Cohesin and Polycomb proteins functionally interact to control transcription at silenced, restrained, and active genes [tiling array data]

Cohesin is crucial for proper chromosome segregation, but also regulates gene transcription and organism development by poorly understood mechanisms. We find that in Drosophila, cohesin functionally interacts with Polycomb group (PcG) silencing proteins at both silenced and active genes. Cohesin unexpectedly facilitates binding of Polycomb Repressive Complex 1 (PRC1) to many active genes. In contrast, cohesin and PRC1 binding are mutually antagonistic at silenced genes. PRC1 depletion decreases phosphorylated RNA polymerase and mRNA at many active genes, but increases them at silenced genes. Cohesin also facilitates long-range interactions between Polycomb Response Elements in the invected-engrailed gene complex where it represses transcription. These multiple distinct cohesin-PcG interactions reveal a previously unrecognized role for PRC1 in facilitating productive gene transcription, and provide new insights into how cohesin and PRC1 control development. ChIP-chip of cohesin, Polycomb group proteins, and RNA Polymerase II was performed in whole wing imaginal discs in developing wing imaginal disc, revealing that cohesin and Polycomb Repressive Complex 1 (PRC1) components co-bind with cohesin proteins at active genes. We then measured cohesin, Pc, and H3K27me3 separately in anterior and posterior wing imaginal discs and compared their binding at the invected-engrailed complex, which is silenced in the anterior disc, and expressed in its posterior. This confirmed that cohesin and PRC1 (Pc) co-bind at inv-en in its active state, and H3K27me3 and PRC1 (Pc) co-target inv-en in its silenced state. Comparison of binding between Pc-RJ and Pc-VP was performed, and revealed that Pc-VP is subject to epitope masking specifically at active genes. Finally, we measured cohesin and Pc binding in Drosophila ML-DmBG3-c2 cells, and found that they co-bind active genes in this cell line in as well as in wing imaginal discs. ChIP-chip of cohesin subunit Rad21 after PRC1 component Ph depletion, and ChIP-chip of PRC1 subunit Pc after Rad21 RNAi depletion, revealed that these two complexes affect one another's binding. Finally, ChIP-chip of Rpb3 (representing total Pol II) and Ser2P-Pol II (representing elongating Pol II) after PRC1 component Ph depletion revealed that PRC1 restrains entry of non-phosphorylated Pol II into gene bodies.黏连蛋白（Cohesin）对正常染色体分离至关重要，同时可通过尚未阐明的分子机制调控基因转录与机体发育。我们在果蝇（Drosophila）中发现，黏连蛋白与多梳家族（Polycomb group, PcG）沉默蛋白在沉默基因与活跃基因上均存在功能互作。出乎意料的是，黏连蛋白可促进多梳抑制复合体1（Polycomb Repressive Complex 1, PRC1）在众多活跃基因位点的结合；反之，在沉默基因位点，黏连蛋白与PRC1的结合却相互拮抗。PRC1敲低会降低众多活跃基因上的磷酸化RNA聚合酶与mRNA水平，却会提升沉默基因上的对应分子水平。黏连蛋白还可促进invected-engrailed基因复合体中多梳应答元件（Polycomb Response Elements）之间的远程相互作用，进而抑制该区域的转录。这类多样且独特的黏连蛋白-PcG互作，揭示了PRC1此前未被认知的促进有效基因转录的功能，并为阐明黏连蛋白与PRC1如何调控机体发育提供了新视角。我们在发育中的完整果蝇翅成虫盘（wing imaginal discs）中，针对黏连蛋白、多梳家族蛋白与RNA聚合酶II开展了染色质免疫沉淀芯片（ChIP-chip）实验，结果显示，在活跃基因位点，黏连蛋白与PRC1组分可共同结合。随后，我们分别在前翅成虫盘与后翅成虫盘内检测了黏连蛋白、Pc以及组蛋白H3赖氨酸27三甲基化（H3K27me3）的结合情况，并对比了二者在invected-engrailed基因复合体上的结合模式：该复合体在前翅成虫盘中处于沉默状态，在后翅成虫盘中则处于表达状态。实验证实，当invected-engrailed复合体处于激活状态时，黏连蛋白与PRC1（Pc）可共同结合该位点；而当其处于沉默状态时，H3K27me3与PRC1（Pc）则共同靶向该复合体。我们还对比了Pc-RJ与Pc-VP的结合情况，结果发现Pc-VP仅在活跃基因位点存在表位遮蔽现象。最后，我们在果蝇ML-DmBG3-c2细胞中检测了黏连蛋白与Pc的结合情况，发现该细胞系与翅成虫盘中一致，二者均可共同结合活跃基因位点。针对PRC1组分Ph敲低后的黏连蛋白亚基Rad21（Rad21）开展染色质免疫沉淀芯片实验，以及针对Rad21 RNA干扰（RNAi）敲低后的PRC1亚基Pc开展染色质免疫沉淀芯片实验，结果显示这两种复合体可相互影响对方的结合模式。此外，我们还在PRC1组分Ph敲低后，针对代表总RNA聚合酶II的Rpb3（Rpb3）以及代表延伸态RNA聚合酶II的丝氨酸2磷酸化RNA聚合酶II（Ser2P-Pol II）开展了染色质免疫沉淀芯片实验，结果表明PRC1可抑制非磷酸化RNA聚合酶II进入基因本体区域。