Normal and Pathologic Functions of DNA-binding shared by

从2005年10月1日到2006年9月1日，我们继续研究了正常细胞和癌细胞染色质中各种功能的全基因组特征，这些功能是通过50 bp长的DNA序列特异性结合两个独特的核因子CTCF和BORIS共享的高度保守的11锌指（11ZF） DNA结合域（DBD）来实现的。CTCF是一种高度保守、多功能、多价的核因子，在16q22位点具有肿瘤抑制因子的特性。它在所有体细胞中普遍表达，参与启动子的激活和抑制，激素诱导的基因沉默，以及构成或甲基化敏感染色质边界的创造。在理解表观遗传机制方面的一个令人兴奋的突破是我们发现了一个被称为BORIS（印迹位点调控因子的兄弟）的CTCF对偶。正常情况下，所有体细胞只表达CTCF，不表达BORIS。在男性生殖细胞分化过程中，这对基因表现出一种互斥的表达模式，与以下模式相关：(1)dna甲基化标记的全基因组重建，(2)某些睾丸特异性染色质重塑因子和dnmt的表达。BORIS通常仅在男性生殖细胞分化过程中以与CTCF互斥的模式表达，而CTCF与表观遗传重编程紧密耦合。我们之前发现CTCF和BORIS具有相同的外显子编码11 ZF DBD，与CTCF/BORIS结合位点的相同谱相互作用，但在氨基端和羧基端存在差异。这两个基因最初是由V. Lobanenkov及其合作者鉴定和分子克隆的。因此，体细胞CTCF和睾丸特异性BORIS可以分别通过表现和重编程父系表观遗传状态作为对抗基因。母体表观遗传标记位点需要具有相同DBD但在胚胎卵巢中表达的第三个因子。事实上，我们发现了BORIS的雌性生殖细胞特异性对应物，我们将其命名为“Natasha”，以突出与雄性生殖细胞特异性BORIS和体细胞CTCF的差异。今年，对Natasha的部分cDNA克隆的研究发现，BORIS和CTCF具有相同的11ZF DBD。娜塔莎的鉴定完成了我们在染色质区域重建和读取父母决定的表观遗传标记所需的所有三个因素的搜索，这些DNA序列被这三个独特因素共享的相同11ZF DBD所识别。我们称这些序列为CTCF/BORIS/ natasha -target sites，或CBN-sites。我们关于这些因素如何运作的工作模型表明；1)体内进入cbn位点的调控高度依赖于细胞类型；2)这些因素在体内占据相同的基因组位点会产生不同的功能结果；3)这些基因的非预定共表达将导致共同结合位点的竞争，从而导致表观遗传状态的全基因组变化，这些变化已被发现是永生化和转化细胞的特征。事实上，我们实验室和其他实验室的研究表明，BORIS在许多不同类型的癌症中异常表达的比例很高，这些癌症也均匀表达CTCF。根据这一假设，我们已经证明BORIS和CTCF在共享目标位点上确实相互竞争。当BORIS占据CTCF通常占据的位点时，可能导致通常仅由一个等位基因表达的基因双等位表达，例如与该位点两侧DNA甲基化改变相关的印迹基因。了解CTCF/BORIS/NATASHA与DNA相互作用的细节的重要性在人类基因决定的异常研究中变得非常明显。第一个是关于贝克威斯-魏德曼综合征（BWS）的家庭。BWS是一种以胎儿和围产期生长异常和儿童实体瘤风险增加为特征的疾病。在两个家族中，BWS与一个小的缺失有关，该缺失消除了关键印迹控制区（ICR）中的两个CTCF靶位点，而在其他家族中，这些位点被甲基化，因此无法结合CTCF。因此，CTCF与这些靶标结合的能力似乎是该位点正常基因表达的主要决定因素。第二个与X染色体失活的异常调控有关。在正常女性中，两条X染色体中的任何一条一个来自父亲，一个来自母亲-将在生理上失活基本上是随机的，CTCF与一条染色体上的关键区域的结合已知会标记该染色体失活。已知这一过程有两种异常变体：1)基本上只有来自父母一方的X基因沉默；2) X基因失活的几率显著降低。我们发现第一种情况是由一个点突变引起的，该突变显著增加了CTCF对该位点的结合亲和力，使其基本上不可逆。第二种是由同一位点的不同碱基突变引起的，但在这种情况下基本上取消了CTCF结合。最后，我们正在努力了解CTCF和BORIS在体内区分功能不同靶点的能力。目前的研究表明，中央11ZF DBD的不同侧翼负责与其他蛋白质（包括参与染色质修饰的因子）的差异配对。

From 10-01-05 to 9-01-06, we continued our efforts on a genome wide characterization of various functions mediated in chromatin of normal and cancer cells by the 50-bp-long DNA sequences capable of specific binding to the highly conserved 11 Zinc Finger (11ZF) DNA-binding domain (DBD) shared in two unique nuclear factors, CTCF and BORIS. CTCF is highly conserved, multifunctional, multivalent, nuclear factor with the properties of a tumor suppressor at 16q22. It is ubiquitously expressed in all somatic cells, and involved in promoter activation and repression, hormone-inducible gene silencing, and creation of constitutive or methylation-sensitive chromatin boundaries. An exciting breakthrough in understanding the epigenetic mechanisms was our discovery of a paralogue of CTCF termed BORIS (for Brother Of Regulator of Imprinted Sites). Normally, only CTCF but not BORIS is expressed in all somatic cells. During male germ cell differentiation, the pair displays a mutually exclusive expression pattern that correlates with the patterns for: (1) genome-wide re-establishment of DNA-methylation marks, and (2) expression of certain testis-specific chromatin-remodeling factors and DNMTs. BORIS is normally expressed only during male germ cell differentiation in a pattern mutually exclusive to that of CTCF, which is tightly coupled with epigenetic reprogramming. We showed earlier that CTCF and BORIS share identical exons encoding the 11 ZF DBD to interact with the same spectrum of CTCF/BORIS-binding sites, but diverge at the amino- and carboxy-termini. Both genes were originally identified and molecularly cloned by V. Lobanenkov and collaborators. Thus, somatic CTCF and testis-specific BORIS could serve as counteracting genes by manifesting and reprogramming paternal epigenetic states, respectively. The sites for maternal epigenetic marking require a third factor with the same DBD but expressed in embryonic ovaries. Indeed, we discovered a female germ cell-specific counterpart of BORIS, that we have named "Natasha" to highlight the differences from the male germ cell-specific BORIS and from somatic CTCF. This year, studies of partial cDNA clones of Natasha revealed the same 11ZF DBD that was earlier shown to be common to BORIS and CTCF. Identification of Natasha has completed our search of all three factors required for re-establishing and reading parentally-determined epigenetic marks at chromatin regions with DNA sequences recognized by the the same 11ZF DBD shared by these three unique factors. We called such sequences CTCF/BORIS/NATASHASHA-target sites, or "CBN-sites". Our working model of how these factors operate suggests; 1) that regulation of access to CBN-sites in vivo is highly cell type dependent; 2) that different functional outcomes are to be expected from the in vivo occupancy of the same genomic sites by each of these factors; and 3) that unscheduled co-expression of these genes would lead to competition for common binding sites leading to genome-wide changes in epigenetic status that have been found to characterize immortalized and transformed cells. Indeed, studies from our laboratory and others have shown that BORIS is aberrantly expressed in a high proportion of cancers of many different types that uniformly express CTCF as well. In accord with this hypothesis, we have shown that BORIS and CTCF do compete with one another at shared target sites. Occupancy by BORIS of sites normally occupied by CTCF can result in biallelic expression of genes normally expressed from only one allele, such as imprinted genes in association with altered methylation of DNA flanking the site. The importance of understanding the fine details of CTCF/BORIS/NATASHA interactions with DNA became strikingly apparent in studies of genetically determined abnormalities in humans. The first revolved around families with Beckwith-Wiedemann syndrome (BWS). BWS is a disorder characterized by fetal and perinatal growth abnormalities and increased risk of childhood solid tumors. It was found two families in which BWS was associated with a small deletion that eliminated two CTCF target sites in a critical imprinting control region (ICR) and other families in which these sites were methylated and thus unable to bind CTCF. The ability of CTCF to bind to these target thus appears to be a major determinant of normal gene expression at this locus. The second relates to aberrant regulation of X chromosome inactivation. In normal women, the chance that either one of the two X chromosomes ? one from the father and one from the mother - will become physiologically inactivated is essentially random The binding of CTCF to a critical region on just one chromosome is known to mark that chromosome for inactivation. Two abnormal variants of this process were known to occur: 1) cases in which essentially only the X from one parent was silenced; and 2) cases in which the chances that either X would be inactivated were markedly diminished. We found that the first scenario was cased by a point mutation that markedly increased the binding affinity of CTCF for the site, making it essentially irreversible. The second was caused by a different base mutation at the same exact site but in this instance essentially abolished CTCF binding. Finally, we are working to understand the ability of CTCF and BORIS to discriminate among functionally distinct targets in vivo. Current studies suggest that the dissimilar flanks to the central 11ZF DBD are responsible for differential partnering with other proteins including factors involved in chromatin modification.