Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

从10-01-06到9-01-07，我们继续研究BORIS （Brother of the Regulator of Imprinted Sites的缩写），这是我们发现的一个独特的ctcf同源基因。在人类和小鼠中，CTCF和BORIS基因编码大小相似的多肽，它们共享一个由11个锌指（11ZF）组成的中心位置接近相同的DNA结合域（DBD）。在20世纪90年代中期，这个显著的11ZF区域被定义为一个多价DBD，涉及到双链DNA中识别的异常延伸（平均50bp）靶序列的多样性。BORIS和CTCF共有的这个DBD区域是由基因组序列编码的，该基因组序列精确地复制了早期哺乳动物CTCF基因中包含所有zf编码外显子的区域。因此，虽然与CTCF共享11ZF DBD的副本允许BORIS特异性结合与CTCF相互作用的相同dna靶序列，但与CTCF结合同一位点相比，BORIS在体内结合到给定的CTCF位点会导致不同的功能结果。正常情况下，CTCF和BORIS兄弟姐妹不在同一细胞中表达，以避免因与11ZF dna靶标的相同谱结合而引起的功能干扰。尽管许多ctcf靶序列并不总是包含哺乳动物甲基化位点（meCpG），但对这种DNA修饰敏感的一部分位点与正常单等位基因DNA甲基化和基因表达的调节和维持、组蛋白修饰模式、非编码rna的存在以及一些统称为表观遗传标记的其他特征有关。一些甲基化敏感的ctcf结合位点与体细胞、胚胎干细胞和生殖细胞的表观遗传调控过程密切相关。这些位点已被普遍定位在印迹控制区（ICR）的差异甲基化区域，该区域调节印迹基因簇中依赖于亲本的单等位基因表达。2000年，三个实验室同时独立地报道了Igf2/H19基因座印迹控制区（ICR）中cpg甲基化调控的一组新的CTCF位点的定位和功能表征。此外，在我们早期关于CTCF和印迹的论文中，我们预测尽管不同印迹位点上的不同ICR之间缺乏明显的同源性，但大多数（如果不是全部）ICR序列应该包含mecpg敏感的CTCF位点。这一预测被证明是正确的，因为我们与几个实验室的合作努力导致了三个最近的出版物（参见本报告和以前的FY报告中的参考文献），描述了ctcf驱动的Rasgrf-1、AWT1/WT1-AS和KvDMR印迹基因位点ICRs中的甲基化敏感绝缘子。利用非洲爪蟾卵母细胞注射的生化系统表达BORIS及其表观遗传辅助因子Jelinic等人发现，BORIS在预组装的染色质背景下招募含有PRMT7和DNMTs的复合体，将CpG靶向H19 ICR的ctcf位点。BORIS在体细胞中通常是严格沉默的，但与一种称为癌睾丸抗原（cta）的特殊基因家族一起被激活。由于编码x连锁CTA基因的启动子区域在男性生殖细胞发育过程中被激活，经历全基因组去甲基化（CTCF水平降低），我们想知道BORIS是否参与了CTA基因激活的协调调节。2005年，我们发现并发表了一类新的含有cpg的CTCF靶序列，它们与CTCF表现出甲基化不敏感而不是甲基化敏感的相互作用。这种类型的CTCF/ boris靶点最初是在MAGE-A1和NY-ISO-1中发现和表征的，这是两个研究得最好的CTA基因的例子。对这类新的CTCF/BORIS靶序列的表征使我们能够解释CTCF如何在体内占据作为失活染色质的一部分甲基化/沉默的DNA区域，以及BORIS如何结合这些DNA序列以将去甲基化招募到高甲基化的CTCF位点，从而导致去甲基化和CTA基因启动子的激活。超过12种CTA和其他生殖/干细胞特异性基因，通常在睾丸中与男性生殖细胞中的BORIS共表达，也可以被正常的原代人成纤维细胞中的异位BORIS表达激活。接下来，我们发现BORIS本身属于CTA家族，在许多类型的人类癌症中异常激活。在许多癌症中，CTCF位于LOH最小重叠区域的16q22位点，而BORIS位于这些癌症中通常复制获得/扩增的20q13位点。因此，我们继续进行研究，旨在了解BORIS作为CTCF肿瘤抑制活性的干扰突变的作用。我们正在进行的研究表明，BORIS与CTCF竞争结合同一组共同的靶点可以解除增殖/凋亡相关的CTCF靶基因的调控，包括c-myc、p27、p21、p53和p19/ARF、PAX6、Wsb1/Nf1、BRCA1、Rb和hTERT，从而具有致癌作用。BORIS高度受限的表达和独特的功能为诊断、免疫治疗或其他靶向治疗提供了巨大的机会。BORIS在包括但不限于乳腺、前列腺、卵巢、肺、胃、肝、子宫内膜、神经胶质、结肠、食道等多种不同组织学来源的肿瘤中异常表达，其发生率远高于其他CT基因，但也能上调其他CT基因的表达。我们之前已经证明，当BORIS在肿瘤中异常表达时，癌症患者会产生抗体反应（正在申请专利）。最近，我们还发现（与英国埃塞克斯大学的E. Klenova博士合作）至少乳腺癌患者在多形核粒细胞中有BORIS表达。这种与肿瘤相关的现象在有损伤、免疫和炎症性疾病的供体中没有观察到。在各类乳腺肿瘤患者中检测到BORIS的比例较高，提示BORIS可作为一种有价值的乳腺癌早期血液标志物。作为一种治疗方法，我们尝试了疫苗疗法。首先，为了获得可靠的可行性证据，Loukinov等人在一种侵袭性强、高转移性、免疫原性差的乳腺癌小鼠模型中测试了BORIS疫苗。在接种4T1细胞的BALB/c小鼠中，用编码小鼠BORIS抗原突变形式（不包括11ZF区域）的DNA进行免疫，可显著延长存活时间，并抑制肿瘤生长。用表达mBORIS的腺病毒载体增强通常更有效，表明疫苗接种方案可以进一步优化。然后，我们与M. Agadjanyan合作，利用BORIS分子的非DNA结合版本，产生了基于DNA和蛋白质的小鼠BORIS抗肿瘤疫苗。为了增强抗boris细胞免疫应答，我们使用了标准的分子佐剂方法。它包括编码小鼠IL-12和IL-18的质粒（用于dna疫苗）和常规Th1型佐剂Quil a（用于蛋白质疫苗）。基于DNA和基于蛋白质的疫苗分别诱导具有Th1和Th2细胞因子谱的ag特异性CD4+ T细胞增殖。BORIS疫苗是基于蛋白质的，而不是基于dna的，可以诱导大量的Ab产生。重要的是，使用基于dna而非基于蛋白质的BORIS疫苗免疫后，产生了有效的抗癌CD8+细胞毒性淋巴细胞。此外，这些细胞溶解反应在多种不同的小鼠癌症中被观察到，包括乳腺腺癌、胶质瘤、白血病和肥大细胞瘤。

From 10-01-06 to 9-01-07, we continued our studies of BORIS (an acronym for Brother of the Regulator of Imprinted Sites) - a unique CTCF-paralogous gene that we discovered. In humans and in mice, CTCF and BORIS genes encode polypeptides of similar size that share a centrally positioned near identical DNA binding domain (DBD) composed of 11 Zn-fingers (11ZF). In the mid 90s, this remarkable 11ZF region was defined as a multivalent DBD with respect to the diversity of unusually extended (average 50bp) target sequences recognized in double-stranded DNA. This DBD region shared in BORIS and CTCF is encoded by genomic sequences displaying an accurate duplication of a region containing all ZF-coding exons of CTCF gene from an early mammal. Therefore, while sharing with CTCF a duplicate of the 11ZF DBD allows BORIS to bind specifically to the same DNA-target sequences that interact with CTCF, in vivo binding of BORIS to a given CTCF-site would result to a different functional outcome compared to CTCF binding to the same site. Normally, CTCF and BORIS siblings are not expressed in the same cell to avoid functional interference caused by competition for binding to the same spectrum of 11ZF DNA-targets. Although numerous CTCF-target sequences do not always contain sites of mammalian methylation (meCpG), a subset of sites sensitive to this DNA modification have been associated with regulation and maintenance of normal mono-allelic DNA methylation and gene expression, histone-modifications patterns, presence of non-coding RNAs, and some additional features collectively known as epigenetic marks. Several methylation-sensitive CTCF-binding sites have been strongly implicated in the processes of epigenetic regulation in somatic cells, in ES cells, and in germline cells. Such sites have been universally mapped in differentially methylated domains of imprinting control regions (ICR) that regulate a parent-of-origin-dependent mono-allelic expression within clusters of imprinted genes. In 2000, three laboratories have simultaneously and independently reported mapping and functional characterization of a novel set of CTCF sites regulated by CpG-methylation in the imprinting control region (ICR) of the Igf2/H19 locus. Moreover, in our early papers on CTCF and imprinting, we predicted that in spite of the lack of any obvious homology between various ICRs mapped in different imprinted loci, most (if not all) of ICR sequences shall contain meCpG-sensitive CTCF-sites. This prediction turned out to be correct, since our collaborative efforts with several labs resulted in three more recent publications (see references in this and previous FY reports) describing CTCF-driven methylation-sensitive insulators in ICRs of Rasgrf-1, AWT1/WT1-AS, and KvDMR imprinted gene-loci. Using a biochemical system of Xenopus oocyte injection for expression of BORIS and its epigenetic co-factors Jelinic et. al. showed that BORIS recruits a complex containing PRMT7 and DNMTs to target CpG to CTCF-sites of the H19 ICR in a pre-assembled chromatin context. BORIS is normally strictly silenced in somatic cells, but activated together with a particular family of genes, called cancer-testis antigens (CTAs). Since promoter regions of genes encoding X-linked CTAs are activated during development of male germ cells undergoing genome-wide demethylation (and having decreased levels of CTCF), we asked if BORIS involved in coordinated regulation of CTA gene activation. In 2005, we discovered and published a novel class of CpG-containing CTCF-target sequences that manifested methylation-insensitive rather than methylation-sensitive interactions with CTCF. This type of CTCF/BORIS-target sites was at first found and characterized in MAGE-A1 and NY-ISO-1 - two best-studied examples of the CTA genes. Characterization of this new class of CTCF/BORIS-target-sequences allowed us to explain how CTCF can occupy in vivo a DNA region that is methylated/silenced as a part of inactivated chromatin, and how BORIS can bind to such DNA sequences to recruit demethylation to a hypermethylated CTCF-site, which results in demethylation and activation of CTA gene-promoters. Over a dozen of additional CTA and other germ/stem-specific genes, normally co-expressed in testis with BORIS in male germ cells, can also be activated by ectopic BORIS expression in normal primary human fibroblasts. Next, we showed that BORIS itself belongs to the CTA family, aberrantly activated in many types of human cancers. While CTCF is located at 16q22 locus at smallest region of overlap for LOH in wide variety of cancers, BORIS is located at 20q13 locus that is commonly copy-gained/amplified in these cancers. Therefore, we continued our studies directed to understanding the role of BORIS as an interfering mutation for tumor-suppressor activity of CTCF. Our ongoing studies suggest that competition of BORIS with CTCF for binding to the same common set of target sites can deregulate proliferation/apoptosis-related CTCF-target genes including c-myc, p27, p21, p53, and p19/ARF, PAX6, Wsb1/Nf1, BRCA1, Rb and hTERT, thus having an oncogenic effect. Highly restricted expression and unique function of BORIS provides a great opportunity for diagnostics, immunotherapeutic, or other targeted therapies. BORIS appears to be aberrantly expressed with a much higher then other CT-genes incidence in many tumors of different histological origin including, but not limited to breast, prostate, ovary, lung, gastric, liver, endometrial, glia, colon, esophagus but also is capable of up-regulating expression of other CT genes. Weve shown previously that BORIS when abnormally expressed in tumors induces antibody response in cancer patients (patent pending). More recently, we also showed (in collaboration with Dr. E. Klenova of the Essex University, UK) that at least breast cancer patients have BORIS expression in polymorphonuclear granulocytes. This tumor-related occurrence is a phenomenon not observed in donors with injuries and immune and inflammatory diseases. Detection of BORIS in a high proportion of patients with various types of breast tumors indicates that BORIS can be a valuable early blood marker of breast cancer. As a therapeutic approach weve tried vaccine approach. First, to obtain a solid proof of feasibility, Loukinov et al., tested BORIS as a vaccine in a very aggressive, highly metastatic, and poorly immunogenic murine model of mammary carcinoma. Immunizations with a DNA encoding the mutant form of murine BORIS antigen (excluding its 11ZF region) significantly prolonged survival, and inhibited tumor growth in BALB/c mice inoculated with 4T1 cells. Boosting with adenoviral vector expressing mBORIS was generally more effective, suggesting that the vaccination protocol could be further optimized. Then in collaboration with M. Agadjanyan we generated DNA- and protein-based mouse BORIS anti-tumor vaccines using again non-DNA-binding version of the BORIS molecule. To enhance anti-BORIS cellular immune responses, we used a standard molecular adjuvant approach. It consisted of plasmids encoding murine IL-12 and IL-18 for a DNA-based vaccine and conventional Th1 type adjuvant, Quil A, for a protein-based vaccine. Both DNA- and protein-based vaccines induced Ag-specific CD4+ T cell proliferation with Th1 and Th2 cytokine profiles, respectively. Protein-based, but not DNA-based, BORIS vaccine induced a significant level of Ab production. Importantly, potent anticancer CD8+-cytotoxic lymphocytes were generated after immunization with the DNA-based, but not protein-based, BORIS vaccine. Moreover, these cytolytic responses were observed across a wide range of different mouse cancers including mammary adenocarcinoma, glioma, leukemia, and mastocytoma.