Normal and Pathologic Functions of CTCF and Its Distinct Classes of DNA-targets

CTCF是一种高度保守的多功能核因子，参与全球基因组结构和基因调控的许多方面，后者的范围从直接的基因抑制/激活到增强子阻断和转录促进沉默。CTCF含有高度保守的11个锌指DNA结合结构域，其介导DNA结合的序列特异性。正如我们所发现的，CTCF通过两种主要机制发挥作用：直接调节CTSes下游基因或通过形成染色质环间接调节。通过CTCF二聚化稳定的染色质环影响启动子、增强子和/或ICR之间的关系。DNA结合的CTCF的二聚化活性可能是其在大多数细胞类型中作为多功能染色质桥接剂和染色质成环剂的活性的核心，是其核心生物学功能的基础。此外，CTCF的成环活性可以自然地延伸到形成局部的体细胞染色体间配对位点，从而获得表观遗传共调节的潜力，如转录工厂、DNA复制工厂和DNA修复灶。许多其他染色质锚定的功能，如印记标记的建立和它们的阅读、X染色体失活和细胞凋亡都受到CTCF的调节。CTCF已经成为间期染色质3D组织的关键促进者，也是细胞增殖控制的主要参与者。在某些情况下，发现CTCF的成环活性伴随/补充了特定基因的更直接调控。这种混合模式调控可能是天然基因调控框架的最合适的代表。我们还鉴定了一种新的CTCF活性，其直接将CTCF与CTCF与Pol II的转录机制结合联系起来。这种新的途径提供了一种机制，用于打开启动子决定的+1位点下游的环非依赖性转录起始位点（在内含子/外显子序列处），或者可能已经特异性地进化以诱导整个基因组的非编码转录物。从机制上讲，Pol II从DNA结合的CTCF复合物中的调节性募集和随后的释放表明CTCF位点本身可以在基因组中的一些位置中充当衰减子和/或启动子。虽然CTCF主要被认为是基因表达的调节剂，但我们关于其在异染色质和中心体中的潜在功能以及其在有丝分裂和减数分裂中的作用的数据表明，CTCF在基因组组织和染色体分离中具有重要的管家作用。CTCF以前被证明经历了各种翻译后修饰，我们扩大了这些研究，以表征新的修饰。启动子靶位点的CTCF占据失调的另一个病理学方面是癌症中的异常DNA甲基化。CTCF的这两种新型生物学作用是MPS正在进行的研究的主题。 我们先前首次分析了全基因组CTCF靶标（Cell 2007，第128卷，第1231 -1245页），并且CTCF在细胞功能中的基本作用通过CTS与人类基因组中基因位置的强相关性来验证。由于具有如此多的重要功能，CTCF成为脊椎动物中的必需基因，因为CTCF敲除小鼠是不能存活的（在非常早期的胚胎阶段是致命的）。 关于人类疾病，CTCF是一个候选的肿瘤抑制基因（TSG）;在原发性癌症中，CTCF的11 ZF DBD中的几个功能性点突变已被表征，与CTCF基因座的洛组合。在过去的一年中，我们研究了几个位点，以了解CTCF CTSes的贡献，他们的监管。它们包括对免疫反应重要的基因，以及具有开发癌症治疗方法潜力的基因。作为一般规则，我们已经发现，如果CTCF结合位点位于转录起始位点的上游，则其倾向于发挥激活剂作用，而位于（+1）下游的CTSes通常表现为阻遏物。端粒酶催化亚基（catalytic subunit of human telomerase，hTERT）基因是本研究中最突出的基因之一。

CTCF is a highly conserved, multi-functional nuclear factor that involved both in global genome architecture and in many aspects of gene regulation, the latter ranging from the direct gene repression/activation to enhancer blocking and hormone-facilitated silencing. CTCF contains a highly conserved 11 Zn-finger DNA-binding domain that mediates sequence-specificity of DNA binding. As we discovered, CTCF functions through two major mechanisms: either direct regulation of a gene downstream of CTSes or indirect regulation via the formation of chromatin loops. The chromatin loops, stabilized by CTCF dimerization, affect relationships among promoters, enhancers and/or ICRs. Dimerization activity of DNA-bound CTCF may potentially be at the core of its activity as a versatile chromatin-bridging and chromatin-looping agent in most cell types, underlying its core biological functions. Furthermore, the loop-forming activity of CTCF can be naturally extended to formation of localized somatic inter-chromosome pairing sites that therefore acquire potential for epigenetic co-regulation such as transcription factories, DNA replication factories, and DNA repair foci. Many other chromatin-anchored functions, such as the establishment of imprinting marks and their reading, X-chromosome inactivation, and apoptosis are regulated by CTCF. CTCF has emerged as a key facilitator of 3D organization of interphase chromatin, as well as a major player in cell proliferation control. In some cases, the loop-forming activity of CTCF was found to be accompanied/complemented by the more direct regulation of a particular gene. This mixed mode regulation is likely the most appropriate representation of a native gene regulation framework. We also identified a novel CTCF activity that directly links CTCF to transcriptional machinery binding of CTCF to Pol II. This novel pathway provides either a mechanism for opening loop-independent transcription start sites downstream of the promoter-determined +1 site (at intron/exon sequences) or may have specifically evolved to induce non-coding transcripts throughout the genome. Mechanistically, the regulated recruitment and the subsequent release of Pol II from a DNA-bound CTCF complex indicates that the CTCF site itself could act as an attenuator and/or promoter in some locations in the genome. While CTCF is mostly known as a regulator of gene expression, our data on its potential functions in heterochromatin and centrosomes, as well as its roles in mitosis and meiosis, suggested a significant housekeeping role of CTCF in genome organization and chromosome segregation. CTCF was previously shown to undergo a variety of posttranslational modifications, and we expanded these studies to characterize novel modifications. Another pathological aspect of the deregulated CTCF occupancy of promoter targets sites is aberrant DNA methylation in cancers. Both of these novel biological roles of CTCF are subjects of ongoing studies in the MPS. We previously analyzed genome-wide CTCF targets for the first time (Cell 2007, vol. 128, pp1231-1245), and the fundamental roles of CTCF in cellular functions were validated by a strong correlation of CTSs with gene positions in human genome. By virtue of having so many vital functions CTCF became an essential gene in vertebrates, as CTCF-knockout mice are non-viable (lethality at the very early embryonic stages). With respect to human disease, CTCF is a candidate tumor suppressor gene (TSG); several functional point mutations in the 11ZF DBD of CTCF have been characterized in primary cancers, in combination with the LOH of the CTCF locus. In the past year, we studied several loci in order to understand contributions of CTCF CTSes to their regulation. They included genes important for immune responses, as well as genes with a potential for the development of approaches for cancer treatment. As a general rule, we have found that if a CTCF binding site is located upstream of the transcriptional start site, it tends to play an activator role, while CTSes located downstream of (+1) usually behave as repressors. The gene for catalytic subunit of human telomerase (hTERT) is one of the most prominent genes in this study.