Germ Cell-specific Transcription Factor Research Articles

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.

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.

Identification of Zfp393, a germ cell-specific gene encoding a novel zinc finger protein

Using the digital differential display program of the National Center for Biotechnology Information, we identified a contig of expression sequence tags (ESTs) which were unique to ovary, testis, and egg libraries. The full-length cDNA of this transcript was deduced and further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA encodes a novel protein of 341 amino acids with a nuclear localization signal. The carboxyl-terminus of the protein contains three C2H2 zinc fingers, and the NH 2-terminus is proline and serine-rich. Based on the conserved zinc finger motifs, we have termed this novel protein as zinc finger protein 393 (ZFP393). Northern blot and RT-PCR analyses revealed that Zfp393 mRNA was exclusively expressed in testis and ovary. The expression sites were further localized by in situ hybridization to step 3–8 spermatids in testis and growing oocytes in ovary. The Zfp393 gene consists of three exons spanning approximately 8 kb on the distal part of mouse chromosome 4. The carboxyl-terminal zinc finger region is highly homologous to several zinc finger-containing proteins, but no proteins were found to share sequence similarity with the NH 2-terminal region of ZFP393. Genomic database mining and Southern blot analysis indicate that Zfp393 is a single copy gene. We hypothesize that ZFP393 functions as a germ cell-specific transcription factor that plays important roles in spermatid differentiation and oocyte development.

FIGalpha, a germ cell-specific transcription factor required for ovarian follicle formation.

Primordial follicles are formed perinatally in mammalian ovaries and at birth represent the lifetime complement of germ cells. With cyclic periodicity, cohorts enter into a growth phase that culminates in ovulation of mature eggs, but little is known about the regulatory cascades that govern these events. FIGalpha, a transcription factor implicated in postnatal oocyte-specific gene expression, is detected as early as embryonic day 13. Mouse lines lacking FIGalpha were established by targeted mutagenesis in embryonic stem cells. Although embryonic gonadogenesis appeared normal, primordial follicles were not formed at birth, and massive depletion of oocytes resulted in shrunken ovaries and female sterility. Fig(&agr;) (the gene for FIGalpha null males have normal fertility. The additional observation that null females do not express Zp1, Zp2 or Zp3 indicates that FIGalpha plays a key regulatory role in the expression of multiple oocyte-specific genes, including those that initiate folliculogenesis and those that encode the zona pellucida required for fertilization and early embryonic survival. The persistence of FIGalpha in adult females suggests that it may regulate additional pathways that are essential for normal ovarian development.

Promoter mapping of the mouse Tcp-10bt gene in transgenic mice identifies essential male germ cell regulatory sequences.

Transgenic mice were generated to localize essential promoter elements in the mouse testis-expressed Tcp-10 genes. These genes are expressed exclusively in male germ cells, and exhibit a diffuse range of transcriptional start sites, possibly due to the absence of a TATA box. A series of transgene constructs containing different amounts of 5' flanking DNA revealed that all sequences necessary for appropriate temporal and tissue-specific transcription of Tcp-10 reside between positions -1 to -973. All transgenic animals containing these sequences expressed a chimeric transgene at high levels, in a pattern that paralleled the endogenous genes. These experiments further defined a 227 bp fragment from -746 to -973 that was absolutely essential for expression. In a gel-shift assay, this 227-bp fragment bound nuclear protein from testis, but not other tissues, to yield two retarded bands. Sequence analysis of this fragment revealed a half-site for the AP-2 transcription factor recognition sequence. Gel shift assays using native or mutant oligonucleotides demonstrated that the putative AP-2 recognition sequence was essential for generating the retarded bands. Since the binding activity is testis-specific, but AP-2 expression is not exclusive to male germ cells, it is possible that transcription of Tcp-10 requires interaction between AP-2 and a germ cell-specific transcription factor.