Genomic and Functional Analyses of Regulatory Regions in Vertebrate Sequences

双向启动子在基因组中含量丰富。它们被定义为一个共同的调节区，位于两个相对定位的基因之间的基因间隙，两个基因之间的距离不超过1,000个碱基对。这些基因是以头对头的方式组织起来的，并相互转录。每对基因转录起始点(TSS)的紧密排列被认为是基因组中的一个非随机事件，事实证明，比预期数量更多的启动子具有这种结构。创造双向基因对的一个看似合理的方案是染色体重组，使两个SSs的末端非常接近。这种结合很可能是不可逆转的，因为基因间启动子区域的破坏将扰乱两个基因的正常调节，深刻影响正常的基因组功能，如DNA修复。我们已经在许多脊椎动物基因组中定位了双向启动子。通过这样做，我们正在创建第一个在脊椎动物谱系中共享的双向启动子序列的高置信度调控图，并识别包括灵长类在内的单个脊椎动物谱系所独有的转录本。 除了确定启动子区域的特征外，我们还对识别新类型的元素感兴趣，例如基因表达的负面调节(NRES；Petrykowska等人，基因组研究，2008年)。与大量关于促进剂和启动子等积极作用元件的文献相反，顺式作用的NRES还没有得到广泛的研究。尽管它们在文献中很稀少，但这些元素在基因组中很可能是丰富的。NRES的例子包括消音器和增强子阻断(EB)元件，前者在其调控下降低基因的表达，后者当被放置在两者之间时阻止增强子对启动子的作用，但不是在其他情况下。通过开发一种实验测试NRES的策略，我们已经在人类基因组中确定了这些功能的新例子。 外显子剪接增强子(ESES)是影响基因表达的第三类调控元件。我们比较了所有以前对ESES的预测方法，以表明一些方法比另一些方法更精确。我们已经开发了一个在线工具包来测试编码序列中出现的多态，以评估它们是否会影响信使核糖核酸剪接(http://research.nhgri.nih.gov/skippy). 所有这些研究项目都在研究顺式作用元件，并集中在鉴定转录因子结合部位或剪接调节因子，它们与反式作用蛋白结合，代表基因调控的基本组成部分。已经为上述每个项目发布了监管主题。在我们的工作过程中，新的基序被认为是消音器，并为调节替代启动子提供了新的生物学见解。已经建立了一个合作努力来定义涉及这些基序的调控网络，特别是在与癌症有关的生物途径中。此外，我们正在建立工具来评估表观遗传事件，这些事件对肿瘤中的基因表达产生异常影响，而不是正常细胞。

Bidirectional promoters are abundant in the genome. They are defined as a shared regulatory region that falls in the intergenic space between two oppositely oriented genes that are separated by no more than 1,000 bp. These genes are organized in a head-to-head arrangement and transcribed away from one another. The closely spaced arrangement of the transcription start sites (TSSs) each pair of genes is recognized as a nonrandom event in the genome, proven by the fact that a greater than expected number of promoters have this architecture. One plausible scenario for the creation of bidirectional gene pairs is chromosomal recombination bringing the ends of two TSSs in close proximity. This union is likely to be irreversible because breakage of the intergenic promoter region would interrupt the normal regulation of two genes, profoundly affecting normal genomic function, such as DNA repair. We have mapped bidirectional promoters in numerous vertebrate genomes. In doing so, we are creating the first high-confidence regulatory map of bidirectional promoter sequences shared in vertebrate lineages as well as identifying transcripts that are unique to single vertebrate lineages, including primates. In addition to characterizing promoter regions, we are interested in identifying novel types of elements such as negative regulators of gene expression (NREs; Petrykowska et al, Genome Research 2008). In contrast to the large body of literature on positively acting elements such as enhancers and promoters, cis-acting NREs have not been extensively studied. Despite their scarceness in the literature, these elements are likely to be abundant in the genome. Examples of NREs include silencers, which decrease expression of a gene under their regulation and enhancer-blocking (EB) elements, which prevent the action of an enhancer on a promoter when placed between the two, but not otherwise. By developing a strategy to experimentally test NREs, we have identified novel examples of these functions in the human genome. Exonic splicing enhancers (ESEs) are a third category of regulatory elements affecting gene expression. We have compared all former predictive methods for ESEs to show that some are more precise than others. We have produced an online toolkit to test polymorphisms that occur in coding sequences to assess whether they may affect mRNA splicing (http://research.nhgri.nih.gov/skippy). All of these research projects examine cis-acting elements and converge on the identification of transcription factor binding sites or splicing regulators, which are bound by trans-acting proteins and represent the basic components of gene regulation. Regulatory motifs have been published for each of the projects above. During the course of our work, novel motifs were implicated as silencers and new biological insights were revealed for the regulation of alternative promoters. A collaborative effort has been established to define the regulatory networks involving these motifs, especially in biological pathways implicated in cancer. Moreover, we are building tools to assess epigenetic events that aberrantly affect gene expression in tumors versus normal cells.