Poly(ADP-ribose) Polymerase-1 (PARP-1) Contributes to the Barrier Function of a Vertebrate Chromatin Insulator
Poly(ADP-ribose) Polymerase-1 (PARP-1) Contributes to the Barrier Function of a Vertebrate Chromatin Insulator
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DOI：
10.1074/jbc.m110.174532
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发表时间：
2010-11-26
影响因子：
4.8
通讯作者：
Emery, David W.
中科院分区：
文献类型：
--
作者：
Aker, Mari;Bomsztyk, Karol;Emery, David W.
原型染色质绝缘子cHS 4已被证明在多种环境中有效减少沉默染色体位置效应。这种屏障绝缘子活性的大部分已经被映射到一个250 bp的核心区域，以及结合该区域的几种蛋白质。然而，我们实验室最近的研究表明，cHS 4元件的延伸的400 bp核心区域是必要的，以实现完整的屏障绝缘子活性时，作为一个单一的副本，在重组γ逆转录病毒和慢病毒载体的背景下使用。在这项研究中，电泳凝胶迁移率变动分析揭示了特定的DNA-蛋白质结合活性与远端部分的这一扩展的核心区域。亲和纯化和串联质谱研究导致鉴定这些蛋白质之一为聚（ADP-核糖）聚合酶-1（PARP-1）。随后通过多种体外生物化学研究和体内染色质免疫沉淀研究验证了这种结合活性与PARP-1的一致性。在细胞系和原代小鼠骨髓祖细胞培养物中使用γ逆转录病毒报告载体的功能研究表明，cHS 4屏障活性在假定的PARP 1结合位点突变或PARP抑制剂治疗后分别被废除。在使用Parp 1缺失小鼠骨髓的研究中，也发现cHS 4元件的屏障活性被消除。总之，该研究表明，PARP-1的结合在延伸的cHS 4绝缘子核心元件的屏障活性中起关键的功能作用。
The prototypic chromatin insulator cHS4 has proven effective in reducing silencing chromosomal position effects in a variety of settings. Most of this barrier insulator activity has been mapped to a 250-bp core region, as well as to several proteins that bind this region. However, recent studies from our laboratory demonstrated that an extended 400-bp core region of the cHS4 element is necessary to achieve full barrier insulator activity when used as a single copy in the context of recombinant gammaretroviral and lentiviral vectors. In this study, electrophoretic gel mobility shift assays revealed specific DNA-protein binding activities associated with the distal portion of this extended core region. Affinity purification and tandem mass spectrometry studies led to the identification of one of these proteins as poly(ADP-ribose) polymerase-1 (PARP-1). The identity of this binding activity as PARP-1 was subsequently verified by a variety of biochemical studies in vitro and by chromatin immunoprecipitation studies in vivo. Functional studies with gammaretroviral reporter vectors in cell lines and primary mouse bone marrow progenitor cultures showed that cHS4 barrier activity was abrogated upon mutation of the putative PARP1-binding site or upon treatment with a PARP inhibitor, respectively. The barrier activity of the cHS4 element was also found to be abrogated in studies using bone marrow from Parp1-null mice. Taken together, this study demonstrates that binding of PARP-1 plays a key functional role in the barrier activity of the extended cHS4 insulator core element.