Gene Expression At The Beginning Of Mammalian Developmen

哺乳动物发育初期的基因表达

• 批准号: 6681731
• 负责人: Melvin DePamphilis
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6681731
• 关键词: DNA replication; developmental genetics; early embryonic stage; embryo implantation; gene expression; genetic regulatory element; laboratory mouse; mammalian embryology; transcription factor; DNA replication; developmental genetics; early embryonic stage; embryo implantation; gene expression; genetic regulatory element; laboratory mouse; mammalian embryology; transcription factor

In previous studies, we utilized microinjection of plasmid DNA into the nuclei of oocytes, fertilized eggs and 2-cell embryos in order to identify cis-acting sequences and trans-acting factors that regulate DNA replication and gene expression at the beginning of mouse development. This work led to the discovery of a novel transcription factor, mTEAD-2, that is expressed at the onset of zygotic gene expression (ZGE) where it is capable of strongly stimulating transcription from promoters or enhancers that contain its sequence-specific binding site. mTEAD-2 is the only member of the TEAD family of transcription factors that is expressed in mouse embryos during the first 7 days of their development. Our objectives are to identify the factors that regulate mTEAD-2 gene expression, to elucidate mechanisms by which mTEAD-2 regulate gene expression, and to identify the role of mTEAD-2 in mammalian development. Investigation of the regulatory region of mTEAD-2 led to the surprising discovery of another gene only 3.8 kb upstream of mTEAD-2. This new gene is a single copy, testis specific gene called Soggy (mSgy) that is transcribed in the direction opposite to mTEAD-2, thus placing the regulatory elements of these two genes in close proximity. mSgy contains three methionine codons that could potentially act as translation start sites, but most mSGY protein synthesis in vitro was initiated from the first Met codon to produce a full length protein, suggesting that mSGY normally consists of 230 amino acids (26.7 kDa). Transcription began at a cluster of nucleotides ~150 bp upstream of the first Met codon using a TATA-less promoter contained within the first 0.9 kb upstream to produce a single, dominant mRNA ~1.3 kb in length. The activity of this promoter was repressed by upstream sequences between -0.9 and ?2.5 kb in cells that did not express mSgy, but this repression was relieved in cells that did express mSgy. mSgy mRNA was detected in embryos only after day 15, and in adult tissues only in the developing spermatocytes of seminiferous tubules, suggesting that mSgy is a spermatocyte-specific gene. Since mTEAD-2 and mSgy were not expressed in the same cells, the mSgy/mTEAD-2 locus provides a unique paradigm for differential regulation of gene expression during mammalian development. We have recently identified the long sought after co-activator of the TEAD family of transcription factors. TEAD-2/TEF-4 protein purified from mouse cells was associated predominantly with a novel TEAD-binding domain at the N-terminus of YAP65, a powerful transcriptional co-activator. YAP65 interacted specifically with the C-terminus of all four TEAD proteins. Both this interaction and sequence-specific DNA binding by TEAD were required for transcriptional activation in mouse cells. Expression of YAP in lymphocytic cells that normally do not support TEAD-dependent transcription (e.g. MPC11) resulted in up to 300-fold induction of TEAD activity. Conversely, TEAD overexpression squelched YAP activity. Therefore, the C-terminal acidic activation domain in YAP is the transcriptional activation domain for TEAD transcription factors. However, while TEAD was concentrated in the nucleus, excess YAP65 accumulated in the cytoplasm as a complex with the cytoplasmic localization protein, 14-3-3. Since TEAD-dependent transcription was limited by YAP65, and YAP65 also binds Src/Yes protein tyrosine kinases, we propose that YAP65 regulates TEAD?dependent transcription in response to mitogenic signals.

在先前的研究中，我们利用质粒DNA的微注射到卵母细胞的核中，受精卵和2细胞胚胎，以鉴定在小鼠发育开始时调节DNA复制和基因表达的顺式作用序列和反式作用因子。这项工作导致发现了一种新型转录因子MTEAD-2，该因子在合子基因表达（ZGE）的开始时表达，在该因素中，它能够强烈刺激包含其序列特异性结合位点的启动子或增强子的转录。 MTEAD-2是Tead家族的转录因子家族的唯一成员，在其发育的前7天中在小鼠胚胎中表达。我们的目标是确定调节MTEAD-2基因表达的因素，以阐明MTEAD-2调节基因表达的机制，并确定MTEAD-2在哺乳动物发育中的作用。 对MTEAD-2的调节区域的调查导致了MTEAD-2上游仅3.8 kb的另一个基因的惊人发现。这个新基因是一个单个副本，睾丸特异性基因，称为湿润（MSGY），它沿与MTEAD-2相反的方向转录，从而将这两个基因的调节元素近距离接近。 MSGY包含三个有可能充当翻译起始位点的蛋氨酸密码子，但是在第一个Met Codon中启动了大多数MSGY蛋白质合成，以产生全长蛋白，这表明MSGY通常由230个氨基酸组成（26.7 kDa）。转录始于第一个METON的核苷酸簇〜150 bp，使用第一个0.9 kb上游的无tata启动子在上游，以产生单个主导的mRNA 〜1.3 kb的长度。该启动子的活性因-0.9和2.5 kb之间的上游序列在未表达MSGY的细胞中被抑制，但是这种抑制作用在表达MSGY的细胞中得到了缓解。仅在第15天之后才在胚胎中检测到MSGY mRNA，并且仅在成年组织中仅在生长小管的发育中的精子细胞中，这表明MSGY是一个精子细胞特异性基因。由于MTEAD-2和MSGY在同一细胞中未表达，因此MSGY/MTEAD-2基因座为哺乳动物发育过程中基因表达的差异调节提供了独特的范例。 我们最近确定了转录因子的TEAD家族的长期追求的共激活因子。从小鼠细胞中纯化的Tead-2/Tef-4蛋白主要与一个强大的转录共激活剂YAP65的N端的新型Tead结合结构域相关。 YAP65与所有四个Tead蛋白的C端特别相互作用。这种相互作用和序列特异性的DNA结合都是小鼠细胞中的转录激活。通常不支持TEAD依赖性转录（例如MPC11）的淋巴细胞中YAP的表达会导致高达300倍的TEAD活性诱导。相反，将过表达的yap活性击退。因此，YAP中的C末端酸性激活结构域是TEAD转录因子的转录激活结构域。然而，虽然tead集中在细胞核中，但与细胞质定位蛋白的复合物中，过量的YAP65在细胞质中积聚，14-3-3。由于TEAD依赖性转录受YAP65的限制，YAP65还结合了SRC/YES蛋白酪氨酸激酶，因此我们建议YAP65调节tead？依赖性转录对有丝分裂信号的响应。