Spag8 (sperm Associated Antigen 8), A New Testis-specific Coactivator,

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.