Determinants of X-chromosome transcriptional regulation in C. elegans

描述（由申请人提供）：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。In C.在线虫中，X染色体剂量通过至少两种不同的机制来补偿。首先，雄性（XO）和雌性（XX）使两性之间的X连锁转录水平相等。这种调节是由剂量补偿复合物（DCC）介导的，该复合物特异性结合XX两性体的两条X染色体，以将表达下调两倍。第二个未表征的机制使每个性别中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中。该项目旨在测试与这两种机制有关的模型。我建议确定的DNA序列和染色质决定因素所需的X-特异性本地化的DCC。DNA序列基序将衍生自最初赋予复合物X特异性的DCC亚基的ChIP-芯片（染色质免疫沉淀，随后进行微阵列分析）鉴定的初始DCC募集位点。此外，将通过FAIRE（甲醛辅助分离调节元件）测量核小体占有率，并且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核小体位置。阻断或辅助局部扩散的因子将通过X-常染色体融合菌株中DCC的ChIP-芯片来鉴定。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对DCC定位的分子基础。最后，我的目标是确定是否发生在X和常染色体之间的剂量补偿的第二种机制发生在转录或翻译水平，通过确定X和常染色体相关基因的RNA聚合酶II水平。X染色体转录的表观遗传调控发生在人类和线虫中，但许多分子机制仍不清楚。已知X连锁转录物在脑组织中特别高表达，并且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C中可用的遗传和分子工具。elegans了解X染色体如何进化来调节其转录，以补偿性别特异性剂量差异和转录单倍不足。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在染色质致密化中具有进化根源的蛋白质复合物如何被增选以微调转录染色体范围。

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.