将检验这些假说。最后，大多数ER结合位点远离最近基因的mRNA起始点的发现提出了一个更普遍的问题，即如何将假定的顺式调控元件分配给特定的靶基因。目标3将解决这个问题。

DESCRIPTION (provided by applicant): Estrogen plays multiple roles in normal physiology and a pathologic one in breast cancer. The completion of the sequencing of the human genome has allowed the near-complete identification of the expressed regions of protein-coding genes, however, little is known concerning the organization of their cis-regulatory elements. In recently published studies we have completed chromosome-scale mapping of estrogen receptor (ER) association with the entire non-repetitive sequence of human chromosomes 21 and 22 by combining chromatin immunoprecipitation (ChIP) with high-density tiled microarrays (ChIP-chip). In these studies we find that ER binds selectively to a limited number of sites on these two chromosomes. Interestingly, the majority of these are at significant distances (>10kb) from the transcription start sites of estrogen-regulated genes. The unbiased sequence interrogation of these genuine ER binding sites suggests that ER binding often involves both estrogen response elements (ERE) and the presence of forkhead factor binding in close proximity. Furthermore, knockdown of the expression of the forkhead factor FoxA1 decreases both the association of ER with these sites and estrogen-induced gene expression. These studies, though limited to only two chromosomes and identifying only 57 binding sites, confirm the feasibility of defining the complete set of the predicted ~4000 ER binding sites across the entire human genome. We hypothesize that once all of the ER binding sites are identified that there will be distinct classes of ER targets based on the transcription factor motifs present within or adjacent to the ER binding regions. In addition, the extension of these studies to include the entire genome, should allow the study of genes both induced and repressed by estrogen. These hypotheses will be addressed in Aim 1. Our findings suggest the hypothesis that FoxA1 is necessary for defining a significant subset of ER targets in breast cancer cells. In addition we hypothesize that the different classes of ER targets defined in Aim 1 will represent different functional classes of target genes. Aim 2 will test these hypotheses. Finally, the finding that most ER binding sites are distant from the mRNA start sites of the nearest genes raises the more general problem of how to assign putative cis-regulatory elements to specific target genes. Aim 3 will address this problem.