The germ cell-specific transcription factor ALF. Structural properties and stabilization of the TATA-binding protein (TBP)-DNA complex. Upadhyaya AB 2002 et al.
The assembly and stability of the RNA polymerase II transcription preinitiation complex on a eukaryotic core promoter involves the effects of TFIIA on the interaction between TATA-binding protein (TBP) and DNA. To extend our understanding of these interactions, we characterized properties of ALF, a germ cell-specific TFIIA-like factor. ALF was able to stabilize the binding of TBP to DNA, but it could not stabilize TBP mutants A184E, N189E, E191R, and R205E nor could it facilitate binding of the TBP-like factor TRF2/TLF to a consensus TATA element. However, phosphorylation of ALF with casein kinase II resulted in the partial restoration of complex formation using mutant TBPs. Studies of ALF-TBP complexes formed on the Adenovirus Major Late (AdML) promoter revealed protection of the TATA box and upstream sequences from -38 to -20 (top strand) and -40 to -22 (bottom strand). The half-life and apparent K(D) of this complex was determined to be 650 min and 4.8 +/- 2.7 nm, respectively. The presence of ALF or TFIIA did not significantly alter the ability of TBP to bind TATA elements from several testis-specific genes. Finally, analysis of the distinct, nonhomologous internal regions of ALF and TFIIAalpha/beta using circular dichroism spectroscopy provided the first evidence to suggest that these domains are unordered, a result consistent with other genetic and biochemical properties. Overall, the results show that while the sequence and regulation of the ALF gene are distinct from its somatic cell counterpart TFIIAalpha/beta, the TFIIAgamma-dependent interactions of these factors with TBP are nearly indistinguishable in vitro. Thus, a role for ALF in the assembly and stabilization of initiation complexes in germ cells is likely to be similar or identical to the role of TFIIA in somatic cells.
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Regulation of ALF gene expression in somatic and male germ line tissues involves partial and site-specific patterns of methylation. Xie W 2002 et al.
ALF (TFIIAalpha/beta-like factor) is a germ cell-specific counterpart of the large (alpha/beta) subunit of general transcription factor TFIIA. Here we isolated homologous GC-rich promoters from the mouse and human ALF genes and used promoter deletion analysis to identify sequences active in COS-7 and 293 cells. Further, bisulfite sequence analysis of the mouse ALF promoter showed that all 21 CpG dinucleotides between -179 and +207 were partially methylated in five somatic tissues, brain, heart, liver, lung, and muscle, and in epididymal spermatozoa from adult mice. In contrast, DNA from prepubertal mouse testis and from purified spermatocytes were unmethylated except at C(+19)G and C(+170)G. We also found that ALF expression correlates with a strong promoter-proximal DNase I-hypersensitive site present in nuclei from testis but not from liver. Finally we show that in vitro methylation of the ALF promoter inhibits activity and that 5-aza-2'-deoxycytidine treatment reactivates the endogenous ALF gene in a panel of seven different mouse and human somatic cell lines. Overall the results show that silencing in somatic cells is methylation-dependent and reversible and that a unique CpG-specific methylation pattern at the ALF promoter precedes expression in pachytene spermatocytes. This pattern is transient as remethylation of the ALF promoter in haploid germ cell DNA has occurred by the time spermatozoa are present in the epididymis.
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Involvement of ALF in human spermatogenesis and male infertility. Huang M 2006 et al.
We conducted this study to explore functions of TF II Aalpha/beta-like factor (ALF) during human spermatogenesis, and the relationship of its expression levels with male infertility. The RT-PCR and Western blot analyses illustrated that ALF was highly expressed in adult testis. Immunohistochemistry and immunoflurescence showed that ALF is located in the spermatid nuclei and in the annulus of spermatozoa. Further, to reveal whether ALF is related to male infertility, we performed the same experiments in infertility patients. The changes in the expression levels of ALF in the male infertility samples lead us to believe that ALF may function in spermatogenesis, especially in spermiogenesis. We also detected the ALF DNA methylation level by real-time methylation-specific PCR (MSP) both in testes of adult, fetal and infertile patient. The differential expression level of ALF gene in different types of testes was regulated by DNA methylation. Our research identified ALF as a human spermatogenesis related gene, the abnormal expression of ALF might be the partial cause for human infertility.
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NCBI Summary:
The assembly and stability of the RNA polymerase II transcription pre-initiation complex on a eukaryotic core promoter involve the effects of transcription factor IIA (TFIIA) on the interaction between TATA-binding protein (TBP) and DNA. This gene encodes a germ cell-specific counterpart of the large (alpha/beta) subunit of general transcription factor TFIIA that is able to stabilize the binding of TBP to DNA and may be uniquely important to testis biology. Alternative splicing for this locus has been observed and two variants, encoding distinct isoforms, have been identified. Co-transcription of this gene and the neighboring upstream gene generates a rare transcript (SALF), which encodes a fusion protein comprised of sequence sharing identity with each individual gene product. [provided by RefSeq, Mar 2014]
General function
DNA binding, Transcription factor
Comment
Cellular localization
Nuclear
Comment
GWAS123
Ovarian function
Oogenesis
Comment
A Short Core Promoter Drives Expression of the ALF Transcription Factor in Reproductive Tissues of Male and Female Mice.
Han S, et al .
The control of gene expression in reproductive tissues involves a number of unique germ cell-specific transcription factors. One such factor, ALF (TFIItau), encodes a protein similar to the large subunit of general transcription factor TFIIA. To understand how this factor is regulated, we characterized transgenic mice that contain the ALF promoter linked to either beta-galactosidase or GFP reporters. The results show that as little as 133 base pairs are sufficient to drive developmentally-accurate and cell-specific expression. Transgene DNA was methylated and inactive in liver but could be reactivated in vivo by systemic administration of 5-aza, 2'-deoxycytidine. Fluorescence activated cell sorting allowed the identification of male germ cells that express the GFP transgene and provides a potential method to collect cells that might be under the control of a non-somatic transcription system. Finally, we find that transcripts from the endogenous ALF gene and derived transgenes can also be detected in whole ovary and in GV-stage oocytes of female mice. The ALF sequence falls into a class of germ cell promoters whose features include small size, high GC content, numerous CpG dinucleotides, and an apparent TATA-like element. Overall, the results define a unique core promoter that is active in both male and female reproductive tissues and suggest mouse ALF may have a regulatory role in male and female gametogenic gene expression programs.
Expression regulated by
Comment
Regulatory factor interactions and somatic silencing of the germ cell-specific ALF gene. Kim M et al. Germ cell-specific genes are active in oocytes and spermatocytes but are silent in all other cell types. To understand the basis for this seemingly simple pattern of regulation, we characterized factors that recognize the promoter proximal region of the germ cell-specific ALF gene. Two of the protein-DNA complexes formed with liver extracts (C4 and C5) are due to the Zn-finger proteins Sp1 and Sp3, respectively, while another complex (C6) is due to the transcription factor RFX1. Two additional complexes (C1 and C3) are due to the multivalent zinc-finger protein CTCF, a factor that plays a role in gene silencing and chromatin insulation. An investigation of CTCF binding revealed a recognition site of only 17 bp that overlaps with the Sp1/Sp3 site. This site is predictive of other genomic CTCF sites, and can be aligned to create a functional concensus. Studies on the activity of the ALF promoter in somatic 293 cells revealed mutations which result in increased reporter activity. In addition, RNAi-mediated downregulation of CTCF is associated with activation of the endogenous ALF gene, and both CTCF and Sp3 repress the promoter in transient transfection assays. Overall, the results suggest a role for several factors, including the multivalent zinc-finger chromatin insulator protein CTCF, in mediating somatic repression of the ALF gene. Release of such repression, perhaps in conjunction with other members of the CTCF, RFX, and Sp1 families of transcription factors, could be an important aspect of germ cell gene activation.
Ovarian localization
Oocyte
Comment
Han SY, et al reported the expression of the germ cell-specific transcription factor ALF in Xenopus
oocytes compensates for silencing of the somatic factor TFIIA.
The discovery of germ cell-specific general transcription factor (GTF) and coactivator variants has
suggested that reproductive tissues control gene expression somewhat differently than somatic
tissues. One of these factors, ALF (TFIIAt), was first described as a testis-specific counterpart of
the large (a/b) subunit of TFIIA. Here we characterize endogenous ALF and TFIIA activities in the
African clawed frog Xenopus laevis. ALF is present in both testis and ovary in this organism and it
completely replaces TFIIA in immature oocytes. When oocytes undergo progesterone-induced
maturation, ALF activity disappears and TFIIA activity is restored. Reactivation occurs through the
translational upregulation of two maternal TFIIAa/b mRNAs and involves polyadenylation of a
conserved 3-UTR module. The effects of ALF overexpression and ALF immunodepletion on a
thymidine kinase promoter construct demonstrate that this factor serves as an active replacement for
TFIIA. In contrast, overexpression of TFIIA inhibits transcription, indicating that the somatic factor
fails to function properly in the context of the oocyte transcription machinery. Overall, the results
show that the translationally regulated reciprocal expression of ALF and TFIIA allows for the
production of an active TFIIA-like GTF throughout oogenesis.
Follicle stages
Comment
Developmental and cell type-specific regulation of core promoter transcription factors in germ cells of frogs and mice Xiao L, et al .
This article reports on the comparative cell type-specific expression profiles of selected core promoter-associated transcription factors during gametogenesis and embryogenesis in frogs and mice. In frogs we tested TBP, TRF2/TLF, TRF3, TFIIAalphabeta, and ALF, as well as variant forms of TAFs 4, 5, and 6. Four of these factors, TRF3, TAF4L, TAF5L, and the previously-characterized ALF gene, are preferentially expressed in testis and ovary. In mice we tested TBP, TRF2/TLF, TRF3, TFIIAalphabeta, and ALF. The results showed that while ALF was present in testis and ovary, as expected, TRF3 could only be detected in the ovary. RT-PCR experiments using RNAs from microdissected ovary tissue, together with in situ hybridization analysis, showed that TRF3 and ALF genes are specifically expressed in oocytes in both adult and prepubertal animals, whereas, their somatic counterparts, TBP and TFIIAalphabeta, are present in oocytes and in surrounding somatic cells of the follicle. Furthermore, both mice and frogs displayed a reduction in TRF3 and ALF transcript levels around the time of fertilization. In mice, transcripts from these genes could again be detected at low levels in embryonic reproductive tissues, but only reached maximal levels in adult animals. Finally, the results of protein-DNA interaction assays show that all combinations of core promoter complexes can be formed in vitro using recombinant TBP, TRF3, TFIIA, and ALF, including a TRF3-ALF complex. Overall, the diverse gene regulatory patterns observed here and in earlier reports indicate precise control over which transcription factor complexes can be formed in vivo during gametogenesis and early embryogenesis.
Phenotypes
PCO (polycystic ovarian syndrome)
Mutations
2 mutations
Species: human
Mutation name: None
type: naturally occurring fertility: subfertile Comment: Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3, 2p21 and 9q33.3. Chen ZJ et al. Polycystic ovary syndrome (PCOS) is a common metabolic disorder in women. To identify causative genes, we conducted a genome-wide association study (GWAS) of PCOS in Han Chinese. The discovery set included 744 PCOS cases and 895 controls; subsequent replications involved two independent cohorts (2,840 PCOS cases and 5,012 controls from northern Han Chinese; 498 cases and 780 controls from southern and central Han Chinese). We identified strong evidence of associations between PCOS and three loci: 2p16.3 (rs13405728; combined P-value by meta-analysis P(meta) = 7.55 ?10???, odds ratio (OR) 0.71); 2p21 (rs13429458, P(meta) = 1.73 ?10???, OR 0.67); and 9q33.3 (rs2479106, P(meta) = 8.12 ?10???, OR 1.34). These findings provide new insight into the pathogenesis of PCOS. Follow-up studies of the candidate genes in these regions are recommended.
At 2p16.3, the most significant SNP is rs13405728. Several known genes are located nearby (?500 kb of rs13405728; Fig. 2a), and a small linkage disequilibrium (LD) block approximately 53.4 kb in size contains both the GTF2A1L (TFIIA-alpha and beta-like factor) andLHCGR genes (Supplementary Fig. 4).
Species: human
Mutation name: type: naturally occurring fertility: subfertile Comment: Evidence for chromosome 2p16.3 polycystic ovary syndrome susceptibility locus in affected women of European ancestry. Mutharasan P et al. (2013) A previous genome-wide association study in Chinese women with polycystic ovary syndrome (PCOS) identified a region on chromosome 2p16.3 encoding the LH/choriogonadotropin receptor (LHCGR) and FSH receptor (FSHR) genes as a reproducible PCOS susceptibility locus. The objective of the study was to determine the role of the LHCGR and/or FSHR gene in the etiology of PCOS in women of European ancestry. This was a genetic association study in a European ancestry cohort of women with PCOS. The study was conducted at an academic medical center. Participants in the study included 905 women with PCOS diagnosed by National Institutes of Health criteria and 956 control women. We genotyped 94 haplotype-tagging single-nucleotide polymorphisms and two coding single-nucleotide polymorphisms mapping to the coding region of LHCGR and FSHR plus 20 kb upstream and downstream of the genes and test for association in the case control cohort and for association with nine quantitative traits in the women with PCOS. We found strong evidence for an association of PCOS with rs7562215 (P = 0.0037) and rs10495960 (P = 0.0046). Although the marker with the strongest association in the Chinese PCOS genome-wide association study (rs13405728) was not informative in the European populations, we identified and genotyped three markers (rs35960650, rs2956355, and rs7562879) within 5 kb of rs13405728. Of these, rs7562879 was nominally associated with PCOS (P = 0.020). The strongest evidence for association mapping to FSHR was observed with rs1922476 (P = 0.0053). Furthermore, markers with the FSHR gene region were associated with FSH levels in women with PCOS. Fine mapping of the chromosome 2p16.3 Chinese PCOS susceptibility locus in a European ancestry cohort provides evidence for association with two independent loci and PCOS. The gene products LHCGR and FSHR therefore are likely to be important in the etiology of PCOS, regardless of ethnicity.//////////////////