The bithorax complex iab-7 Polycomb response element has a novel role in the functioning of the Fab-7 chromatin boundary.
The bithorax complex iab-7 Polycomb response element has a novel role in the functioning of the Fab-7 chromatin boundary.
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DOI：
10.1371/journal.pgen.1007442
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发表时间：
2018-08
期刊：
影响因子：
4.5
通讯作者：
Georgiev P
中科院分区：
文献类型：
--
作者：
Kyrchanova O;Kurbidaeva A;Sabirov M;Postika N;Wolle D;Aoki T;Maksimenko O;Mogila V;Schedl P;Georgiev P
这三个双胸复合体同源异型基因的表达是由九个副段特异的调节域协调的。每个域的自主性由边界元素(绝缘体)授予。在这里，我们使用了一种原位替换策略来重新分析Fab-7这一特征最好的苍蝇边界之一的功能所需的序列。它最初是通过缺失Fab-71来鉴定的，该缺失将副分段(PS)11转变为PS12的复制副本。FAB-71缺失了位于IAB-6和IAB-7调节域之间的四个核酸酶敏感位点HS*、HS1、HS2和HS3。转基因和P元件切除实验将边界定位为HS*+HS1+HS2，而HS3被证明是IAB-7多梳反应元件(Pre)。最近的替换实验表明，当HS3也存在于替换结构中时，HS1对于边界活动既是必要的也是充分的。令人惊讶的是，虽然HS1+HS3组合具有完全的边界活性，但我们发现仅HS1单独具有最小的功能。此外，当与HS3结合时，只需要HS1的远端半部分，即dHS1。一个~1,000 kD的包含GAF蛋白的多蛋白复合体被称为LBC，与dHS1序列结合，我们发现dHS1的突变破坏了核提取液中的LBC结合，在体内消除了边界活性和GAF结合。HS3具有预先沉默所需的GAF和Pho蛋白的结合位点。相反，HS3边界活性只需要GAF结合位点。细胞核提取物中LBC与HS3的结合以及体内GAF的结合依赖于HS3的GAF位点，而不是Pho位点。与LBC在HS3边界活动中的作用一致，当果蝇的GAF基因突变为杂合子时，dHS1+HS3mPho组合的边界功能丢失。综上所述，这些结果揭示了IAB-7 PreS在染色体结构中的一种新功能。多梳蛋白(PcG)是所有高等真核生物发育基因的重要表观遗传调控因子。在果蝇中，这些蛋白质与特定的调控DNA元件结合，称为聚梳组反应元件(PreS)。果蝇PreS由一系列复杂的DNA结合蛋白的结合位点组成，包括GAF和Pho。在双胸复合体(BX-C)的调节区中，边界/绝缘体元件组织自治的调节域，它们的激活或抑制状态受PreS的调节。在这里，我们研究了构成FAB-7边界和相邻IAB-7 PRE的序列的功能性质。以前认为IAB-7 Pre的唯一功能是在PS12之前的副节段中招募PcG蛋白，并沉默IAB-7结构域。然而，我们发现IAB-7 Pre也作为Fab-7边界的一个组成部分。IAB-7前序列的边界活性取决于一种称为LBC的大型复合体。我们表明，通过将HS1中的LBC结合序列与IAB-7 PRE相结合，有可能重建一个完全功能的边界。此外，它的边界函数与它的PcG沉默活动无关。
Expression of the three bithorax complex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used an in situ replacement strategy to reanalyze the sequences required for the functioning of one of the best-characterized fly boundaries, Fab-7. It was initially identified by a deletion, Fab-71, that transformed parasegment (PS) 11 into a duplicate copy of PS12. Fab-71 deleted four nuclease hypersensitive sites, HS*, HS1, HS2, and HS3, located between the iab-6 and iab-7 regulatory domains. Transgenic and P-element excision experiments mapped the boundary to HS*+HS1+HS2, while HS3 was shown to be the iab-7 Polycomb response element (PRE). Recent replacement experiments showed that HS1 is both necessary and sufficient for boundary activity when HS3 is also present in the replacement construct. Surprisingly, while HS1+HS3 combination has full boundary activity, we discovered that HS1 alone has only minimal function. Moreover, when combined with HS3, only the distal half of HS1, dHS1, is needed. A ~1,000 kD multiprotein complex containing the GAF protein, called the LBC, binds to the dHS1 sequence and we show that mutations in dHS1, that disrupt LBC binding in nuclear extracts, eliminate boundary activity and GAF binding in vivo. HS3 has binding sites for GAF and Pho proteins that are required for PRE silencing. In contrast, HS3 boundary activity only requires the GAF binding sites. LBC binding with HS3 in nuclear extracts, and GAF association in vivo, depend upon the HS3 GAF sites, but not the Pho sites. Consistent with a role for the LBC in HS3 boundary activity, the boundary function of the dHS1+HS3mPho combination is lost when the flies are heterozygous for a mutation in the GAF gene. Taken together, these results reveal a novel function for the iab-7 PREs in chromosome architecture. Polycomb group proteins (PcG) are important epigenetic regulators of developmental genes in all higher eukaryotes. In Drosophila, these proteins are bound to specific regulatory DNA elements called Polycomb group Response Elements (PREs). Drosophila PREs are made up of binding sites for a complex array of DNA binding proteins, including GAF and Pho. In the regulatory region of the bithorax complex (BX-C), the boundary/insulator elements organize the autonomous regulatory domains, and their active or repressed states are regulated by PREs. Here, we studied functional properties of sequences that constitute the Fab-7 boundary and the adjacent iab-7 PRE. It was previously thought that the sole function of the iab-7 PRE is to recruit PcG proteins in parasegments anterior to PS12 and silence the iab-7 domain. However, we found that the iab-7 PRE also functions as a component of the Fab-7 boundary. The boundary activity of the iab-7 PRE sequence depends upon a large complex called the LBC. We show that it is possible to reconstitute a fully functional boundary by combining the LBC binding sequences in HS1 with the iab-7 PRE. Moreover, its boundary function is independent of its PcG silencing activity.
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