中国科学院昆明动物研究所机构知识库

真核生物的染色体由编码蛋白质的基因和不编码蛋白质的非编码DNA组成。另外，真核基因组也被高度组装成不同的功能单元，这些功能单元由基因及相应的调控元件组成。而非编码DNA可以作为调控元件增强子、启动子、绝缘子或转录因子结合位点，发挥调控蛋白编码基因表达的作用。同时，转录因子、顺式调控元件、染色质修饰及基因间的相互作用也决定了基因表达和调控的时空特异性。染色质绝缘子通过与其结合蛋白互作，避免基因受到临近基因结构域中增强子或沉默子 (也叫多梳响应元件PREs)的影响。在Abd-B位点，删除Fab-7或Fab-8等边界元件，能导致显性的获得功能表型，推测是由于染色质屏障丢失导致这些表型的产生。果蝇中，同源染色体是处于配对状态的，这产生了许多配对依赖的现象，如transvection。但是不知道transvection是否有助于获得功能表型。本研究中，我们研究了Fab-8绝缘子的染色质屏障活性和转基因合子状态如何影响Fab-8的染色质屏障活性。我们发现Fab-8绝缘子能以果蝇CTCF蛋白结合位点依赖的方式阻止Ubx PRE元件对DsRed报告基因的沉默效应。但是，该阻止效应也依赖于转基因的合子状态，因为染色质屏障活性只在纯合子转基因果蝇中存在；当转基因处于杂合子状态时，转基因果蝇没有表现出明显的染色质屏障活性。为了分析所观察到的效应，我们在纯合子转基因卵中进行染色质免疫沉淀ChIP-荧光定量qPCR实验，发现抑制性H3K27me3和H3K9me3修饰为Fab-8所限制；当Fab-8上两个CTCF蛋白结合位点发生突变时，H3K27me3和H3K9me3修饰从转基因的Fab-8绝缘子处扩散到DsRed报告基因处。与此相对应的，果蝇CTCF结合位点发生突变也降低了DsRed基因所结合的活性H3K4me3修饰和RNA聚合酶II的量，进而导致DsRed表达下降。更重要的是，在杂合子果蝇卵中，Fab-8绝缘子不能避免H3K27me3和H3K9me3修饰扩散到DsRed报告基因，这说明在杂合子果蝇中，其Ubx PRE能顺式绕过间隔的未配对的Fab-8绝缘子。这些结果表明，在Abd-B位点，一拷贝染色体上的绝缘子删除会导致同源染色体上绝缘子活性的丢失，而在其它位点，染色体删除会使基因组处于半合子状态，导致染色质屏障活性丢失。本研究表明同源染色体配对在果蝇基因组的基因表达调控中发挥作用。启动子靶向序列PTS是一种在果蝇Abd-B位点中发现的新型顺式调控元件，是位于绝缘子内的非编码DNA序列；PTS元件具有反绝缘子活性，能介导长距离的增强子-启动子相互作用。另外，PTS元件能介导增强子竞争并调控远端增强子表达时间差。更重要的是，PTS元件介导了表观遗传学效应。但是，到目前为止，人们还不清楚PTS互作的蛋白及PTS互作蛋白是如何参与PTS元件的反绝缘子功能的？已有文章报道称结合DNA的蛋白质能在表观遗传学上调控小RNAs起始的基因表达；且果蝇绝缘子复合物的活性与哺乳动物CTCF-Cohesin复合物的活性都能为RNAs分子或RNA结合蛋白所调控。果蝇的DNA-RNA双结合蛋白Hrb87F是人异质性核蛋白hnRNP A/B家族成员hnRNP A1蛋白的同源类似物，是核小体的组成成分，由两个串联的RNA识别基序RRMs及C端高度灵活的甘氨酸富集区 (该区域含有能结合RNA的RGG盒子结构域)组成。本研究中，我们通过DNA-蛋白亲和纯化偶联质谱分析、凝胶迁移阻滞实验EMSA及生物素-链亲和素pulldown实验，发现Hrb87F是PTS元件的互作蛋白质。进一步通过免疫共沉淀和GST pulldown实验发现Hrb87F蛋白能与绝缘子核心蛋白CTCF相互作用，其互作区域是Hrb87F蛋白的C端结构域及CTCF蛋白的锌指结构域与C端结构域。最后，染色质免疫沉淀ChIP和RNA干扰实验表明Hrb87F蛋白有可能是体内PTS元件发挥反绝缘子活性的功能组分。总之，我们初步证明Hrb87F蛋白很可能参与了PTS元件的反绝缘子功能在本论文中，我们对纯合子和杂合子果蝇中Fab-8绝缘子的染色质屏障功能作用机制进行了研究，同时也对Abd-B位点中PTS元件的结合蛋白进行了初步探索，这对理解真核生物的基因表达调控具有一定的理论意义和应用价值。

Eukaryotic chromosomes consist of genes that encode proteins and non-coding DNA that does not encode proteins. In addition, eukaryotic genomes are also highly organized into various functional units composed of genes and the corresponding regulatory elements. Non-coding DNA is responsible for appropriate expression and regulation of protein-coding genes by functioning as enhancers, promoters, insulators or transcription factors binding sites. Meanwhile, the interactions among transcription factors, cis-regulatory elements, chromatin modifications and genes also determine the spatiotemporal specificity of gene expression and regulation.Chromatin insulators, through their binding proteins protect genes from enhancers or silencers/ Polycomb response elements (PREs) of neighboring genes. In the Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdomianl (Fab)-7 or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. However, homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may contribute to the phenotypes is not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is fully present when the transgene is homozygous, but there is little or no blocking effect when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from acrossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights a role of homologous chromosome pairing in the regulation of gene expression in the Drosophila genome.Promoter targeting sequence (PTS), a novel cis-regulatory element and non-coding DNA located within the insulators in Drosophila Abd-B locus, could mediate anti-insulator activity and long-range enhancer-promoter interactions. In addition, the PTS could mediate enhancer competition and regulate the timing of a distant enhancer. Most of all, the PTS mediates epigenetic inheritance of transcriptional memory. However, both the PTS-interacting proteins and how PTS-interacting proteins are involved in PTS’s anti-insulator activity are still poorly understood. It is reported that the proteins bound to DNA could maintain epigenetically regulation of gene expression initiated by small RNAs and that the activities of Drosophila insulator complex and mammalian CTCF-cohesin complex could be modulated by RNAs or RNAs-binding proteins. DNA-RNA-binding protein Hrb87F, a Drosophila homolog of human heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family member hnRNP A1 protein and a component of ribonucleosomes, contains two tandem RNA recognition motif (RRMs) domain and C terminal highly flexible glycine-rich (Gly-rich) region with an RGG box RNA binding domain. Here, we described that Hrb87F is a PTS-interacting protein by performing DNA-protein affinity purification-Mass spectrometric analysis, electrophoretic mobility shift assay (EMSA) and biotin-avidin pulldown assays. Further assays found that Hrb87F could interact with insulator core protein CTCF with the interacting regions being C termini of Hrb87F and 1-11 Zinc fingers domain and C termini of CTCF using co-immunoprecipitaion (Co-IP) and GST pulldown experiments. Finally, Chromatin immunoprecipitation (ChIP) and RNA interference (RNAi) revealed that Hrb87F protein may be a functional partner of PTS anti-insulator activity in vivo. In conclusion, Our study initially demonstrated that Hrb87F protein is likely to be involved in PTS anti-insulator activity.In this dissertation, we studied the mechanisms of chromatin barrier function of the Fab-8 insulator in homozygous and heterozygous Drosophila, and also initially explored the binding proteins of the PTS element from the Abd-B locus. All the obtained results have certain theoretical significance and application value for understanding the gene expression and regulation in eukaryotes.