A 50-bp enhancer of the mouse acrosomal vesicle protein 1 gene activates round spermatid-specific transcription in vivo†.

Enhancers are cis-elements that activate transcription and play critical roles in tissue- and cell type-specific gene expression. During spermatogenesis, genes coding for specialized sperm structures are expressed in a developmental stage- and cell type-specific manner, but the enhancers responsible for their expression have not been identified. Using the mouse acrosomal vesicle protein (Acrv1) gene that codes for the acrosomal protein SP-10 as a model, our previous studies have shown that Acrv1 proximal promoter activates transcription in spermatids; and the goal of the present study was to separate the enhancer responsible. Transgenic mice showed that three copies of the -186/-135 fragment (50 bp enhancer) placed upstream of the Acrv1 core promoter (-91/+28) activated reporter expression in testis but not somatic tissues (n = 4). Immunohistochemistry showed that enhancer activity was restricted to the round spermatids. The Acrv1 enhancer failed to activate transcription in the context of a heterologous core promoter (n = 4), indicating a likely requirement for enhancer-core promoter compatibility. Chromatin accessibility assays showed that the Acrv1 enhancer assumes a nucleosome-free state in male germ cells (but not liver), indicating occupancy by transcription factors. Southwestern assays (SWA) identified specific binding of the enhancer to a testis nuclear protein of 47 kDa (TNP47). TNP47 was predominantly nuclear and becomes abundant during the haploid phase of spermatogenesis. Two-dimensional SWA revealed the isoelectric point of TNP47 to be 5.2. Taken together, this study delineated a 50-bp enhancer of the Acrv1 gene for round spermatid-specific transcription and identified a putative cognate factor. The 50-bp enhancer could become useful for delivery of proteins into spermatids.

TDP-43 is a transcriptional repressor: the testis-specific mouse acrv1 gene is a TDP-43 target in vivo.

TDP-43 is an evolutionarily conserved ubiquitously expressed DNA/RNA-binding protein. Although recent studies have shown its association with a variety of neurodegenerative disorders, the function of TDP-43 remains poorly understood. Here we address TDP-43 function using spermatogenesis as a model system. We previously showed that TDP-43 binds to the testis-specific mouse acrv1 gene promoter in vitro via two GTGTGT-motifs and that mutation of these motifs led to premature transcription in spermatocytes of an otherwise round spermatid-specific promoter. The present study tested the hypothesis that TDP-43 represses acrv1 gene transcription in spermatocytes. Plasmid chromatin immunoprecipitation demonstrated that TDP-43 binds to the acrv1 promoter through GTGTGT motifs in vivo. Reporter gene assays showed that TDP-43 represses acrv1 core promoter-driven transcription via the N-terminal RRM1 domain in a histone deacetylase-independent manner. Consistent with repressor role, ChIP on physiologically isolated germ cells confirmed that TDP-43 occupies the endogenous acrv1 promoter in spermatocytes. Surprisingly, however, TDP-43 remains at the promoter in round spermatids, which express acrv1 mRNA. We show that RNA binding-defective TDP-43, but not splice variant isoforms, relieve repressor function. Transitioning from repressive to active histone marks has little effect on TDP-43 occupancy. Finally, we found that RNA polymerase II is recruited but paused at the acrv1 promoter in spermatocytes. Because mutation of TDP-43 sites caused premature transcription in spermatocytes in vivo, TDP-43 may be involved in pausing RNAPII at the acrv1 promoter in spermatocytes. Overall, our study shows that TDP-43 is a transcriptional repressor and that it regulates spatiotemporal expression of the acrv1 gene during spermatogenesis.

Histone H2A.Z acid patch residues required for deposition and function.

The incorporation of histone variants is one mechanism used by the eukaryotic cell to alter the generally repressive chromatin template. However, the exact molecular mechanisms that direct this incorporation are not well understood. The SWR1 chromatin remodeling complex that binds to and directs incorporation of histone variant H2A.Z into chromatin has been characterized, but significantly less information is available concerning the requirements on the H2A.Z target molecule. We performed an unbiased mutagenic screen designed to elucidate the function of H2A.Z in Saccharomyces cerevisiae. The screen identified residues within the conserved acidic patch of H2A.Z as being important for the function of the variant. We characterized single point mutations in the patch that are phenotypically sensitive to a variety of growth conditions and are expressed at lower protein levels, but are functionally defective (htz1-D99A, htz1-D99K, and htz1-E101K). The mutants were significantly less detectable by chromatin immunoprecipitation at PHO5, a gene previously described to be enriched for H2A.Z. These results identify acidic patch residues of H2A.Z that are critical for mediating deposition and function in chromatin, and represent potential candidates for the interaction of H2A.Z with its deposition and/or targeting machinery.

Targeted deletion of Tssk1 and 2 causes male infertility due to haploinsufficiency.

Targeted deletion of Tssk1 and 2 resulted in male chimeras which produced sperm/spermatogenic cells bearing the mutant allele, however this allele was never transmitted to offspring, indicating infertility due to haploinsufficiency. Morphological defects in chimeras included failure to form elongated spermatids, apoptosis of spermatocytes and spermatids, and the appearance of numerous round cells in the epididymal lumen. Characterization of TSSK2 and its interactions with the substrate, TSKS, were further investigated in human and mouse. The presence of both kinase and substrate in the testis was confirmed, while persistence of both proteins in spermatozoa was revealed for the first time. In vivo binding interactions between TSSK2 and TSKS were established through co-immunoprecipitation of TSSK2/TSKS complexes from both human sperm and mouse testis extracts. A role for the human TSKS N-terminus in enzyme binding was defined by deletion mapping. TSKS immunoprecipitated from both mouse testis and human sperm extracts was actively phosphorylated. Ser281 was identified as a phosphorylation site in mouse TSKS. These results confirm both TSSK 2 and TSKS persist in sperm, define the critical role of TSKS' N-terminus in enzyme interaction, identify Ser 281 as a TSKS phosphorylation site and indicate an indispensable role for TSSK 1 and 2 in spermiogenesis.

TSKS concentrates in spermatid centrioles during flagellogenesis.

Centrosomal coiled-coil proteins paired with kinases play critical roles in centrosomal functions within somatic cells, however knowledge regarding gamete centriolar proteins is limited. In this study, the substrate of TSSK1 and 2, TSKS, was localized during spermiogenesis to the centrioles of post-meiotic spermatids, where it reached its greatest concentration during the period of flagellogenesis. This centriolar localization persisted in ejaculated human spermatozoa, while centriolar TSKS diminished in mouse sperm, where centrioles are known to undergo complete degeneration. In addition to the centriolar localization during flagellogenesis, mouse TSKS and the TSSK2 kinase localized in the tail and acrosomal regions of mouse epididymal sperm, while TSSK2 was found in the equatorial segment, neck and the midpiece of human spermatozoa. TSSK2/TSKS is the first kinase/substrate pair localized to the centrioles of spermatids and spermatozoa. Coupled with the infertility due to haploinsufficiency noted in chimeric mice with deletion of Tssk1 and 2 (companion paper) this centriolar kinase/substrate pair is predicted to play an indispensable role during spermiogenesis.

Role of an insulator in testis-specific gene transcription.

Testis-specific promoters are unique in that relatively short proximal promoters of several genes have been shown to be capable of directing tissue- and cell-type-specific expression in transgenic mice. How such small promoter fragments perform the dual functions of maintaining a silenced state in somatic tissues and activating gene expression in the correct germ-cell type in testis remains poorly understood. Studies from our laboratory using the round spermatid-specific SP-10 gene as an experimental model have provided some insights into the mechanisms involved. It was found that the proximal promoter of the SP-10 gene acts as a chromatin insulator or boundary element in somatic tissues and prevents transcription of the SP-10 gene. In round spermatids, the insulator function is relieved, thus facilitating the SP-10 gene transcription. Insulators act as enhancer blockers and/or barriers to heterochromatin to protect the programmed expression of a gene. Typically, insulators are separable from promoters. In the case of the SP-10 gene, however, the insulator overlaps the promoter and operates in a facultative manner. We hypothesize that the proximal promoters of some testis-specific genes have adapted the insulator function to maintain transcriptional silence in the somatic tissues.

A novel CpG-free vertebrate insulator silences the testis-specific SP-10 gene in somatic tissues: role for TDP-43 in insulator function.

Regulation of cell type-specific gene transcription is central to cellular differentiation and development. During spermatogenesis, a number of testis-specific genes are expressed in a precise spatiotemporal order. How these genes remain silent in the somatic tissues is not well understood. Our previous studies using the round spermatid-specific mouse SP-10 gene, which codes for an acrosomal protein, revealed that its proximal promoter acts as an insulator and prevents expression in the somatic tissues. Here we report that the insulator tethers the SP-10 gene to the nuclear matrix in somatic tissues, sequestering the core promoter in the process, thus preventing transcription. In round spermatids where the SP-10 gene is expressed, this tethering is released. TAR DNA-binding protein of 43 kDa (TDP-43), previously shown to interact with the SP-10 insulator, was found to be in the 2 m NaCl-insoluble nuclear matrix fraction. TDP-43 prevented enhancer-promoter interactions when artificially recruited between the two by Gal4 strategy. Knockdown of TDP-43 using small interfering RNA released the enhancer-blocking effect of the SP-10 insulator in a stable cell culture model. Mutation of TDP-43 binding sites abolished this effect. Finally, a 50-bp subfragment of the SP-10 insulator, which includes TDP-43 binding sites, functioned as a minimal insulator in transgenic mice and silenced an otherwise ectopically expressed transgene in somatic tissues. The SP-10 insulator lacks CpG dinucleotides or CTCF binding sites. Thus, the present study characterized a novel vertebrate insulator in a physiological context and showed for the first time how a testis-specific gene is silenced in the somatic tissues by an insulator.