Kobelyatskaya Anastasia A, Morgunova Valeria, Kalinkin Aleksey, Shchukina Anna A, Sokolova Olesya, Kwon Dmitry A, Funikov Sergei, Kalmykova Alla
Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Moscow, 119334, Russia.
Syntol LLC Company, Moscow, 127434, Russia.
Epigenetics Chromatin. 2025 Jul 28;18(1):49. doi: 10.1186/s13072-025-00613-6.
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. The Drosophila insulator BEAF32 (Boundary Element-Associated Factor of 32 kD) is a component of chromatin complexes found in open chromatin regions containing promoters of housekeeping genes. BEAF32 knockout impairs oogenesis and female fertility suggesting its specific functions during oogenesis.
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. We discovered that BEAF32 occupied many ovary-specific gene promoters and acted as a positive regulator of expression for the cell-cycle regulatory kinase, Polo. To investigate the possible role of BEAF32 in the Piwi-interacting RNAs (piRNAs) pathway we analyzed ovarian small RNAs in BEAF32 null mutants and found a strong decrease in the production of piRNAs from the 3R subtelomeric region. Our data suggest that the 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 to localize upstream of flamenco, a major piRNA source locus in follicular cells, and to be required for cell-specific transcription of the flamenco locus.
Our findings suggest that BEAF32 coordinates multiple transcriptional regulatory functions important for Drosophila oogenesis. BEAF32 represses the ectopic expression of developmental and tissue-specific genes in the ovaries. BEAF32 regulates polo kinase and other oogenesis-related genes. We demonstrate here that BEAF32 play a specific ovarian role in the maintenance of piRNA-producing loci. Our results support an important role for the BEAF32 insulator protein in determining the proper landscape of tissue-specific gene expression.
绝缘子是多功能DNA结合蛋白，具有构建功能并调节基因转录。尽管绝缘子在基因组组织中具有明确的作用，但绝缘子蛋白如何影响组织特异性过程的控制仍不清楚。果蝇绝缘子BEAF32（32kD边界元件相关因子）是染色质复合物的一个组成部分，存在于包含管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和雌性生育能力，表明其在卵子发生过程中具有特定功能。
为了更好地了解BEAF32在卵子发生中的作用，我们首先检测了其在卵巢中的结合靶点，发现其定位位点在管家基因和组织特异性基因的启动子中均有富集。差异表达基因分析表明，BEAF32基因敲除导致卵巢中非卵巢组织特异性基因的异常激活，这意味着BEAF32调节基因表达的组织特异性模式。我们发现BEAF32占据了许多卵巢特异性基因的启动子，并作为细胞周期调节激酶Polo表达的正调控因子。为了研究BEAF32在与Piwi相互作用的RNA（piRNA）途径中的可能作用，我们分析了BEAF32基因缺失突变体中的卵巢小RNA，发现3R亚端粒区域的piRNA产生量大幅下降。我们的数据表明，位于亚端粒重复序列上游的含BEAF32染色质复合物维持了生殖系中该区域的转录和染色质完整性。还发现BEAF32定位于滤泡细胞中主要的piRNA来源位点flamenco上游，并对flamenco位点的细胞特异性转录是必需的。
我们的研究结果表明，BEAF32协调了对果蝇卵子发生重要的多种转录调控功能。BEAF32抑制卵巢中发育和组织特异性基因的异位表达。BEAF32调节polo激酶和其他与卵子发生相关的基因。我们在此证明，BEAF32在维持piRNA产生位点方面发挥着特定的卵巢作用。我们的结果支持BEAF32绝缘子蛋白在确定组织特异性基因表达正确格局方面发挥重要作用。
