Springhetti Evelyn M, Istomina Natalia E, Whisstock James C, Nikitina Tatiana, Woodcock Chris L, Grigoryev Sergei A
Department of Biochemistry and Molecular Biology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
J Biol Chem. 2003 Oct 31;278(44):43384-93. doi: 10.1074/jbc.M307635200. Epub 2003 Aug 19.
MENT is a developmentally regulated heterochromatin-associated protein that condenses chromatin in terminally differentiated avian blood cells. Its homology to the serpin protein family suggests that the conserved serpin reactive center loop (RCL) and the unique M-loop are important for its function. To examine the role of these domains, we studied the interaction of wild-type and mutant MENT with naked DNA and biochemically defined nucleosome arrays reconstituted from 12-mer repeats containing nucleosome positioning sequences. Wild-type MENT folded the naked DNA duplexes into closely juxtaposed parallel structures ("tramlines"). Deletion of the M-loop, but not inactivation of the RCL, prevented tramline formation and the cooperative interaction of MENT with DNA. Reconstitution of wild-type MENT with nucleosome arrays caused their tight folding and self-association. M-loop deletion inhibited nucleosome array folding, whereas the inactive RCL mutant was competent to fold the nucleosome arrays, but had a significantly impaired ability to cause their self-association. Bifunctional chemical cross-linking of MENT revealed oligomerization of wild-type MENT in the presence of chromatin and DNA. This oligomerization was severely reduced in the RCL mutant. We propose that the mechanism of MENT-induced heterochromatin formation involves two independent events: bringing together nucleosome linkers within a chromatin fiber and formation of protein bridges between chromatin fibers. Ordered binding of MENT to linker DNA via its unique M-loop domain promotes the folding of chromatin, whereas bridging of chromatin fibers is facilitated by MENT oligomerization mediated by the RCL.
MENT是一种在发育过程中受调控的异染色质相关蛋白，它能使终末分化的禽类血细胞中的染色质浓缩。它与丝氨酸蛋白酶抑制剂（serpin）蛋白家族的同源性表明，保守的丝氨酸蛋白酶抑制剂反应中心环（RCL）和独特的M环对其功能很重要。为了研究这些结构域的作用，我们研究了野生型和突变型MENT与裸DNA以及由含有核小体定位序列的12聚体重复序列重构的生化定义的核小体阵列之间的相互作用。野生型MENT将裸DNA双链折叠成紧密并列的平行结构（“电车轨道”）。M环的缺失而非RCL的失活阻止了电车轨道的形成以及MENT与DNA的协同相互作用。野生型MENT与核小体阵列的重构导致它们紧密折叠和自我结合。M环缺失抑制了核小体阵列的折叠，而无活性的RCL突变体能够折叠核小体阵列，但导致其自我结合的能力明显受损。MENT的双功能化学交联显示在染色质和DNA存在的情况下野生型MENT发生寡聚化。在RCL突变体中这种寡聚化严重减少。我们提出MENT诱导异染色质形成的机制涉及两个独立事件：使染色质纤维内的核小体连接子聚集在一起以及在染色质纤维之间形成蛋白质桥。MENT通过其独特的M环结构域与连接子DNA的有序结合促进染色质的折叠，而由RCL介导的MENT寡聚化促进染色质纤维的桥接。
