Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, USA.
Biol Reprod. 2012 Feb 9;86(2):30. doi: 10.1095/biolreprod.111.095661. Print 2012 Feb.
Generally, knowledge of the mechanism regulating gene expression in primary spermatocytes is incomplete. We have used the lactate dehydrogenase gene (Ldhc) as a model to explore these mechanisms during spermatogenesis. Its 100-bp core promoter contained two essential elements common to many genes, a GC box and a CRE site. Here we report results that support a model in which transcription factor MYBL1 acts as a coactivator directing tissue-specific expression via the CRE cis element. We hypothesize that this is a common mechanism involving activation of multiple genes in the primary spermatocyte. MYBL1 is expressed predominantly as a tissue-specific transcription factor in spermatocytes and breast epithelial cells. Our finding that LDHC expression is lost in 21-day testes of MYBL1 mutant mice supports our hypothesis. In the GC1-spg germ cell line exogenous MYBL1 induces activity 4- to 8-fold, although extracts from these cells do not show MYBL1 binding activity for the Myb consensus sequences in the Ldhc promoter by EMSA. Rather, MYBL1 stimulates expression from a synthetic promoter containing only CRE elements, suggesting MYBL1 activates the promoter by interacting with protein that binds to a CRE element. Mutation of three Myb sites does not affect Ldhc promoter activity significantly (P > 0.05). CREB-binding protein (CBP) is a coactivator that interacts with CRE-binding protein CREB. We show that the transactivation domain (TAD) in MYBL1 interacts with the KIX domain in CBP, and the TAD domain and DNA binding domain in MYBL1 each interact with the CREB N-terminal domain. MYBL1 also stimulated expression from testis-specific genes Pgk2 (phosphoglycerate kinase 2) and Pdha2 (pyruvate dehydrogenase alpha 2) promoters, each of which contains CRE promoter elements and is expressed in primary spermatocytes. We propose that MYBL1 directs germ cell-specific activation via the CRE site of certain genes that are activated specifically in the primary spermatocyte, although other, more indirect effects of MYBL1 remain a possible explanation for our results.
一般来说，人们对调节初级精母细胞中基因表达的机制知之甚少。我们使用乳酸脱氢酶基因（Ldhc）作为模型，探索了在精子发生过程中这些机制。它的 100bp 核心启动子包含两个常见于许多基因的必需元件，一个 GC 盒和一个 CRE 位点。在这里，我们报告的结果支持了这样一种模型，即转录因子 MYBL1 作为一种共激活因子，通过 CRE 顺式元件指导组织特异性表达。我们假设这是一种涉及激活初级精母细胞中多个基因的常见机制。MYBL1 在精母细胞和乳腺上皮细胞中主要作为组织特异性转录因子表达。我们发现，在 MYBL1 突变小鼠的 21 天睾丸中，LDHC 的表达丧失，这支持了我们的假设。在 GC1-spg 生殖细胞系中，外源性 MYBL1 诱导活性增加 4-8 倍，尽管来自这些细胞的提取物在 EMSA 中没有显示出 MYBL1 对 Ldhc 启动子中 Myb 共有序列的结合活性。相反，MYBL1 刺激仅含有 CRE 元件的合成启动子的表达，表明 MYBL1 通过与结合 CRE 元件的蛋白相互作用来激活启动子。三个 Myb 位点的突变对 Ldhc 启动子活性没有显著影响（P>0.05）。CREB 结合蛋白（CBP）是一种与 CRE 结合蛋白 CREB 相互作用的共激活因子。我们表明，MYBL1 的转录激活结构域（TAD）与 CBP 的 KIX 结构域相互作用，MYBL1 的 TAD 结构域和 DNA 结合结构域分别与 CREB N 端结构域相互作用。MYBL1 还刺激了包含 CRE 启动子元件并在初级精母细胞中表达的睾丸特异性基因 Pgk2（磷酸甘油酸激酶 2）和 Pdha2（丙酮酸脱氢酶 alpha 2）启动子的表达。我们提出，MYBL1 通过某些基因的 CRE 位点指导生殖细胞特异性激活，这些基因在初级精母细胞中特异性激活，尽管 MYBL1 还有其他更间接的影响，但这仍是我们结果的一种可能解释。
