摘要
精子发生是指从精原干细胞经过一系列的有丝分裂和减数分裂产生单倍体的圆形精子细胞,这些细胞再经过巨大的形态重构而最终分化成成熟精子的过程。这一复杂过程受到多种阶段特异性和细胞特异性蛋白质的调节。因而,揭示生精细胞中参与基因调控的、睾丸特异性表达的蛋白质的特征及其功能对于阐明精子发生的机理具有极其重要的价值。
本实验室曾应用含抗精子抗体、可引起精子凝集的不孕妇女患者的血清筛选人睾丸λgtll cDNA表达型文库获得了一个新基因,命名为HSD-1,其GenBank接受号为U12978。该基因编码523个氨基酸,编码蛋白质被命名为hSMP-1。2003年人类基因命名数据库将其统一命名为SPAG8 (Sperm associated antigen)。荧光原位杂交实验显示SPAG8基因位于人类第9号染色体短臂1区2带至1区3带,由7个外显子和6个内含子组成,其在mRNA水平上存在多种剪接方式。多组织Northern blot分析显示SPAG8基因仅仅在睾丸组织中特异性高表达,大鼠睾丸组织的免疫组织化学结果显示SPAG8蛋白定位于圆形精子细胞和长形精子细胞内,这些结果提示SPAG8可能参与了精子发生过程。以SPAG8蛋白的C端序列作为诱饵,筛选人睾丸cDNA表达文库,获得多个与SPAG8蛋白可能存在相互作用的有意义克隆,其中一个克隆经序列比对后分析发现,与activator of CREM in testis (ACT)基因吻合。
在雄性生殖细胞分化过程中,减数分裂后活化的许多基因的启动子区都含有cAMP应答元件(CREs),这些元件可以募集转录因子CREB (CRE-binding protein)家族中的一个成员cAMP应答元件调节子(CREM)。由于选择性的转录起始点和选择性的剪接等因素,crem基因编码的产物包括激活型CREM(CREMτ)和抑制型的CREM。CREMτ从粗线期精母细胞开始大量表达,它调控多种减数分裂后特异的、关键基因的转录。已证实CREM靶向突变小鼠的精子发生完全停滞于圆形精子细胞阶段,说明了它在生殖细胞的分化过程发挥了极其重要的作用。CREMτ的活化方式在体细胞和在生殖细胞内是不同的：在体细胞中它的活化依赖其P-box结构域中117Ser被蛋白激酶A磷酸化后与CBP (CREB-binding protein)或p300等共激活子结合而激活下游基因的转录；在生殖细胞中它则是通过与其睾丸特异性表达的激活子ACT结合形成转录激活物来调节特异性靶基因的转录,这种活化过程并不依赖CREMτ的磷酸化以及与CBP等蛋白质的结合,从而为CREMτ提供了一条组织特异性的活化途径。
本论文在酵母双杂交结果的基础上,着重通过对SPAG8与ACT蛋白之间关系的研究来探讨SPAG8蛋白在ACT调节的CREM的转录激活过程中发挥的功能。
研究显示,SPAG8在睾丸发育过程中的表达以及在生精细胞中的分布与ACT蛋白存在着时间和空间上的吻合。SPAG8蛋白在小鼠出生后第三周的睾丸中开始出现表达,第四周时急剧增多并一直升高到成年时期。ACT蛋白主要从第四周时大量表达直至成年。这表明两者在发育时间上是有重合的。SPAG8蛋白定位在圆形精子细胞的胞浆和胞核内、长形精子细胞的胞浆内,这与ACT蛋白在精子细胞中的表达分布也有重合。但SPAG8并不仅限于ACT蛋白所存在的细胞内,我们还发现SPAG8蛋白在成熟精子的头部背侧以及尾部均有表达,这说明SPAG8在成熟精子中也发挥着作用。
在进一步的研究中,我们分别选用了体外和体内两种体系确证了SPAG8和ACT的相互作用。首先,利用原核表达系统表达和纯化了融合蛋白GST-ACT,将等量的小鼠睾丸组织抽提液分别与纯化的GST-ACT和GST进行孵育,再加入Glutathione Sepharose4B树脂进行GST pulldown实验,结果发现睾丸组织中的SPAG8蛋白与GST-ACT能够同时被Glutathione Sepharose 4B树脂结合下来,说明这两种蛋白质在体外能发生相互作用。为了在体内系统中验证SPAG8和ACT蛋白的相互作用,我们将Myc-SPAG8和Flag-ACT的表达质粒共同转染HEK293T细胞。分别用抗Flag和抗Myc的抗体进行沉淀,结果显示过表达的SPAG8能与ACT蛋白相结合。通过将SPAG8与ACT分别进行缺失突变后寻找彼此的结合位点,免疫共沉淀结果显示,SPAG8蛋白除了N端的140个氨基酸长度区域不参与同ACT蛋白的结合外,其它区域均与ACT蛋白结合。而ACT蛋白中的第2个LIM结构域是介导ACT与SPAG8蛋白结合的主要部位。
由于ACT通过与CREMτ蛋白结合而有力地激活了CREMτ的转录活性,我们又证实SPAG8能够与ACT相互结合,所以我们推测SPAG8可能影响ACT-CREMτ复合物的转录调控过程。为了支持这一观点,我们用双荧光素酶报告基因分析的方法进行验证。以Gal4DBD-CREMτAD可自动结合并激活含Gal4结合元件的报告基因活性建立报告基因体系平台。在ACT存在的条件下加入剂量逐渐增加的SPAG8表达质粒,结果发现SPAG8能够以剂量依赖的方式增强ACT调节的Gal4DBD-CREMτ的转录活性。将报告基因上游的启动子区换成CREMτ靶基因上游的特异性结合元件CREs时,SPAG8同样能够增强ACT-CREMτ的转录激活活性。这些结果提示SPAG8具有增强ACT-CREMτ转录激活的能力。我们进一步用免疫共沉淀的方法研究了SPAG8蛋白是如何发挥作用的?通过免疫共沉淀的方法发现SPAG8与CREMτ之间并不相互结合；当SPAG8, ACT和CREMτ三者共存在时,SPAG8能够明显地增强ACT和CREMτ之间的结合。结果提示SPAG8可能是通过增强ACT与CREMτ蛋白质的结合而使ACT-CREMτ复合物的转录激活活性增强的。由于CREMτ在体细胞内的活化方式与生殖细胞不同,因此为了观察在体细胞内过表达的SPAG8对ACT与CREMτ的转录激活作用是否受到CREMτ的磷酸化影响,我们将CREMτ的117位丝氨酸位点突变成甘氨酸后进行研究。报告基因分析结果提示SPAG8增强ACT与CREMτ的转录激活作用并不依赖于CREMτ磷酸化位点。免疫共沉淀的结果同样提示SPAG8对ACT与CREMτ的结合增强也不受CREMτ磷酸化位点突变的影响。综合上述实验结果,我们推测在生殖细胞内SPAG8蛋白可能通过增强ACT-CREMτ蛋白复合物的转录激活活性而引起它们调控的特异基因的转录增多,以满足精子形成过程中对这些基因的大量需求。因此,这个发现使我们能够更深入地了解生殖细胞内复杂的转录调控机制。
Mammalian spermatogenesis is a highly complex and ordered process in which stem cells undergo mitotic proliferation proceed into meiosis, followed by a remodeling of the haploid spermatids to form mature spermatozoa, associated with the expression of multiple genes to form essential proteins under stringent temporal and spatial regulation. So, the identification of an increasing number of transcription factors and coactivators that play specific roles during germ cells differentiation will constantly extend the understanding of spermatogenesis.
SPAG8 (sperm-associated antigene 8), also known as HSD-1, was isolated from the human testis cDNA expression library by using the antiserum of an infertile woman which contained anti-sperm antigen antibody and led to spermatozoa agglutination. SPAG8 gene was a new gene and assigned the accession number U12978 by GeneBank and named by HUGO Gene Nomenclature Committee in 2003. It is located on the 9th chromosome by using FISH assay. There are 7 exons and 6 introns in the SPAG8 gene. The results of northern blot for sixteen different mRNAs prepared from human tissues showed that the SPAG8 mRNA was only present in human testis, and immunohistochemistry results also showed that SPAG8 protein localizes in round and elongated spermatids in rat testes. These previous results hint us that SPAG8 might involve in the program of spermatogenesis. Using yeast two-hybrid system with the C-terminal sequence of SPAG8 gene as bait to screen the human testis expression library, several potential interaction proteins with SPAG8 were obtained. One nucleotide sequence among these clones was blasted as that of activator of CREM in testis (ACT).
During germ cell differentiation, many genes activated postmeiotically contain CREs (cAMP-responsive elements), which recruit a member of the CREB family of transcription factors, CREM. Due to alternative transcriptional initiation and alternative splicing process, the products coded by crem gene contain activator CREM (CREMτ) and repressor CREM. From the pachytene spermatocyte stage onward, the transcript corresponding to CREMτis expressed at very high level. CREMτregulates the transcription of many specific and important genes postmeiotically. Crem-null mice completely blocks spermatogenesis at early spermatid stage, indicating that CREM is essential for germ cell differentiation. There is a difference on CREMτactivation in somatic cells and germ cells:activated CREMτin somatic calls is dependent on the phosphorylation of Ser117 in P-box by PKA and consequently binding to CBP or p300, wherase in germ cells CREMτbinds to testis-specific ACT to act as a powerful transcriptional activator in a phosphorylation and CBP-independent manner, which points to a tissue-specific modulation mechanism of CREM transcriptional activity.
In this paper, we focus on the function of SPAG8 protein by studing the association of SPAG8 with ACT, basing on the previous yeast two-hybrid result about SPAG8.
During testis development, the expression of SPAG8 partially overlaps with that of ACT in a spatial and temporal manner. SPAG8 protein is very low in mouse testes at the age of 3 weeks after birth, whereas a robust increase occurs at the fourth week, at the time of accumulation of ACT expression. Using cell immunofluorescence assay, we find that SPAG8 protein localizes in both cytoplasm and nucleus of round spermatid and the cytoplasm of elongated sptermatid. The distribution of SPAG8 protein in spermatids partially overlaps with that of ACT. However, SPAG8 expression in male germ cells is not restricted to the stages involving ACT function, but it is also present later in elongating spermatids and mature sperm at a time when the expression of ACT has already disappeared. At these stages of sperm development, SPAG8 accumulates in the region of sperm head and tail, indicating that SPAG8 might have other function in these cells.
In vitro and in vivo systems had been used to comfirm the interaction between SPAG8 and ACT. GST-ACT fusion protein had been expressed in E coli cells, and purified by affinity chromatography. GST pull-down assay was performed by incubating GST-ACT fusion protein or GST with testis lysate. The result showed that SPAG8 protein can be co-precipitated with GST-ACT using Glutathione Sepharose 4B agarose, but not with GST alone. To further examine whether SPAG8 can interact with ACT in physiological condition, pcDNA6-HisB-Myc-SPAG8 and p3×Flag-CMV-14-ACT plasmids were co-transfected into HEK293T cells. Lysates of transfected cells had been immunoprecipitated by anti-Flag and anti-Myc antibody respectively. The result showed that overexpressed SPAG8 protein could interact with ACT in vivo. Besides, we also found that 140-412aa region of SPAG8 involves in association with ACT, and that the second LIM domain of ACT plays an important role in the binding of ACT to SPAG8.
Since ACT powerfully activates the transcriptional activity of CREMτand ACT can interact with SPAG8, we wondered whether SPAG8 can influence the transcriptional activation of ACT-CREMτcomplex. To confirm this hypothesis, we used dual luciferase reporter gene assay. We constructed Gal4DBD-CREMτAD expressing vector encoding chimeric CREMτAD protein, provided with an autonomous DNA-binding capability. In the presence of ACT, it seems to be that SPAG8 could enhance the transcription activation of ACT-medicated CREMτin a dose-dependent manner. Analogical result was obtained with full length CREMτon a CRE-driven somatostatin reporter promoter.These results suggested that SPAG8 possesses a potential ability to enhancing the transcriptional activation of ACT-CREMτ. To further study the mechanism of transcriptional activation enhanced by SPAG8, we firstly observed the association of SPAG8 with CREMτby cell co-immunoprecipitation assay. The result showed that SPAG8 does not interact with CREMτ. ACT, SPAG8 and CREMτexpression vectors were co-transfected into HEK293T cells to perform co-immunoprecipitation experiments. The result showed that SPAG8 could obviously enhance the binding of ACT to CREMτ. So, we suggest that the transcriptional activity of ACT-medicated CREMτenhanced by SPAG8 is due to the binding of ACT to CREMτenhanced by SPAG8. In order to exclude that enhanced transcriptional activation by SPAG8 was influenced by the phosphorylation of CREMτSer117 in somatic cells, serine117 in CREMτwas mutanted into alanine. CREMτSer117Ala was used in luciferase reporter gene assay and co-immunoprecipitation experiments. The results indicated that SPAG8 enhanced the transcriptional activation of ACT-medicated CREMτindependent on the the phosphorylation of CREMτSer117 and the enhancement of the binding of ACT to CREMτby SPAG8 is also not influenced as the phosphorylation site mutant. In closing, we suggest that SPAG8 plays roles in the transcription regulation of cell-stage specific genes by ACT-CREMτcomplex by interaction with ACT in germ cells, which provides a deeper understanding for the complex transcriptional mechanism in post-meiosis germ cells.
引文
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