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

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.

在之前的研究中，我们将质粒DNA显微注射到卵母细胞、受精卵和2细胞胚胎的细胞核中，以鉴定在小鼠发育初期调节DNA复制和基因表达的顺式作用序列和反式作用因子。这项工作发现了一种新型转录因子 mTEAD-2，它在合子基因表达 (ZGE) 开始时表达，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 转录因子家族的唯一成员，在小鼠胚胎发育的前 7 天内表达。我们的目标是确定调节 mTEAD-2 基因表达的因素，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 调控区的研究令人惊讶地发现了位于 mTEAD-2 上游仅 3.8 kb 的另一个基因。这个新基因是一个单拷贝的睾丸特异性基因，称为 Soggy (mSgy)，其转录方向与 mTEAD-2 相反，从而使这两个基因的调控元件非常接近。 mSgy 包含三个可能充当翻译起始位点的蛋氨酸密码子，但大多数 mSGY 蛋白质体外合成是从第一个 Met 密码子开始的，以产生全长蛋白质，表明 mSGY 通常由 230 个氨基酸 (26.7 kDa) 组成。转录从第一个 Met 密码子上游约 150 bp 的核苷酸簇开始，使用上游第一个 0.9 kb 内包含的无 TATA 启动子，产生长度约 1.3 kb 的单个显性 mRNA。在不表达 mSgy 的细胞中，该启动子的活性被 -0.9 至 2.5 kb 之间的上游序列抑制，但在表达 mSgy 的细胞中，这种抑制得到缓解。仅在第 15 天后才在胚胎中检测到 mSgy mRNA，而在成人组织中仅在生精小管的发育精母细胞中检测到 mSgy mRNA，这表明 mSgy 是精母细胞特异性基因。由于 mTEAD-2 和 mSgy 不在同一细胞中表达，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。 我们最近发现了长期追捧的转录因子 TEAD 家族的共激活因子。从小鼠细胞中纯化的 TEAD-2/TEF-4 蛋白主要与 YAP65（一种强大的转录共激活剂）N 末端的新型 TEAD 结合结构域相关。 YAP65 与所有四种 TEAD 蛋白的 C 末端特异性相互作用。这种相互作用和 TEAD 的序列特异性 DNA 结合都是小鼠细胞中转录激活所必需的。 YAP 在通常不支持 TEAD 依赖性转录的淋巴细胞（例如 MPC11）中表达，导致 TEAD 活性诱导高达 300 倍。相反，TEAD 过度表达抑制了 YAP 活性。因此，YAP中的C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，但过量的 YAP65 作为与细胞质定位蛋白 14-3-3 的复合物积聚在细胞质中。由于TEAD依赖性转录受到YAP65的限制，并且YAP65还结合Src/Yes蛋白酪氨酸激酶，因此我们推测YAP65响应有丝分裂信号来调节TEAD依赖性转录。