Testis-specific Transcription Factor Research Articles

Loss of the importin Kpna2 causes infertility in male mice by disrupting the translocation of testis-specific transcription factors

Karyopherins mediate the movement between the nucleus and cytoplasm of specific proteins in diverse cellular processes. Through a loss-of-function approach, we here examine the role of Karyopherin Subunit Alpha 2 (Kpna2) in spermatogenesis. Knockout male mice exhibited reduced body size and sperm motility, increased sperm abnormalities, and led to the dysregulation of testis gene expression and ultimately to infertility. Impaired mRNA expression mainly affected clusters of genes expressed in spermatids and spermatocytes. Downregulated genes included a set of genes that participate in cell adhesion and extracellular matrix (ECM) organization. We detected both the enrichment of some transcription factors that bind to regions around transcription start sites of downregulated genes and the impaired transport of specific factors to the nucleus of spermatid cells. We propose that Kpna2 is essential in the seminiferous tubules for promoting the translocation of testis-specific transcription factors that control the expression of genes related to ECM organization.

The transcription factor TCFL5 responds to A-MYB to elaborate the male meiotic program in mice.

The testis-specific transcription factor, TCFL5, expressed in pachytene spermatocytes regulates the meiotic gene expression program in collaboration with the transcription factor A-MYB. In male mice, the transcription factors STRA8 and MEISON initiate meiosis I. We report that STRA8/MEISON activates the transcription factors A-MYB and TCFL5, which together reprogram gene expression after spermatogonia enter into meiosis. TCFL5 promotes the transcription of genes required for meiosis, mRNA turnover, miR-34/449 production, meiotic exit, and spermiogenesis. This transcriptional architecture is conserved in rhesus macaque, suggesting TCFL5 plays a central role in meiosis and spermiogenesis in placental mammals. Tcfl5em1/em1 mutants are sterile, and spermatogenesis arrests at the mid- or late-pachytene stage of meiosis. Moreover, Tcfl5+/em1 mutants produce fewer motile sperm.

A novel high throughput screen to identify candidate molecular networks that regulate spermatogenic stem cell functions†.

Spermatogenic regeneration is key for male fertility and relies on activities of an undifferentiated spermatogonial population. Here, a high-throughput approach with primary cultures of mouse spermatogonia was devised to rapidly predict alterations in functional capacity. Combining the platform with a large-scale RNAi screen of transcription factors, we generated a repository of new information from which pathway analysis was able to predict candidate molecular networks regulating regenerative functions. Extending from this database, the SRCAP-CREBBP/EP300 (Snf2-related CREBBP activator protein-CREB binding protein/E1A binding protein P300) complex was found to mediate differential levels of histone acetylation between stem cell and progenitor spermatogonia to influence expression of key self-renewal genes including the previously undescribed testis-specific transcription factor ZSCAN2 (zinc finger and SCAN domain containing 2). Single cell RNA sequencing analysis revealed that ZSCAN2 deficiency alters key cellular processes in undifferentiated spermatogonia such as translation, chromatin modification, and ubiquitination. In Zscan2 knockout mice, while spermatogenesis was moderately impacted during steady state, regeneration after cytotoxic insult was significantly impaired. Altogether, these findings have validated the utility of our high-throughput screening approach and have generated a transcription factor database that can be utilized for uncovering novel mechanisms governing spermatogonial functions.

Gene promoter polymorphisms in boar spermatozoa differing in freezability

Single nucleotide polymorphisms (SNPs) in the 5′-flanking regulatory regions of genes could affect their expression levels. This is a follow-up study aimed to identify polymorphic variants in the 5′-flanking regulatory regions of genes expressed in boar spermatozoa, and to predict the interactions of such variants with transcription factors (TFs) on the gene promoter activity, using bioinformatics. Five and six boars were classified as having good and poor semen freezability (GSF and PSF, respectively) according to post-thaw (PT) assessment of sperm motility and membrane integrity characteristics. The 5′-flanking region sequences of the 14 genes (FOS, NFATC3, EAF2, FGF-14, BAMBI, RAB33B, CKS2, LARS2, SLC25A16, ACADM, CPT2, CCT3, DTD2 and CCDC85A) were PCR amplified and analyzed by Sanger sequencing method. A total of 32 polymorphic variants were identified in the 5′-flanking regions of the genes, including 4 insertion/deletion (indel) polymorphisms, and 8 unknown (novel) SNPs. Multiple sequence alignment analysis revealed a 26-bp indel variant in the 5′-flanking region of the LARS2 gene, which showed greater protein expression in spermatozoa from boars of the PSF group. It was found that 17 polymorphic variants, observed in the differentially expressed (DE) genes, showed significant allele frequency differences between the GSF and PSF groups. Polymorphic variants in the 5′-flanking regulatory regions of the genes contributed to the decrease or increase in the binding affinity for different testis-specific TFs, such as SMAD1, NF-1, FOXMI, RXRA, STAT4 and C/EBPβ. This study provides more insights into the mechanisms responsible for variations in transcriptional activity in promoters of genes expressed in boar spermatozoa. The allelic variants are promising genetic markers for predicting the freezability of boar spermatozoa.

The testis protein ZNF165 is a SMAD3 cofactor that coordinates oncogenic TGFβ signaling in triple-negative breast cancer.

Cancer/testis (CT) antigens are proteins whose expression is normally restricted to germ cells yet aberrantly activated in tumors, where their functions remain relatively cryptic. Here we report that ZNF165, a CT antigen frequently expressed in triple-negative breast cancer (TNBC), associates with SMAD3 to modulate transcription of transforming growth factor β (TGFβ)-dependent genes and thereby promote growth and survival of human TNBC cells. In addition, we identify the KRAB zinc finger protein, ZNF446, and its associated tripartite motif protein, TRIM27, as obligate components of the ZNF165-SMAD3 complex that also support tumor cell viability. Importantly, we find that TRIM27 alone is necessary for ZNF165 transcriptional activity and is required for TNBC tumor growth in vivo using an orthotopic xenograft model in immunocompromised mice. Our findings indicate that aberrant expression of a testis-specific transcription factor is sufficient to co-opt somatic transcriptional machinery to drive a pro-tumorigenic gene expression program in TNBC.

The arginine methyltransferase CARM1 represses p300•ACT•CREMτ activity and is required for spermiogenesis.

CARM1 is a protein arginine methyltransferase (PRMT) that has been firmly implicated in transcriptional regulation. However, the molecular mechanisms by which CARM1 orchestrates transcriptional regulation are not fully understood, especially in a tissue-specific context. We found that Carm1 is highly expressed in the mouse testis and localizes to the nucleus in spermatids, suggesting an important role for Carm1 in spermiogenesis. Using a germline-specific conditional Carm1 knockout mouse model, we found that it is essential for the late stages of haploid germ cell development. Loss of Carm1 led to a low sperm count and deformed sperm heads that can be attributed to defective elongation of round spermatids. RNA-seq analysis of Carm1-null spermatids revealed that the deregulated genes fell into similar categories as those impacted by p300-loss, thus providing a link between Carm1 and p300. Importantly, p300 has long been known to be a major Carm1 substrate. We found that CREMτ, a key testis-specific transcription factor, associates with p300 through its activator, ACT, and that this interaction is negatively regulated by the methylation of p300 by Carm1. Thus, high nuclear Carm1 levels negatively impact the p300•ACT•CREMτ axis during late stages of spermiogenesis.

Epigenetic regulation of the histone-to-protamine transition during spermiogenesis

In mammals, male germ cells differentiate from haploid round spermatids to flagella-containing motile sperm in a process called spermiogenesis. This process is distinct from somatic cell differentiation in that the majority of the core histones are replaced sequentially, first by transition proteins and then by protamines, facilitating chromatin hyper-compaction. This histone-to-protamine transition process represents an excellent model for the investigation of how epigenetic regulators interact with each other to remodel chromatin architecture. Although early work in the field highlighted the critical roles of testis-specific transcription factors in controlling the haploid-specific developmental program, recent studies underscore the essential functions of epigenetic players involved in the dramatic genome remodeling that takes place during wholesale histone replacement. In this review, we discuss recent advances in our understanding of how epigenetic players, such as histone variants and histone writers/readers/erasers, rewire the haploid spermatid genome to facilitate histone substitution by protamines in mammals.

MEF2 Is Restricted to the Male Gonad and Regulates Expression of the Orphan Nuclear Receptor NR4A1

Leydig cell steroidogenesis is controlled by the pituitary gonadotropin LH that activates several signaling pathways, including the Ca(2+)/calmodulin kinase I (CAMKI) pathway. In other tissues, CAMKI regulates the activity of the myocyte enhancer factor 2 (MEF2) transcription factors. MEF2 factors are essential regulators of cell differentiation and organogenesis in numerous tissues but their expression and role in the mammalian gonad had not been explored. Here we show that MEF2 factors are expressed in a sexually dimorphic pattern in the mouse gonad. MEF2 factors are present in the testis throughout development and into adulthood but absent from the ovary. In the testis, MEF2 was localized mainly in the nucleus of both somatic lineages, the supporting Sertoli cells and the steroidogenic Leydig cells. In Leydig cells, MEF2 was found to activate the expression of Nr4a1, a nuclear receptor important for hormone-induced steroidogenesis. In these cells MEF2 also cooperates with forskolin and CAMKI to enhance Nr4a1 promoter activity via two MEF2 elements (-318 and -284 bp). EMSA confirmed direct binding of MEF2 to these elements whereas chromatin immunoprecipitation revealed that MEF2 recruitment to the proximal Nr4a1 promoter was increased following hormonal stimulation. Modulation of endogenous MEF2 protein level (small interfering RNA-mediated knockdown) or MEF2 activity (MEF2-Engrailed active dominant negative) led to a significant decrease in Nr4a1 mRNA levels in Leydig cells. All together, our results identify MEF2 as a novel testis-specific transcription factor, supporting a role for this factor in male sex differentiation and function. MEF2 was also positioned upstream of NR4A1 in a regulatory cascade controlling Leydig cell gene expression.

Sequence-specific promoter elements regulate temporal-specific changes in chromatin required for testis-specific activation of the Pgk2 gene

The phosphoglycerate kinase-2 (Pgk2) gene is regulated in a tissue-, cell type-, and developmental stage-specific manner during spermatogenesis and is required for normal sperm motility and fertility in mammals. Activation of Pgk2 transcription is regulated by testis-specific demethylation of DNA and binding of testis-specific transcription factors to enhancer and core promoter elements. Here, we show that chromatin remodeling including reconfiguration of nucleosomes and changes in histone modifications is also associated with transcriptional activation of the Pgk2 gene during spermatogenesis. Developmental studies indicate that the order of events involved in transcriptional activation of the Pgk2 gene includes demethylation of DNA in T₁- and T₂-prospermatogonia, binding of a factor to the CAAT box in type A and B spermatogonia, followed by recruitment of chromatin remodeling factors, displacement of a nucleosome from the Pgk2 promoter region, binding of factors to the Pgk2 core promoter and enhancer regions, and, finally, initiation of transcription in primary spermatocytes. Transgene studies show that Pgk2 core promoter elements are required to direct demethylation of DNA and reconfiguration of nucleosomes, whereas both enhancer and core promoter elements are required to direct changes in histone modifications and initiation of transcription. These results provide novel insight into the developmental order of molecular events required to activate tissue-specific transcription of the Pgk2 gene, the distinct elements in the 5'-regulatory region of the Pgk2 gene that regulate each of these events, and the relationship among these events in that each step in this process appears to be a necessary prerequisite for the subsequent step.

GRTH: A Key to Understanding Androgen-Mediated Germ Cell Signaling

GRTH: A Key to Understanding Androgen-Mediated Germ Cell Signaling

Dose-Dependent Activation of Putative Oncogene SBSN by BORIS

Testis-specific transcription factor BORIS (Brother of the Regulator of Imprinted Sites), a paralog and proposed functional antagonist of the widely expressed CTCF, is abnormally expressed in multiple tumor types and has been implicated in the epigenetic activation of cancer-testis antigens (CTAs). We have reported previously that suprabasin (SBSN), whose expression is restricted to the epidermis, is epigenetically derepressed in lung cancer. In this work, we establish that SBSN is a novel non-CTA target of BORIS epigenetic regulation. With the use of a doxycycline-inducible BORIS expressing vector, we demonstrate that relative BORIS dosage is critical for SBSN activation. At lower concentrations, BORIS induces demethylation of the SBSN CpG island and disruption and activation of chromatin around the SBSN transcription start site (TSS), resulting in a 35-fold increase in SBSN expression in the H358 human lung cancer cell line. Interestingly, increasing BORIS concentrations leads to a subsequent reduction in SBSN expression via chromatin repression. In a similar manner, increase in BORIS concentrations leads to eventual decrease of cell growth and colony formation. This is the first report demonstrating that different amount of BORIS defines its varied effects on the expression of a target gene via chromatin structure reorganization.

Zmynd15 Encodes a Histone Deacetylase-dependent Transcriptional Repressor Essential for Spermiogenesis and Male Fertility

Spermatogenesis is a complex process through which male germ line stem cells undergo a multi-step differentiation program and sequentially become spermatogonia, spermatocytes, spermatids, and eventually spermatozoa. In this process, transcription factors act as switches that precisely regulate the expression of genes that in turn control the developmental program of male germ cells. Transcription factors identified to be essential for normal haploid gene expression all display transcription-activating effects and thus serve as the "on" switch for haploid gene expression. Here, we report that ZMYND15 acts as a histone deacetylase-dependent transcriptional repressor and controls normal temporal expression of haploid cell genes during spermiogenesis. Inactivation of Zmynd15 results in early activation of transcription of numerous important haploid genes including Prm1, Tnp1, Spem1, and Catpser3; depletion of late spermatids; and male infertility. ZMYND15 represents the first transcriptional repressor identified to be essential for sperm production and male fertility.

Male‐specific expression of Aldh1a1 in mouse and chicken fetal testes: Implications for retinoid balance in gonad development

Balanced production and degradation of retinoids is important in regulating development of several organ systems in the vertebrate embryo. Among these, it is known that retinoic acid (RA), and the retinoid-catabolyzing enzyme CYP26B1 together regulate the sex-specific behavior of germ cells in developing mouse gonads. We report here that the gene encoding a cytosolic class-1 aldehyde dehydrogenase, ALDH1A1, a weak catalyst of RA production, is strongly expressed in a male-specific manner in somatic cells of the developing mouse testis, beginning shortly after Sry expression is first detectable. This expression pattern is conserved in the developing male gonad of the chicken and is dependent on the testis-specific transcription factor SOX9. Our data suggest that low levels of RA may be required for early developmental events in the testis, or that Aldh1a1 expression in the fetus may prefigure a later requirement for ALDH1A1 in regulating spermatogenesis postnatally.

Cloning and Character Analysis of A Novel Testis-specific Gene, TSF22, in Mice

Objective To identify genes that involved in spermatogenesis. Methods In order to screen the testis-specific genes, testes cDNA samples from BALB/c mice of different postnatal days (days 4, 9, 18, 35, 54 and 6 months) were performed with mouse whole genome Affymetrix chip. The characteristics of the selected gene were analyzed by various bioinformatic tools. The expression profile of the selected gene was identified by RT-PCR. Results By analyzing the hybridization signals, a gene with a differential expression in the developmental stages of testis was identified. This gene was designated as TSF22.The full length cDNA of 1 597 bp contained an open reading frame of 570 bp which encoded a putative protein of 190 amino acids and a molecular weight of 22.106 kD. RT-PCR analysis revealed that TSF22 mRNA was exclusively expressed in mice testis. Conclusions TSF22, functions as a testis-specific transcription factor, may play important roles during spermatogenesis.

Identification of a novel mutation in the SRY gene in a 46, XY female patient

Background The SRY gene encodes for a testis-specific transcription factor (TDF, testis determining factor) that plays a key role in sexual differentiation and development in males. Several SRY mutations have been described in patients with gonadal dysgenesis, accounting for 10–15% of the sex reversal cases. The reported mutations are both point mutations and deletions, mostly involving the high mobility group (HMG) box domain of SRY, which is a conserved region through the evolution, suggesting that SRY function strictly depends on the HMG box. Case presentation Here we describe the clinical, endocrinological and molecular data of a patient with complete 46, XY gonadal dysgenesis caused by SRY mutation located within the conserved HMG box. Using DNA direct sequencing of the SRY coding region, we identified a single nucleotide insertion at codon 89 with subsequent frameshift of the reading frame sequence, which results in a truncated protein as consequence of an introduction of a stop codon at the position 103. Conclusion A novel SRY mutation has been described in a female with a gonadal dysgenesis associated with a 46, XY karyotype. The described case is of importance for genetic counseling.

Sohlh1 is essential for spermatogonial differentiation

Spermatogonia are adult germline stem cells that can both self-renew and differentiate into spermatocytes. Little is known about factors necessary for spermatogonial differentiation. We identified a novel germ-cell-specific transcription factor that we named Sohlh1 (spermatogenesis and oogenesis specific basic helix–loop–helix (bHLH) transcription factor). In males, Sohlh1 is preferentially expressed in prespermatogonia and Type A spermatogonia. Loss of Sohlh1 causes infertility by disrupting spermatogonial differentiation into spermatocytes. Seven-day-old testes without Sohlh1 still express the testis-specific transcription factors Etv5, Taf4b, Zfp148, and Plzf, overexpress a novel Sohlh2 bHLH transcription factor, but lack LIM homeobox gene Lhx8 and show reduced expression of Ngn3. Sohlh1 represents the first testis-specific bHLH transcription factor that is essential for spermatogonial differentiation.

Epigenetic Regulation of Testis‐Specific Gene Expression

Cellular differentiation is mediated by differential gene expression. The cells of the testis are no exception. Indeed, recent studies based on microarray and expressed sequence tag analyses have revealed dynamic changes in gene expression during spermatogenesis. The autosomal phosphoglycerate kinase gene Pgk2 is an example of a gene that is tightly regulated during spermatogenesis, with transcription initiating in primary spermatocytes and ceasing in postmeiotic spermatids. Our studies show that this tissue-specific transcription is regulated at multiple levels, including binding of ubiquitous and testis-specific transcription factors, reconfiguration of nucleosomes, decondensation of chromatin, modifications of specific residues in histones, and demethylation of DNA. Our ability to isolate relatively pure populations of different spermatogenic cell types has allowed us to determine the order of these events that lead up to initiation of transcription of the Pgk2 gene. By examining similar parameters in transgenic mice carrying various portions of Pgk2 regulatory sequence, we have localized specific signals responsible for each of these regulatory events. Together these results demonstrate that activation of testis-specific transcription of the Pgk2 gene involves a multifaceted, ordered cascade of epigenetic events that potentiates this locus in preparation for the initiation of transcription.

Basic helix-loop-helix transcription factor Tcfl5 interacts with the Calmegin gene promoter in mouse spermatogenesis

In mouse spermatogenesis, differentiating germ line cells initiate expression of specific genes at subsequent developmental steps. The Calmegin (Clgn) gene is first expressed in meiotic prophase, in primary spermatocytes, and encodes a protein that acts as a chaperone. To identify testis-specific transcription factors that control expression of the Clgn gene in spermatogenesis, we performed a yeast one-hybrid screening with a Clgn promoter sequence as bait DNA. This screening resulted in the identification of mouse Tcfl5 as a candidate Clgn promoter-binding protein. Tcfl5 is a member of the basic helix-loop-helix (bHLH) family of transcription factors, and mouse Tcfl5 shows 83% amino acid sequence identity with human TCFL5. Gel-shift and yeast one-hybrid experiments showed that Tcfl5 interacts with a non-canonical CACGCG site that is present in the Clgn promoter. By using northern blot, RT-PCR and in situ hybridization, mouse Tcfl5 mRNA was detected only in testis, with the highest expression level in primary spermatocytes and round spermatids. The highest level of Tcfl5 protein was found in primary spermatocytes at the diplotene stage of meiotic prophase, where the protein colocalizes with transcriptionally active chromatin.

Two new novel point mutations localized upstream and downstream of the HMG box region of the SRY gene in three Indian 46,XY females with sex reversal and gonadal tumour formation.

The Y chromosome-specific gene SRY is one of the key genes involved in human sex determination. The SRY gene encodes a testis-specific transcription factor that plays a key role in sexual differentiation and development in males and is located on the distal region of the short arm of the Y chromosome. Mutations in SRY gene result in XY sex reversal and pure gonadal dysgenesis. SRY expression initiates a network of gene activity that transforms the undifferentiated gonad, genital ridge into testis. Mutations in the SRY gene have been considered to account for only 10-15% of 46,XY gonadal dysgenesis cases, whereas the majority of the remaining cases may have mutation(s) in the SRY regulatory elements or other genes involved in the sex differentiation pathway. Patients both with gonadal dysgenesis and Y-chromosome presence are at high risk of developing gonadoblastoma. Using PCR, single strand conformational polymorphism (SSCP) and automated DNA sequencing, we analysed the mutations in the SRY gene in three 46,XY sex reversal patients. Two patients demonstrated nucleotide substitution (A-->G) within the open reading frame just outside and upstream of the conserved DNA-binding motif called the high-mobility group (HMG) box, replacing glutamine at codon 57 with arginine. Altered SSCP patterns were also observed in these patients. Histological examination of gonads in patient 1 revealed the formation of gonadoblastoma. Patient 3 demonstrated A-->T substitution which replaces serine at codon 143 with cysteine, just outside but downstream of the HMG box. Results suggest the involvement of SRY gene in sex reversal which further supports the relationship between SRY alterations, gonadal dysgenesis and/or primary infertility.

Molecular cloning and characterization of a mouse spermatogenesis-related ring finger gene znf230

Complete cDNA of mouse gene znf230 was cloned by rapid amplification of cDNA ends (RACE). This cDNA is 982 base pairs (bp) in length and encodes a 230 amino acids (aa) protein with a ring finger domain at its C-terminus. Ninety-one and 98% nucleotide (nt) and aa sequence identity are observed with its human homolog. Revealed by Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR), this cDNA is only detected in testicular tissue, whereas the longer transcripts of 2.4 and 4.4 kb are ubiquitously expressed. The expression of znf230 in testis is developmentally regulated and first detected at day 6 postnatal (pn). It reaches adult level between day 14 and 21 pn during which round spermatids appear in seminiferous tubule. The protein of znf230 exhibits DNA binding activity and its ring finger domain may function as an activator module in transcription. Therefore, it is postulated that znf230 may function as a testis specific transcription factor during mouse spermatogenesis.