Insulator BEAF32 regulates expression of tissue-specific genes and piRNA source loci in the Drosophila ovaries

Background: Insulators are the multifunctional DNA binding proteins that perform architectural functions and regulate gene transcription. Although insulators have a well-established role in genome organization, it is still unclear how insulator proteins affect the control of tissue-specific processes. Drosophila insulator BEAF32 (Boundary Element-Associated Factor of 32 kD) is a component of chromatin complexes found in the open chromatin regions containing promoters of housekeeping genes. BEAF32 knockout impairs oogenesis and female fertility suggesting its specific functions during oogenesis Results: To get a better understanding of BEAF32 roles in oogenesis, we first examined its ovarian binding targets and discovered an enrichment of its localization sites in the promoters of both housekeeping and tissue-specific genes. Differential expression gene analysis revealed that BEAF32 knockout resulted in abnormal activation of non-ovarian tissue-specific genes in the ovaries, implying that BEAF32 regulates tissue-specific patterns of gene expression. Among upregulated genes there were not only BEAF32-associated genes suggesting direct and indirect mechanisms of BEAF32-mediated expression control. We discovered that BEAF32 occupied many ovary-specific gene promoters and acted as a positive regulator of the cell-cycle regulatory kinase Polo expression. To reveal the possible role of BEAF32 in the pathway mediated by Piwi-interacting RNAs (piRNAs) we performed an analysis of ovarian small RNAs in the BEAF32 null mutants and revealed a strong decrease in the production of piRNAs from the 3R subtelomeric region. Our data suggest that BEAF32-containing chromatin complex located upstream of the subtelomeric repeats preserves transcriptional and chromatin integrity of this domain in the germline. BEAF32 was also found upstream of flamenco, a largest piRNA source locus in the follicular cells, and is required for the cell-specific transcription of the flam locus. Conclusions: These findings suggest that BEAF32 represses the ectopic expression of developmental and tissue-specific genes in the ovaries. BEAF32 regulates polo kinase and other oogenesis-related genes. BEAF32 insulators have been revealed to play a specific ovarian role in the maintenance of piRNA-producing loci. Our results provide strong support for the importance of BEAF32 insulator protein in determining the proper landscape of tissue-specific gene expression and locus-specific function realization. Deep-sequencing of chromatin-associated RNAs of Drosophila melanogaster ovaries

背景：绝缘子（insulator）是一类多功能DNA结合蛋白，兼具基因组架构调控功能与基因转录调控作用。尽管绝缘子在基因组组织中的功能已得到广泛证实，但绝缘子蛋白如何调控组织特异性进程仍未明确。果蝇绝缘子BEAF32（32 kDa边界元件相关因子，Boundary Element-Associated Factor of 32 kD）是染色质复合物的组分之一，存在于包含持家基因启动子的开放染色质区域。BEAF32基因敲除会损害卵子发生与雌性生育能力，提示其在卵子发生过程中发挥特异性功能。
结果：为深入解析BEAF32在卵子发生中的作用，我们首先对其在黑腹果蝇（Drosophila melanogaster）卵巢中的结合靶点进行了检测，发现其定位位点在持家基因与组织特异性基因的启动子区域均存在富集。差异表达基因分析显示，BEAF32敲除会导致卵巢中非卵巢组织特异性基因的异常激活，表明BEAF32可调控基因表达的组织特异性模式。在上调基因中，不仅包含BEAF32结合的基因，这提示BEAF32介导的基因表达调控存在直接与间接两种机制。我们还发现，BEAF32可结合多个卵巢特异性基因的启动子，并作为细胞周期调控激酶Polo表达的正调控因子。为探究BEAF32在Piwi互作RNA（Piwi-interacting RNAs, piRNAs）介导通路中的潜在作用，我们对BEAF32纯合缺失突变体的卵巢小RNA进行了分析，发现3R亚端粒区域产生的piRNAs产量显著下降。本研究数据表明，定位于亚端粒重复序列上游的含BEAF32染色质复合物，可在生殖细胞中维持该区域的转录与染色质完整性。此外，BEAF32还富集于滤泡细胞中最大piRNA来源位点flamenco的上游区域，且对flam位点的细胞特异性转录至关重要。
结论：上述研究结果表明，BEAF32可抑制卵巢中发育相关及组织特异性基因的异位表达。BEAF32能够调控Polo激酶及其他卵子发生相关基因的表达。本研究还揭示，BEAF32绝缘子在维持piRNA生成位点的卵巢特异性功能中发挥特定作用。本研究结果为BEAF32绝缘子蛋白在塑造正确的组织特异性基因表达模式及实现位点特异性功能方面的重要性提供了有力支撑。
黑腹果蝇卵巢染色质结合RNA的深度测序