Determinants of X-chromosome transcriptional regulation in C. elegans

描述(申请人提供)：基于染色体的性别决定机制导致性别之间X染色体转录剂量的不平衡。在线虫中，X染色体的剂量至少由两种不同的机制补偿。首先，男性(XO)和女性(XX)的X连锁转录水平在性别之间持平。这种调节是由剂量补偿复合体(DCC)介导的，该复合体与XX两性体的两条X染色体特异性结合，以2倍的比例下调表达。第二种未知的机制使性别间X和常染色体之间的平均转录水平相等。第二种机制存在于包括人类在内的其他后生动物身上。该项目旨在测试与这两种机制相关的模型。我建议确定DCC的X特异性定位所需的DNA序列和染色质决定因素。DNA序列基序将从最初赋予复合体X特异性的DCC亚单位的ChlP-ChIP(染色质免疫沉淀随后进行微阵列分析)确定的初始DCC招募位点获得。此外，核小体占有率将通过FAIRE(甲醛辅助分离调节元件)来测量，核小体位置将通过微球菌核酸酶消化产生的单核小体DNA的微阵列分析来确定。DCC的ChlP-芯片将在X-常染色体融合株中识别阻止或帮助局部传播的因素。我建议通过确定DPY-30在组蛋白H3赖氨酸4转录相关甲基化中的作用来确定DPY-30需要DCC定位的分子基础。最后，我的目的是通过检测X和常染色体连锁基因上的RNA聚合酶II水平来确定X和常染色体之间发生的第二种剂量补偿机制是发生在转录水平还是在翻译水平。X染色体转录的表观遗传调控在人类和线虫中都存在，但许多分子机制仍未确定。众所周知，X连锁转录本在脑组织中高度表达，X连锁基因的表观遗传学可能与精神疾病有关。我们计划使用线虫中可用的遗传和分子工具来了解X染色体是如何进化来调节其转录的，以补偿性别特定的剂量差异和转录单倍性不足。此外，确定DCC活性和凝聚素结合的分子机制将有助于我们理解染色体上具有进化根源的蛋白质复合体是如何被用于微调整个染色体的转录的。

DESCRIPTION (provided by applicant): Chromosome-based mechanisms of sex determination cause an imbalance in the transcript dosage of the X chromosomes between the sexes. In C. elegans, X chromosome dose is compensated by at least two distinct mechanisms. First males (XO) and females (XX) equalize X-linked transcript levels between the sexes. This regulation is mediated by the Dosage Compensation Complex (DCC) that binds specifically to both X chromosomes of XX hermaphrodites to down-regulate expression by a factor of two. A second uncharacterized mechanism equalizes average transcript levels between X and autosomes within each sex. This second mechanism is present in other metazoans including humans. This project aims to test models that pertain to both mechanisms. I propose to identify the DNA sequence and chromatin determinants required for X-specific localization of the DCC. DNA sequence motifs will be derived from initial DCC recruitment sites identified by ChlP-chip (Chromatin ImmunoPrecipitation followed by microarray analysis) of the DCC subunit that initially confers X specificity to the complex. In addition, nucleosome occupancy will be measured by FAIRE (Formaldehyde- Assisted Isolation of Regulatory Elements) and nucleosome positions will be determined by microarray analysis of mononucleosomal DNA generated by micrococcal nuclease digestion. Factors that block or assist local spreading will be identified by ChlP-chip of DCC in X-to-autosome fusion strains. I propose determine the molecular basis of DPY-30 requirement for DCC localization by identifying its role in transcription related methylation of histone H3 lysine 4. Finally, I aim to determine whether the second mechanism of dosage compensation that occurs between X and autosomes occurs at the level of transcription or translation by determining RNA Polymerase II levels on X and autosomal-linked genes. Epigenetic regulation of X-chromosome transcription occurs both in humans and C elegans, yet many of the molecular mechanisms remain uncharacterized. It is known that X-linked transcripts are particularly highly expressed in brain tissues and that the epigenetics of X-linked genes maybe linked to mental diseases. We plan to use the genetic and molecular tools available in C. elegans to understand how the X chromosome evolved to regulate its transcription to compensate for both sex-specific dosage differences and transcriptional haploinsufficiency. Additionally, determining the molecular mechanism of DCC activity and condensin binding will contribute to our understanding of how a complex of proteins with evolutionary roots in chromatin compaction have been co-opted to fine-tune transcription chromosome-wide.