Regulatory Factor Interactions and Somatic Silencing of the Germ Cell-specific ALF Gene

Abstract

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␣/␤-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.Germ cells express a unique set of core promoter-associated transcription factors that are not found in somatic cells. These include members of the TBP family as well as TBP-associated factors, such as TFIIA and TAFs (1-3). One of these germ cell variants, TFIIA␣/␤-like factor (ALF) 2 (TFIIA), encodes a counterpart of the large subunit of the general transcription factor TFIIA (4 -6). ALF interacts with the small TFIIA␥ subunit to form a complex that can stabilize binding of TBP-like factors to promoter DNA, thereby facilitating formation of a transcription preinitiation complex in germ cells (7,8). The loss of ALF expression is associated with male infertility in humans (9).An intriguing feature of ALF and other germ cell-specific genes is that the sequences sufficient to exert correct control of expression are very short, on the order of 100 base pairs. For instance, a region of the ALF gene between Ϫ133 and ϩ21 relative to the transcription start site is able to recapitulate accurate tissue-specific expression and silencing of a linked reporter in transgenic mice (10). The most notable sequences in this region include a TTCAAA element, a GC-rich region with a number of CpG dinucleotides, and an upstream TC-rich region. Although broadly similar features can be observed in several other germ cell-specific promoters (10), individual sequences of germ cell promoters are not precisely homologous, and the identification of factors responsible for their regulation remains an i...

Expression of a Testis-Specific Form of Gal3st1 (CST), a Gene Essential for Spermatogenesis, Is Regulated by the CTCF Paralogous Gene BORIS

“…Since the amino and carboxy termini of BORIS have no homology with the parallel sequences in CTCF, the distinct molecular functions of BORIS may be attributable to the unique protein-protein interactions specified by these sequences. Our observations on the role of BORIS binding site in activation of the CST form F TS transcription are remarkably consistent with results on regulation of testis-specific TFIIA ␣/␤-like factor (ALF) gene promoter obtained by DeJong and coworkers, who showed that unlike CTCF, expression of BORIS had an activating effect on the ALF promoter-reporter construct (9,26). However, ALF only starts to express at the time point we performed microarray analysis, and that may be why we missed it as a target (15).…”

“…Methylation interference assays followed by EMSA with mutated fragments revealed that the exact BORIS binding site in the promoter is highly homologous to the TAGGGGG-containing CTCF/BORIS binding sequence mapped by DeJong and coworkers in the testis-specific ALF gene promoter (8,26), which has been termed the consensus CTCF binding site as identified by genome-wide ChIP analysis (27). Consistent with the finding of a BORIS/CTCF target site in the promoter, ectopic expression of BORIS activated the CST form F TS promoter, but not the promoter mutated in the BORIS/CTCF target site.…”

A Novel CpG-free Vertebrate Insulator Silences the Testis-specific SP-10 Gene in Somatic Tissues

“…Previous studies established a correlation between the methylation status of the promoter and gene expression for some testis-specific genes (42). A recent study implicated CTCF and Sp3 in mediating somatic repression of a testis-specific gene (43). Our study shows how an insulator located in a CpG-free proximal promoter can regulate testis-specific gene transcription.…”

Does CTCF mediate between nuclear organization and gene expression?

“…The importance of such experimental validation is reinforced by the recent discovery of several functional CTSs that do not match any in-silico-generated CTSs consensus motif. Examples include CTSs in the hTERT (44) and ALF (45) gene promoters, and numerous methylation-sensitive and methylation-resistant CTSs in the promoter regions of the human BORIS gene (46) and in pericentromeric gamma-satellite DNA arrays. (18) Finally, the data on CTSs provided by genome-wide screening have limitations as although these studies present indications of the binding position, they only give approximations of the length of the region of DNA bound by CTCF.…”