Epigenetic Regulation of Normal and Pathologic CTCF Functions by BORIS

在过去的一年里，我们继续研究BORIS (the Brother of the Regulator of imprint Sites)，这是我们在2002年发现并发表的ctcf同源基因。CTCF和BORIS蛋白具有几乎相同的11锌指（11ZF） DNA结合域（DBD），其两侧有不同的N端和c端区域。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，与双链DNA中识别的异常延伸（平均50bp）靶序列的多样性有关。BORIS和CTCF蛋白共享的DBD区域是由包含CTCF基因所有zf编码外显子的保守基因组区域的精确复制序列编码的。我们首先提出BORIS的非病理功能之一是基于对CTCF因子的明显需求，该因子可以在男性生殖细胞中重建CTCF位点时识别特定CTCF位点的父本特异性DNA甲基化模式，这些DNA甲基化模式最初由我们和其他人在Igf2/H19位点的印迹控制区（ICR）定位和表征。最近对BORIS在羊膜动物中的进化和表达的研究证实了我们的发现，即在鸟类或任何其他缺乏亲本起源特异性基因印迹的低等脊椎动物中不存在睾丸型BORIS基因表达，并发现BORIS在小袋鼠和牛中具有性腺特异性表达，两者都具有IGF2/H19基因的印迹。这表明睾丸限制性BORIS在生殖系细胞中的表达进化确实与IGF2/H19和其他基因座的基因组印迹进化相关，正如我们在描述BORIS的原始论文中首次提出的那样。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BORIS在体内结合到给定的CTCF位点将导致与CTCF结合到相同位点不同的功能结果。正常情况下，CTCF和BORIS不在同一个细胞中表达，以避免由于竞争结合到它们都能识别的目标序列的同一谱而造成的功能干扰。因此，我们从以往对CTCF和印迹的研究中预测，尽管不同印迹位点的印迹控制区（ICR）中没有明显的序列同源性，但大多数（如果不是全部）ICR序列都包含mecpg敏感的CTCF靶位点。这一预测被证明是正确的，并导致一些出版物描述了在不相关的印迹位点上新的ctcf驱动的ICRs。BORIS在体细胞中通常是严格沉默的，但在癌症中与一个特殊的基因家族（称为癌睾丸（CT）基因）一起被激活。为了了解BORIS表达的调控机制，我们对人类基因的5侧区域进行了表征，并鉴定了三个启动子，分别对应于3个转录起始位点，分别命名为A、B和C。发现替代启动子的使用与至少5个可选择剪接的BORIS mrna的产生有关，这些mrna具有不同的半衰期，由不同的5个UTR序列决定。在正常睾丸中，BORIS由所有三个启动子转录而成，而在大多数癌细胞类型中（分析的30个细胞系中的80%），只有启动子A和/或启动子C被激活，而在其余细胞系中，启动子B和C最活跃。虽然DNA甲基化有助于每个启动子的负调控，但去甲基化的BORIS启动子可以通过抑制CTCF表达或下调p53来激活。此外，在正常细胞中阻断CTCF可导致BORIS启动子的去甲基化和下调。这些结果为理解CTCF的减少或丧失与功能性p53的丧失和BORIS启动子沉默的放松之间的两种与癌症相关的功能联系提供了机制基础。接下来，我们发现BORIS在许多不同组织来源的原发肿瘤中似乎异常表达，其发生率远高于其他CT基因。最近发表的两项关于BORIS基因/蛋白在女性恶性肿瘤中的异常表达的研究是与Risinger博士（NCI）合作进行的BORIS在子宫癌中的研究和与Essex大学的Klenova博士合作进行的BORIS在乳腺肿瘤中的研究。在第一项研究中，我们使用全基因组基因表达阵列查询了已知和推测的CT基因转录本的表达，并通过反转录PCR和定量PCR检测了BORIS转录本，因为转录本没有在阵列上表示。微阵列分析检测子宫癌中表达的多个CT基因；然而，没有任何一种CT基因在超过25%的癌症中表达。与之形成鲜明对比的是，BORIS在95例子宫内膜癌中的73例（77%）和31例子宫混合中胚层肿瘤中的24例（77%）中表达。BORIS在子宫癌中的高频率表达再次提示了利用BORIS作为这些恶性肿瘤的免疫或诊断靶点的巨大潜力。在第二项研究中，我们评估了BORIS在乳腺癌中的表达水平与临床/病理参数之间的关系。BORIS在所有18个乳腺细胞系中检测到，但在单个原代正常乳腺细胞培养中未检测到。58例原发性乳腺肿瘤中有41例（71%）表达BORIS。我们还发现，乳腺癌中BORIS的高水平与孕酮和雌激素受体的高水平相关。我们之前表明，当BORIS在肿瘤中异常表达时，可以诱导在癌症患者血清样本中检测到的抗体反应（正在申请专利）。我们与L.L Hansen博士（丹麦奥胡斯大学）在一项针对乳腺癌患者和健康供体的盲法研究中进一步证实了这一点，我们现在正努力通过CRADA协议推动这种诊断方法在临床实践中得到适当的应用。最后，我们与M. Agadjanyan博士（加利福尼亚州亨廷顿海滩分子医学研究所）合作，利用小鼠模型进一步开展抗癌疫苗开发。先前，作为原理证明，我们发现BORIS蛋白在多种小鼠癌症中表达，并且可以使用DNA疫苗和重组蛋白产生对BORIS的免疫应答。在最近的一项研究中，我们比较了采用强th1型佐剂配制的BORIS重组蛋白、QuilA或以DNA形式编码相同免疫原的DNA以及表达il - 12和il - 18的质粒作为分子佐剂的疫苗策略。对两组接种疫苗的小鼠进行肿瘤特异性免疫诱导（抗体反应、t细胞增殖、细胞因子产生、t细胞毒性）以及抑制侵袭性乳腺癌细胞系生长和延长接种疫苗动物存活的能力检测。我们确定DNA，而不是重组蛋白疫苗，能诱导强效th1样t细胞反应，显著抑制肿瘤生长并延长接种小鼠的生存期。这些研究为针对广泛表达的肿瘤抗原的癌症疫苗的临床开发提供了明确的指导。

During the last year, we continued our studies of BORIS (Brother Of the Regulator of Imprinted Sites) - a CTCF-paralogous gene we discovered and published in 2002. Both CTCF and BORIS proteins share a nearly identical 11 Zn-finger (11ZF) DNA binding domain (DBD) that is flanked with dissimilar N- and C-terminal regions. 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 the double-stranded DNA. This DBD region shared by BORIS and CTCF proteins is encoded by sequences displaying an accurate duplication of a conserved genomic region containing all ZF-coding exons of CTCF gene. One of the non-pathological functions that we first proposed for BORIS was based on the apparent need for a different from CTCF factor that could nevertheless recognize CTCF sites during re-establishment in male germ cells of paternal-specific DNA methylation patterns at particular CTCF sites initially mapped and characterized by us and others in the imprinting control region (ICR) of the Igf2/H19 locus. Recent study of BORIS evolution and expression in amniotes confirmed our finding that a testicular-type of BORIS gene expression was absent in birds or in any other lower vertebrates that lack parent-of-origin specific gene imprinting, and uncovered that BORIS expression became gonad-specific in wallaby and cattle, both of which exhibit imprinting of IGF2/H19 genes. This implies that evolution of the testis-restricted BORIS expression in the germline cells does correlate with the evolution of genomic imprinting at IGF2/H19 and other loci exactly as we had first suggested in the original paper describing BORIS. 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 in a different functional outcome than binding of CTCF to the same site. Normally, CTCF and BORIS are not expressed in the same cell to avoid functional interference caused by competition for binding to the same spectrum of target sequences they can both recognize. Therefore, we predicted from previous studies of CTCF and imprinting that in spite of the lack of any obvious sequence homologies in the imprinting control regions (ICR) from different imprinted loci, most, if not all ICR sequences would contain meCpG-sensitive CTCF target sites. This prediction turned out to be correct and resulted in several publications describing novel CTCF-driven ICRs in unrelated imprinted loci. BORIS is normally strictly silenced in somatic cells, but activated in cancers together with a particular family of genes, called cancer-testis (CT) genes. To understand the mechanisms governing BORIS expression, we characterized the 5-flanking region of the human gene, and identified three promoters designated A, B, and C, that correspond to 3 transcription start sites. Alternative promoter usage was found to be associated with the generation of at least five alternatively spliced BORIS mRNAs having different half lives determined by varying 5 UTR sequences. While in normal testis BORIS is transcribed from all three promoters, in the majority of cancer cell types ( 80 % of 30 cell lines analyzed) only promoter A and/or promoter C were activated, and in the remaining cell lines, promoters B and C were most active. While DNA methylation contributes to the negative regulation of each promoter, demethylated BORIS promoters are activated by suppression of CTCF expression or by downregulation of p53. Moreover, blocking CTCF in normal cells resulted in demethylation and derepression of BORIS promoters. These results provide a mechanistic basis for understanding two cancer-associated functional connections between decrease or loss of CTCF and loss of functional p53 and relaxation of BORIS promoters silencing. Next, we showed that BORIS appears to be aberrantly expressed with a much higher incidence than other CT genes in many primary tumors of different tissue origins. Two recently published studies of abnormal BORIS gene/protein expresssion in female malignancies were performed in collaboration with Dr. Risinger (NCI) on BORIS in uterine cancers and with Dr. Klenova of the Essex University on BORIS in breast tumors. In the first study we queried the expression of known and putative CT gene transcripts using whole genome gene expression arrays, and examined BORIS transcripts by reverse transcription PCR and quantitative PCR since the transcript was not represented on the array. Microarray analysis detected many CT genes expressed in uterine cancers; however, no individual CT gene was expressed in more than 25% of any cancer. In stark contrast, BORIS was expressed in 73 of 95 (77%) endometrial cancers and 24 of 31 (77%) uterine mixed mesodermal tumors. The high frequency of BORIS expression in uterine cancers suggest again of a great potential of utilizing BORIS as an immunologic or diagnostic target for these malignancies. In the second study, we assessed the relation between BORIS expression levels in breast cancer with clinical/pathological parameters. BORIS was detected in all of 18 breast cell lines tested, but not in a single primary normal breast cell culture. BORIS was expressed in 41 of 58 cases (71%) of primary breast tumors. We also showed that high BORIS levels in breast cancer correlates with high levels of progesterone and estrogen receptor. We showed previously that BORIS, when abnormally expressed in tumors, can induce antibody responses detectable in serum samples from cancer patients (patent pending). We further confirmed this in collaboration with Dr. L.L Hansen (University of Aarhus, Denmark) in a blind study of breast cancer patients and healthy donors, and we are now working to push forward this diagnostic approach to reach its appropriate application in clinical practice through a CRADA agreement. Finally, we have further pursued anti-cancer vaccine development using a mouse models in collaboration with Dr. M. Agadjanyan (Institute for Molecular Medicine, Huntington Beach, CA). Previously, as proof of principle, we showed that BORIS protein is expressed in various mouse cancers and that it is possible to generate immune responses to BORIS using both DNA vaccines and recombinant protein. In a recent study, we compared vaccine strategies employing BORIS recombinant protein formulated in a strong Th1-type adjuvant, QuilA, or DNA encoding this same immunogen in the form of DNA along with plasmids expressing IL12 and IL18 as molecular adjuvants. Both groups of vaccinated mice were examined for induction of tumor-specific immunity (antibody response, T-cell proliferation, cytokine production, T-cell cytotoxicity) as well as the ability to inhibit growth of an aggressive breast cancer cell line and to prolong survival of vaccinated animals. We determined that DNA, but not recombinant protein vaccine, induced potent Th1-like T-cell responses that significantly inhibited tumor growth and prolonged the survival of vaccinated mice. These studies provide a clear guidance for clinical development of a cancer vaccine targeting what appears to be a widely expressed tumor antigen.