期刊：
The International Journal of Biochemistry & Cell Biology（Q2, 5.652）
NBPF is a potential DNA-binding transcription factor that is directly regulated by NF-B
NBPF 是一种潜在的 DNA 结合转录因子，由 NF-B 直接调节
a b s t r a c t
The neuroblastoma breakpoint family (NBPF) has been reported to play potential roles in the development of neuroblastoma and human evolution. However, the exact regulation and function of this family is still unknown. In this study, the genes of NBPF family were found to be densely covered by many highconfidence ChIP-Seq peaks of NF-B. The expressions of NBPF genes were thus deduced to be regulated by this transcription factor. The activities of NF-B in HeLa, HepG2 and ECa109 cells were then manipulated with NF-B activator (TNFa) and inhibitors (BAY11-7082 or p65 siRNA), and the expressions of NBPF genes in these cells were checked. As result, it was found that the expressions of NBPF genes were regulated by NF-B in HeLa and HepG2 cells. Therefore, the genes of NBPF family were identified as new bona fide direct target genes of NF-B. In addition, NBPF was also identified as a nuclear protein by in silico prediction and immunolocalization. Finally, the bioinformatics analysis revealed that most of NBPF proteins contained classical nuclear localization signals (NLSs) and a conserved DNA-binding domain similar to the transcription factor stat3b/dna complex or stat-1/dna complex in their N-terminals. Therefore, this study concluded that NBPF was nuclear protein that contained classical NLSs and conserved known DNA-binding domain, and its expression was regulated by another important transcription factor, NF-B. These findings suggest that NBPF may function as DNA-binding transcription factor in nucleus, which provides important new insight into the functions of NBPF genes in the human cells.
据报道，神经母细胞瘤断点家族 (NBPF) 在神经母细胞瘤的发展和人类进化中发挥潜在作用。然而，这个家族的确切调控和功能仍然未知。在这项研究中，发现 NBPF 家族的基因被许多 NF-B 的高置信度 ChIP-Seq 峰密集覆盖。因此推断 NBPF 基因的表达受该转录因子的调节。然后用 NF-B 激活剂 (TNFa) 和抑制剂 (BAY11-7082 或 p65 siRNA) 操纵 HeLa、HepG2 和 ECa109 细胞中 NF-B 的活性，并检查这些细胞中 NBPF 基因的表达。结果发现NBPF基因在HeLa和HepG2细胞中的表达受NF-B调控。因此，NBPF家族的基因被鉴定为NF-B的新的真正直接靶基因。此外，通过计算机预测和免疫定位，NBPF 也被鉴定为核蛋白。最后，生物信息学分析表明，大多数 NBPF 蛋白含有经典的核定位信号 (NLS) 和一个保守的 DNA 结合结构域，类似于转录因子 stat3b/dna 复合物或 stat-1/dna 复合物的 N 末端。因此，本研究得出结论，NBPF是含有经典NLSs和保守的已知DNA结合结构域的核蛋白，其表达受另一个重要转录因子NF-B的调控。这些发现表明，NBPF 可能在细胞核中作为 DNA 结合转录因子发挥作用，这为了解 NBPF 基因在人类细胞中的功能提供了重要的新见解。
1. Introduction
Neuroblastoma is the most common extracranial solid malignancies in children (Maris and Matthay, 1999). A constitutional translocation t(1; 17) (p36.2; q11.2) in the patient (Laureys et al., 1990, 1995) resulted in the disruption of a novel gene named NBPF1 (Vandepoele et al., 2005), which belongs to a novel gene family, neuroblastoma breakpoint family. The genes of this family were primarily located on the duplicated regions of chromosome1 (Fig. S1), and had numerous low-copy repetitive elements. The genes of this family showed high intragenic and intergenic sequence similarity in both coding and noncoding regions ( Vandepoele et al., 2005). Due to the intricate gene structures, it is a great challenge to distinguish each member from all the members of the family for analyzing their expression and function.
一、简介
神经母细胞瘤是儿童中最常见的颅外实体恶性肿瘤（Maris 和 Matthay，1999）。 患者体内的体质易位 t(1; 17) (p36.2; q11.2) (Laureys et al., 1990, 1995) 导致了一个名为 NBPF1 的新基因的破坏 (Vandepoele et al., 2005)， 它属于一个新的基因家族，神经母细胞瘤断点家族。 该家族的基因主要位于染色体1的重复区域（图S1），并具有许多低拷贝重复元件。 该家族的基因在编码区和非编码区都表现出高度的基因内和基因间序列相似性（Vandepoele 等，2005）。 由于复杂的基因结构，将每个成员与所有家庭成员区分开来分析它们的表达和功能是一个巨大的挑战。
Previous study revealed that the regulatory region of NBPF1 shared extensive homology with the upstream region of an unrelated gene, EVI5, and they showed the similar expression patterns in a panel of neuroblastoma cell lines (Vandepoele et al., 2009). This finding indicated that these two genes might have similar molecular function in cellular processes. The EVI5 gene encodes a centrosomal protein that plays a key role in the cell mitosis and its disruption leads to incomplete cell division and formation of multinucleate cells. It was reported that NBPF1 also had effect on cell proliferation. For example, the overexpression of NBPF1 was found to suppress the formation of soft agar colony of human colorectal cells (Vandepoele et al., 2008).
先前的研究表明，NBPF1 的调节区域与无关基因 EVI5 的上游区域具有广泛的同源性，并且它们在一组神经母细胞瘤细胞系中显示出相似的表达模式（Vandepoele 等，2009）。 这一发现表明这两个基因在细胞过程中可能具有相似的分子功能。 EVI5基因编码一种在细胞有丝分裂中起关键作用的中心体蛋白，其破坏导致细胞分裂不完全和多核细胞的形成。 据报道，NBPF1对细胞增殖也有影响。 例如，发现 NBPF1 的过表达可抑制人结肠直肠细胞软琼脂集落的形成（Vandepoele 等，2008）。
The genes of NBPF family were found widely expressed in variety of tissues and cell types, including embryonic stem cells, fetal and adult tissues, and normal and cancerous tissues (Vandepoele et al., 2005). However, they showed remarkable species-specific duplications, their orthologues could not be identified in the completed genomes of fruit fly, nematode and rodent, and just had low sequence homology to canine and bovine genomes (Vandepoele et al., 2005). This gene family was proposed to evolve rapidly in the primates (Vandepoele et al., 2005), and had undergone a rapid recent expansion reflected in copy number variation in humans (Sudmant et al., 2010). Therefore, this gene family was thought to play a role in the physiological divergence of primate species, such as brain size (Dumas and Sikela, 2009), and also considered as an important player in human evolution (Vandepoele et al., 2005).
发现 NBPF 家族的基因在多种组织和细胞类型中广泛表达，包括胚胎干细胞、胎儿和成人组织以及正常和癌组织 (Vandepoele et al., 2005)。 然而，它们表现出显着的物种特异性重复，它们的直系同源物在果蝇、线虫和啮齿动物的完整基因组中无法识别，并且与犬和牛基因组的序列同源性较低（Vandepoele 等，2005）。 该基因家族被提议在灵长类动物中快速进化（Vandepoele 等人，2005），并且最近经历了快速扩张，反映在人类的拷贝数变异中（Sudmant 等人，2010）。 因此，该基因家族被认为在灵长类物种的生理分化中发挥作用，例如大脑大小 (Dumas and Sikela, 2009)，也被认为是人类进化的重要参与者 (Vandepoele et al., 2005)。
A copy number variation is an important sources of genetic diversity, and play a vital role in several diseases (Redon et al., 2006), such as child neuroblastoma (Diskin et al., 2009), autism (Sebat et al., 2007), and psoriasis (Hollox et al., 2008). The genes of NBPF family exhibit high copy number variation, suggesting that the genes of this family are likely involved in pathology of human disease (Diskin et al., 2009). NBPF1 has been proposed as a tumor suppressor because it inhibited the formation of soft agar colony of human colorectal cells (Vandepoele et al., 2008). Moreover, in the human 1p36 region where NBPF was located, several tumor suppressor genes had also been identified, which suggested that NBPF might act as a tumor suppressor gene ( Vandepoele et al., 2008, 2010). Based on Oncomine database analysis, it was found that the decrease of NBPF expression levels in neuroblastomas contributed to the relapse of this tumor within five years (Vanessa Andries, 2012). Conversely, the overexpression of NBPF transcripts (NBPF12) has been found in several cancers including sarcomas (Meza-Zepeda et al., 2002), and non-small-cell lung cancer (Petroziello et al., 2004).
拷贝数变异是遗传多样性的重要来源，在多种疾病（Redon et al., 2006）中起着至关重要的作用，例如儿童神经母细胞瘤（Diskin et al., 2009）、自闭症（Sebat et al., 2007） ）和牛皮癣（Hollox 等人，2008 年）。 NBPF 家族的基因表现出高拷贝数变异，表明该家族的基因可能与人类疾病的病理学有关（Diskin 等，2009）。已提出 NBPF1 作为肿瘤抑制因子，因为它抑制人结肠直肠细胞软琼脂集落的形成（Vandepoele 等，2008）。此外，在NBPF所在的人类1p36区域，还发现了多个抑癌基因，提示NBPF可能作为抑癌基因（Vandepoele et al., 2008, 2010）。基于 Oncomine 数据库分析，发现神经母细胞瘤中 NBPF 表达水平的降低导致该肿瘤在五年内复发（Vanessa Andries，2012）。相反，已在包括肉瘤（Meza-Zepeda 等人，2002）和非小细胞肺癌（Petroziello 等人，2004）在内的几种癌症中发现了 NBPF 转录物（NBPF12）的过表达。
The previous studies revealed that NBPF1 protein could interact with chibby and clusterin. It was found that NBPF1 could form a tri-molecular complex with these two proteins (Vandepoele et al., 2010). Chibby was a documented inhibitor of canonical Wnt-()-catenin pathway (Takemaru et al., 2003). Dysregulation of Wnt/()-catenin signaling has been significantly association with several diseases, such as disturbances in skeletal bone mass, colon cancer, hair follicle tumors and leukemia (Clevers, 2006). Clusterin, also known as apolipoprotein J, associated with the clearance of Q2 cellular debris (Jones and Jomary, 2002), could inhibit cancer cell apoptosis (Zhang et al., 2005), and promote the survival of prostate cancer cell via activating the canonical NF-B pathway (Zoubeidi et al., 2010). NF-B is an inducible ubiquitous nuclear transcription factor that regulates expressions of a large number of genes that are critical for the regulation of apoptosis, viral replication, tumorigenesis, inflammation, and various autoimmune diseases. Thus, it seems that NBPF may involve in the pathologies of some diseases by its interactions with proteins linking to some important signaling pathways such as the Wnt and NF-B signaling pathways. In a word, the previous studies have already revealed that the NBPF genes played important roles in the physiological and pathological processes, however, the exact function and regulation of this gene family is still unknown.
先前的研究表明NBPF1蛋白可以与chibby和clusterin相互作用。发现 NBPF1 可以与这两种蛋白质形成三分子复合物 (Vandepoele et al., 2010)。 Chibby 是经典 Wnt-()-catenin 通路的有文献记载的抑制剂（Takemaru et al., 2003）。 Wnt/()-连环蛋白信号的失调与多种疾病显着相关，例如骨骼骨量紊乱、结肠癌、毛囊肿瘤和白血病 (Clevers, 2006)。凝聚素，也称为载脂蛋白 J，与 Q2 细胞碎片的清除有关 (Jones and Jomary, 2002)，可以抑制癌细胞凋亡 (Zhang et al., 2005)，并通过激活典型的前列腺癌细胞促进前列腺癌细胞的存活。 NF-B 通路（Zoubeidi 等，2010）。 NF-B 是一种可诱导的普遍存在的核转录因子，它调节大量基因的表达，这些基因对调节细胞凋亡、病毒复制、肿瘤发生、炎症和各种自身免疫性疾病至关重要。因此，似乎 NBPF 可能通过与连接一些重要信号通路（如 Wnt 和 NF-B 信号通路）的蛋白质相互作用而参与某些疾病的病理过程。总之，以往的研究已经揭示了NBPF基因在生理和病理过程中发挥了重要作用，但该基因家族的确切功能和调控机制尚不清楚。
In a recent study that characterized the genome-wide binding profile of NF-B (Xing et al., 2013), we found that the genes of NBPF family were densely covered by many high-confidence ChIP-Seq peaks of NF-B. Therefore, we deduced that the genes of this family may be the direct target genes of this transcription factor. Because it was reported that NF-B was constitutively activated in neuroblastoma (Bian et al., 2002; Brown et al., 2007), and NBPF played potential roles in the development of neuroblastoma (Vandepoele et al., 2008; Vanessa Andries, 2012), therefore, if NBPF is proved to be the target genes of NF-B, it would provide new insight into the mechanism that NF-B functions in this cancer. For this purpose, this study manipulated the activities of NF-B in HeLa, HepG2 and ECa109 cells with NF-B activator (TNF（）) and inhibitors (BAY11-7082 or p65 siRNA), and checked the expressions of NBPF genes in these cells. As a result, the expressions of NBPF genes were found to be regulated by NF-B in HeLa and HepG2 cells. Therefore, the genes of NBPF family were identified as new bona fide direct target genes of NF-B. In addition, NBPF was also identified as a nuclear protein by in silico prediction and immunolocalization. Finally, the bioinformatics analysis revealed that most of NBPF proteins contained classical nuclear localization signals (NLSs) and a conserved DNA-binding domain similar to the transcription factor stat3b/dna complex or stat-1/dna complex in their N-terminals.
在最近一项表征 NF-B 的全基因组结合谱的研究中（Xing 等人，2013 年），我们发现 NBPF 家族的基因被 NF-B 的许多高置信度 ChIP-Seq 峰密集覆盖。因此，我们推断该家族的基因可能是该转录因子的直接靶基因。因为据报道，NF-B 在神经母细胞瘤中被组成型激活（Bian 等人，2002；Brown 等人，2007），而 NBPF 在神经母细胞瘤的发展中发挥了潜在作用（Vandepoele 等人，2008；Vanessa Andries， 2012），因此，如果证明 NBPF 是 NF-B 的靶基因，它将为 NF-B 在这种癌症中的作用机制提供新的见解。为此，本研究用 NF-B 激活剂（TNF（））和抑制剂（BAY11-7082 或 p65 siRNA）操纵 HeLa、HepG2 和 ECa109 细胞中 NF-B 的活性，并检查这些细胞中 NBPF 基因的表达。细胞。结果发现NBPF基因在HeLa和HepG2细胞中的表达受NF-B调控。因此，NBPF家族的基因被鉴定为NF-B的新的真正直接靶基因。此外，通过计算机预测和免疫定位，NBPF 也被鉴定为核蛋白。最后，生物信息学分析表明，大多数 NBPF 蛋白含有经典的核定位信号 (NLS) 和一个保守的 DNA 结合结构域，类似于转录因子 stat3b/dna 复合物或 stat-1/dna 复合物的 N 末端。
3. Results
3.1. ChIP-seq peaks in the gene region of NBPF genes
To identify the genes bound by NF-B in TNF()-stimulated HeLa cells, we recently performed ChIP-Seq assays and found that many genes were bound by this transcription factor (Xing et al., 2013). Among these genes, the genes of NBPF family attracted our attention because they were covered by many ChIP-Seq peaks of high confidence (with enrichment fold over 20) (Table 1; Figs. S2 and S3). For example, the gene region of NBPF10 was occupied by 36 peaks of this kind, including 14 peaks in 5' distal region, 18 peaks in gene and 4 peaks in 3' region (Figs. 1A and S4). These peaks demonstrated that NBPF genes were bound by NF-B in the TNF()-stimulated HeLa cells and their expression might be regulated by this transcription factor.
3. 结果
3.1。 NBPF 基因的基因区域中的 ChIP-seq 峰
为了鉴定 TNF() 刺激的 HeLa 细胞中 NF-B 结合的基因，我们最近进行了 ChIP-Seq 分析，发现许多基因与这种转录因子结合（Xing et al., 2013）。 在这些基因中，NBPF 家族的基因引起了我们的注意，因为它们被许多高置信度的 ChIP-Seq 峰覆盖（富集倍数超过 20）（表 1；图 S2 和 S3）。 例如，NBPF10的基因区域被36个此类峰占据，其中5'远端区域有14个峰，基因中有18个峰，3'区域有4个峰（图1A和S4）。 这些峰表明 NBPF 基因在 TNF() 刺激的 HeLa 细胞中与 NF-B 结合，并且它们的表达可能受该转录因子的调节。
To confirm these peaks were truly resulted from NF-B binding, ten B motifs described in the Materials and Methods were searched in the DNA sequences of these peaks. It was found that each of these peaks contained multiple B sites (Table 2). For example, the NBPF10-related peaks contained a total of 502 B sites, and the peak with the highest enrichment fold contained 60 B sites (Fig. 1.B). These data demonstrated that the ChIP-Seq peaks related to NBPF genes were produced by the binding of NF-B to these genes. These data also suggested that the expressions of NBPF genes might be regulated by NF-B in the TNF()-mediated HeLa cells.
为了确认这些峰确实是由 NF-B 结合产生的，在这些峰的 DNA 序列中搜索了材料和方法中描述的十个 B 基序。 发现这些峰中的每一个都包含多个 B 位点（表 2）。 例如，NBPF10 相关峰共包含 502 个 B 位点，而富集倍数最高的峰包含 60 个 B 位点（图 1.B）。 这些数据表明，与 NBPF 基因相关的 ChIP-Seq 峰是由 NF-B 与这些基因的结合产生的。 这些数据还表明 NBPF 基因的表达可能受 TNF() 介导的 HeLa 细胞中的 NF-B 调节。
。。。乏了，不翻了，笔者直接自己看了。。。感兴趣的小伙伴自行下载阅读吧。
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