Ryan R F, Schultz D C, Ayyanathan K, Singh P B, Friedman J R, Fredericks W J, Rauscher F J
The Wistar Institute, Philadelphia, Pennsylvania, USA.
Mol Cell Biol. 1999 Jun;19(6):4366-78. doi: 10.1128/MCB.19.6.4366.
Krüppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments.
克勒ppel相关盒（KRAB）结构域存在于所有人类锌指蛋白（ZFP）的约三分之一中，是强大的转录抑制模块。我们之前克隆了一种KRAB结构域的共抑制因子KAP-1，它是KRAB在体内介导的抑制作用所必需的。为了表征KAP-1所利用的抑制机制，我们分析了KAP-1与HP1蛋白家族的小鼠（M31和M32）和人类（HP1α和HP1γ）同源物相互作用的能力，HP1蛋白家族是一类非组蛋白异染色质相关蛋白，在果蝇中具有成熟的表观遗传基因沉默功能。体外研究证实，KAP-1能够直接与通常存在于着丝粒异染色质中的M31和hHP1α相互作用，以及与常染色质中均存在的M32和hHP1γ相互作用。对KAP-1中HP1相互作用所需区域的定位表明，在体外消除HP1结合的氨基酸取代会降低KAP-1在体内介导的抑制作用。我们观察到KAP-1与M31和M32在间期核中共定位，这支持了M31和M32直接与KAP-1相互作用的生化证据。KAP-1与M31的共定位有时出现在潜在的着丝粒周围异染色质的亚核区域，而KAP-1和M32的共定位则出现在整个细胞核中的点状常染色质区域。这项工作提出了一种机制，即KRAB-ZFP-KAP-1复合物通过形成沉默基因活性的异染色质样复合物，将HP1样基因产物募集到基因组内的特定位点。我们推测基因特异性抑制可能是此类复合物形成的结果，最终导致新形成的异染色质染色体环境中的基因沉默。
