Placenta specific and ribosomal RNA genes: structure and function

对于PLAC1，为了确定其异常选择性的组织特异性表达的基础，我们已经证明该基因是由两个启动子P1和P2表达的，它们相距105千碱基，并被交替剪接。通过克隆小鼠和人的启动子，确定了最小启动子区域。最小启动子区域与核受体维甲酸X受体α(RXR-α)、维甲酸X受体-β(LXR-β)和类固醇生成因子1(SF1)/雌激素相关受体β(ERR-β)在特定的位置结合，在它们各自的激动剂存在下，它们的结合具有刺激转录10倍的正效应。 在一篇后续发表的《Oncogensis》(2013)中，通过建立癌细胞系的经典方法评估了Plac1在癌细胞中的表达；SV40介导原代细胞WI38和IMR90细胞的转化。我们发现，在SV40介导的转化后，原代细胞诱导PLAC1和一系列步骤是由SV40早期区域编码的大T抗原催化的，这些T抗原改变了通常与启动子区域结合的TP53抑制物特性，使其失去抑制能力，使染色质从封闭状态转变为开放状态，促进Plac1转录。然后在核受体存在的情况下进一步刺激转录，如果存在额外的辅助激活因子NCOA2(核受体辅助激活因子2)，它就招募RB，导致该基因的额外上调。因此，我们已经确定了该基因在癌细胞中被激活的主要方式，从而提供了一条抑制该基因活性的途径。 目前，我们已经在生化水平上证明Plac1与桥粒相互作用，特别是桥粒蛋白的c-末端部分。桥粒芯糖蛋白的C末端被切割，并作为外体的一部分分泌，因此可能会在怀孕期间作为PLAC1进入血液的载体，在怀孕期间检测到高水平的PLAC1。分娩后，母亲血液中PLAC1水平急剧下降。描述这项工作的手稿现已发表在《胎盘杂志》上。 在rDNA结构分析方面，通过与另外两个NIH小组的合作，解决了克隆和分析问题。NCI的J.H.Kim和Vladimir Larionov创建了一种基于转化相关重组(TAR)的先进克隆方法，提供了具有多达2个重复单位的rDNA的稳定克隆；NHGRI的Adam Phillippy和Alex Dilthee采用了先进的长读测序技术(PacBio和Nanopore)来促进序列恢复和组装；我们为分析提供了注释和背景。到目前为止的主要发现是，核糖体DNA以及包括5个启动子和3个启动子的外部转录序列和内部转录序列的转录区域，所有这些都在组装过程中被消除，但对于形成成熟的核糖体是必不可少的，含有许多变异，其中一部分深入人类进化。13个克隆，约为21号染色体rDNA补体的0.32倍(0.82Mb)，揭示了先前缺失的2kb区域、几个回文结构和300多个变体；85个变体落在成熟的18S/28S rRNA序列中。在独立的全基因组或RNA-Seq数据中也发现了回文断点和80%的45S变异等位基因，表明许多变异在人类群体中长期存在。我们已经开发了一个更新的44,838个碱基对的rDNA参考序列，并用检测到的变体进行了注释，这表明了一条完成人类基因组rDNA成分分析的可能途径。大量的变异体揭示了人类核糖体DNA中越来越多的普遍异质性，这为核糖体动力学中相应的变异打开了可能性。此外，我们还将研究扩展到包含人22号染色体的人-鼠杂交细胞系22号染色体上的rDNA单元，从22号染色体上收集了额外的克隆，测序、组装、注释并保存在Genbank中。这些分离株包括22号染色体rDNA重复序列两侧的端粒和着丝粒边界。描述这项工作的手稿现在发表在《科学报告》上。 我们现在已经将rDNA分析扩展到非人类灵长类动物和小鼠。现在已经分析了来自黑猩猩、大猩猩、猩猩和恒河猴细胞系的基于焦油克隆的克隆，以及来自小鼠的53个克隆。序列分析帮助我们提炼了数据库中现有的rDNA基因座序列，识别和分类了新的变异体。结果表明，小鼠和非人类灵长类动物的45S RNA含有比以前报道的更多的变体，并提供了一种手段，在正在进行的工作中测试这些变体对酵母细胞的影响。

For PLAC1, to determine the basis for its extraordinarily selective tissue-specific expression, we have shown that the gene is expressed from two promoters, P1 and P2, spaced 105 Kilobases apart and is alternatively spliced. By cloning both promoters from mouse and human, defined the minimal promoter regions. The minimal promoter region binds nuclear receptors Retinoic Acid X Receptor alpha (RXR-alpha), LXR-beta, and Steroidogenic factor 1 (SF1)/ Estrogen related receptor beta (ERR-beta) at specific sites and their binding has a positive effect stimulating transcription >10 fold, in the presence of their respective agonists. In a follow up publication, in Oncogenesis (2013), Plac1 expression in cancer cells was evaluated by a classical approach establishing cancer cell lines; SV40 mediated transformation of primary cells WI38 and IMR90 cells. We found that following SV40 mediated transformation the primary cells induced PLAC1 and a series of steps are catalyzed by Large T antigen encoded by SV40 early regions that modify Tp53 repressor properties normally bound to the promoter region such that it loses its repressive ability, bring about changes in chromatin from closed to open status facilitating Plac1 transcription. The transcription is then further stimulated in the presence of nuclear receptors and if an additional coactivator NCOA2 (nuclear receptor co-activator2) is present, it recruits RB, leading to additional up-regulation of the gene. Thus, we have defined a major way in which the gene is activated in cancer cells, which thereby provides a route to repress the gene activity. Currently, we have shown at the biochemical level that Plac1 interacts with desmosomes, specifically c-terminal portion of desmoglein. The c-terminal end of desmoglein is cleaved and is secreted as part of exosomes and thus could potentially act as a carrier for PLAC1 into the blood stream during pregnancy, where it is detected at high levels during pregnancy. The levels of PLAC1 decreases dramatically after delivery in the blood stream of mother. A manuscript describing this work is now published in Placenta Journal. For rDNA structure analysis the cloning and analysis problems were resolved with collaborations with 2 other NIH groups. J.H. Kim and Vladimir Larionov at NCI created an advanced approach to cloning based on transformation associated recombination (TAR), that provided stable clones with up to 2 repeat units of rDNA; Adam Phillippy and Alex Dilthey at NHGRI adapted advanced long-read sequencing techniques (PacBio and Nanopore) to facilitate sequence recovery and assembly; and we supplied annotation and context for the analyses. The major findings thus far are that ribosomal DNA, and transcribed regions that included 5 prime and 3 prime external transcribed sequences, and internal transcribed sequences, all of which are eliminated during assembly but are essential for formation of mature ribosomes harbor many variants, with a fraction of them deeply seated in human evolution. Thirteen clones, about 0.32-fold coverage (0.82 Mb) of the chromosome 21 rDNA complement, revealed a previously missed 2 kb tract, several palindromic structures, and over 300 variants; 85 variants fall in mature 18S/28S rRNA sequences. Palindromic breakpoints and >80% of 45S variant alleles were also found in independent whole-genome or RNA-Seq data, indicating that many variants are long established in human populations. We have developed an updated 44,838 bp rDNA reference sequence annotated with detected variants, suggesting a possible route to complete analysis of the rDNA component of the human genome. The large number of variants reveal more and more universal - heterogeneity in human ribosomal DNA than previously considered, opening the possibility of corresponding variations in ribosome dynamics. Further, we have extended the study to rDNA units in chromosome 22 from mouse-human hybrid cell-line containing human chromosome 22, collected additional clones from chromosome 22, sequenced, assembled, annotated and deposited in Genbank. These isolates include the telomeric and centromeric borders flanking the rDNA repeats from chromosome 22. A manuscript describing this work is now published in Science Reports. We have now extended the rDNA analysis to non-human primates and mouse. TAR cloning based clones from chimp, gorilla, orangutan, and rhesus monkey cell lines along with 53 clones from mouse have been now analyzed. The sequence analysis has helped us to refine the current rDNA locus sequence present in the database, identify and catalog new variants. The results have shown that the mouse and non-human primate 45S RNA contains significant number of variants than reported before and provides a means to test the effect of these variants in yeast cells in ongoing work.