Organization and transcriptional output of a novel mRNA-like piRNA gene (mpiR) located on mouse chromosome 10.

This letter describes the architecture and transcriptional output of a novel noncoding RNA gene in mouse and rat. The mRNA-like piRNA (mpiR) gene, lies between the Perp and KIAA1244 genes on mouse chromosome 10 and rat chromosome 1. In mouse, the mpiR gene is associated with the production of at least 13 different alternatively spliced and polyadenylated transcripts ranging from 500 nt to over 6 kb. Although these transcripts are structurally similar to conventional mRNAs, only short polypeptides are predicted on each of the three possible reading frames. Intron 2 is unique in that it harbors a novel low copy repeat with homology with the 3'-UTR of the lin-28 gene, while Exon 4 contains an unusual cluster of nine sequence modules that are dispersed throughout the mouse genome. The mpiR gene is expressed at low levels in somatic tissues, but is transcriptionally up-regulated in the testis at day 14 post-partum, a time that coincides with the pachytene stage of meiosis I. Bisulfite methylation analysis shows that expression in brain, liver, and testis is correlated with the methylation status of the promoter region. In addition to mRNA-like transcripts, the mpiR gene is also a precursor to testis-specific piRNAs, and these can be detected by both Northern and PCR-based approaches. Remarkably, piRNAs originate from two specific regions of the gene, one corresponding to Intron 2 and the other to Exon 4. Overall, this work provides a picture of a novel, lineage-specific, noncoding RNA gene and describes its processing into both mRNA-like and piRNA products.

Basic mechanisms for the control of germ cell gene expression.

The patterns of gene expression in spermatocytes and oocytes are quite different from those in somatic cells. The messenger RNAs produced by these cells are not only required to support germ cell development but, in the case of oocytes, they are also used for maturation, fertilization, and early embryogenesis. Recent studies have begun to provide an explanation for how germ-cell-specific programs of gene expression are generated. Part of the answer comes from the observation that germ cells express core promoter-associated regulatory factors that are different from those expressed in somatic cells. These factors supplement or replace their somatic counterparts to direct expression during meiosis and gametogenesis. In addition, germ cell transcription involves the recognition and use of specialized core promoter sequences. Finally, transcription must occur on chromosomal DNA templates that are reorganized into new chromatin-packaging configurations using alternate histone subunits. This article will review recent advances in our understanding of the factors and mechanisms that control transcription in ovary and testis and will discuss models for germ cell gene expression.

Developmental and cell type-specific regulation of core promoter transcription factors in germ cells of frogs and mice.

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.

Expression of human TFIIA subunits in Saccharomyces cerevisiae identifies regions with conserved and species-specific functions.

The transcription factor TFIIA stabilizes the interaction between the TATA-binding protein (TBP) and promoter DNA and facilitates activator function. In yeast, TFIIA is composed of large (TOA1) and small (TOA2) subunits that interact to form a beta-barrel domain and a helix bundle domain. Here we report plasmid shuffle experiments showing that the human subunits (TFIIAalpha/beta, ALF, and TFIIAgamma) are not able to support growth in yeast and that the failure is associated with morphological abnormalities related to cell division. To determine the regions responsible for species specificity, we examined a series of chimeric yeast-human subunits. The results showed that yeast-human hybrids that contained the N-termini of TFIIAgamma or TFIIAalpha/beta were viable, presumably because they could form a functional interspecies alpha-helical bundle. Likewise, a TOA1 hybrid that contained the nonconserved internal region from TFIIAalpha/beta also had no effect on TFIIA function. However, hybrids that contained the acidic region III or C-terminal region IV from TFIIAalpha/beta grew more slowly than the wild-type TOA1 subunit, and if both regions were exchanged, this effect was far more severe. Although these hybrids exchanged sequences which are involved in beta-barrel formation and interactions with TBP, they were all active in a TBP-dependent mobility shift assay. The results suggest that the growth phenotypes of these hybrids might be due to a failure to interact with components of the yeast transcription machinery other than TBP. Finally, we show that sequences from region III of TFIIA large subunits fall into classes that are either highly acidic or that are divergent and nonacidic, and provide the first evidence to suggest that, at least in yeast, this region is important for TFIIA function.

Regulation of ALF promoter activity in Xenopus oocytes.

BACKGROUND: In this report we evaluate the use of Xenopus laevis oocytes as a matched germ cell system for characterizing the organization and transcriptional activity of a germ cell-specific X. laevis promoter. PRINCIPAL FINDINGS: The promoter from the ALF transcription factor gene was cloned from X. laevis genomic DNA using a PCR-based genomic walking approach. The endogenous ALF gene was characterized by RACE and RT-PCR for transcription start site usage, and by sodium bisulfite sequencing to determine its methylation status in somatic and oocyte tissues. Homology between the X. laevis ALF promoter sequence and those from human, chimpanzee, macaque, mouse, rat, cow, pig, horse, dog, chicken and X. tropicalis was relatively low, making it difficult to use such comparisons to identify putative regulatory elements. However, microinjected promoter constructs were very active in oocytes and the minimal promoter could be narrowed by PCR-mediated deletion to a region as short as 63 base pairs. Additional experiments using a series of site-specific promoter mutants identified two cis-elements within the 63 base pair minimal promoter that were critical for activity. Both elements (A and B) were specifically recognized by proteins present in crude oocyte extracts based on oligonucleotide competition assays. The activity of promoter constructs in oocytes and in transfected somatic Xenopus XLK-WG kidney epithelial cells was quite different, indicating that the two cell types are not functionally equivalent and are not interchangeable as assay systems. CONCLUSIONS: Overall the results provide the first detailed characterization of the organization of a germ cell-specific Xenopus promoter and demonstrate the feasibility of using immature frog oocytes as an assay system for dissecting the biochemistry of germ cell gene regulation.

Regulatory factor interactions and somatic silencing of the germ cell-specific ALF gene.

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 TFIIA alpha/beta-like factor (ALF) gene. Two of the protein-DNA complexes formed with liver extracts (C4 and C5) are due to the zinc finger proteins Sp1 and Sp3, respectively, whereas 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 consensus. Studies on the activity of the ALF promoter in somatic 293 cells revealed mutations that result in increased reporter activity. In addition, RNAi-mediated down-regulation 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.

Regulation of ALF gene expression in somatic and male germ line tissues involves partial and site-specific patterns of methylation.

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.

Expression of the germ cell-specific transcription factor ALF in Xenopus oocytes compensates for translational inactivation of the somatic factor TFIIA.

The discovery of germ cell-specific general transcription factor and coactivator variants has suggested that reproductive tissues control gene expression somewhat differently than somatic tissues. One of these factors, ALF (TFIIAtau), was first described as a testis-specific counterpart of the large (alpha/beta) 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 up-regulation of two maternal TFIIAalpha/beta mRNAs and involves polyadenylation of a conserved 3'-untranslated region 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 general transcription factor throughout oogenesis.

A short core promoter drives expression of the ALF transcription factor in reproductive tissues of male and female mice.

The control of gene expression in reproductive tissues involves a number of unique germ cell-specific transcription factors. One such factor, ALF (TFIIA tau), 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 green fluorescent protein (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 system 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 nonsomatic transcription system. Finally, we found that transcripts from the endogenous ALF gene and derived transgenes can also be detected in whole ovary and in germinal vesicle-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.

The germ cell-specific transcription factor ALF. Structural properties and stabilization of the TATA-binding protein (TBP)-DNA complex.

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.