RFX2 is a candidate downstream amplifier of A-MYB regulation in mouse spermatogenesis.

BACKGROUND: Mammalian spermatogenesis involves formation of haploid cells from the male germline and then a complex morphological transformation to generate motile sperm. Focusing on meiotic prophase, some tissue-specific transcription factors are known (A-MYB) or suspected (RFX2) to play important roles in modulating gene expression in pachytene spermatocytes. The current work was initiated to identify both downstream and upstream regulatory connections for Rfx2. RESULTS: Searches of pachytene up-regulated genes identified high affinity RFX binding sites (X boxes) in promoter regions of several new genes: Adam5, Pdcl2, and Spag6. We confirmed a strong promoter-region X-box for Alf, a germ cell-specific variant of general transcription factor TFIIA. Using Alf as an example of a target gene, we showed that its promoter is stimulated by RFX2 in transfected cells and used ChIP analysis to show that the promoter is occupied by RFX2 in vivo. Turning to upstream regulation of the Rfx2 promoter, we identified a cluster of three binding sites (MBS) for the MYB family of transcription factors. Because testis is one of the few sites of A-myb expression, and because spermatogenesis arrests in pachytene in A-myb knockout mice, the MBS cluster implicates Rfx2 as an A-myb target. Electrophoretic gel-shift, ChIP, and co-transfection assays all support a role for these MYB sites in Rfx2 expression. Further, Rfx2 expression was virtually eliminated in A-myb knockout testes. Immunohistology on testis sections showed that A-MYB expression is up-regulated only after pachytene spermatocytes have clearly moved away from the tubule wall, which correlates with onset of RFX2 expression, whereas B-MYB expression, by contrast, is prevalent only in earlier spermatocytes and spermatogonia. CONCLUSION: With an expanding list of likely target genes, RFX2 is potentially an important transcriptional regulator in pachytene spermatocytes. Rfx2 itself is a good candidate to be regulated by A-MYB, which is essential for meiotic progression. If Alf is a genuine RFX2 target, then A-myb, Rfx2, and Alf may form part of a transcriptional network that is vital for completion of meiosis and preparation for post-meiotic differentiation.

The rat histone H1d gene has intragenic activating sequences that are absent from the testis-specific variant H1t.

In some cases core histone genes in the mouse depend on intragenic sequence elements for high level expression [Gene 176 (1996) 1]. Here we report that the highly expressed gene for rat linker histone H1d also contains an intragenic activating region (IAR). Using transient transfection assays in mouse fibroblast NIH3T3 cells, we showed that rat H1d contains a downstream region (+21 to +116) that imparts a two- to threefold up-regulation of fused reporters. This region also activated expression when moved to the promoter region, though the effect was dependent on its distance from other promoter elements. The IAR contains sequence homologies to the core alpha and Omega elements identified as functional protein binding sites within the mouse H3.2 coding region activating sequence (CRAS). A pair of Omega elements (+32 and +66) accounts for the activating effect of the H1d intragenic region as shown by targeted mutations as well as stepwise deletions. The H1d and H3.2 Omega sequences bound similar and perhaps identical proteins by gel shift analysis. The H1d alpha-like sequence at +56 overlaps the translational start codon and was therefore not mutated. Like the mouse H3.2 alpha element, it bound transcription factor YY1 in gel shift assays. H1t, the gene for the testis-specific linker histone, did not demonstrate an IAR. While H1t has a similar alpha sequence and did bind YY1, it lacks the Omega homologies of H1d. Sequence comparison shows that the YY1/alpha site as well as the adjacent Omega site are likely present in genes for other standard H1 variants, but that the +32 Omega site in the 5' untranslated region (UTR) of H1d is unique. We conclude that the +32 and +66 Omega sequences of the rat H1d gene contribute significantly to its high-level expression.

Differential expression of Rfx1-4 during mouse spermatogenesis.

The regulatory factor X (RFX) family of transcription factors has been recently implicated in gene regulation during spermatogenesis. However, the relative expression of individual members during this developmental process is not completely characterized, particularly in the case of Rfx4, which has multiple transcript variants in the testis. We used reverse transcriptase-dependent real-time PCR, 5'-RACE cloning, and Western blotting to compare transcripts and protein levels for this family in cell populations from the three major phases of spermatogenesis (mitotic, meiotic, and haploid). Transcripts for Rfx1-4 were present at trace to low levels in spermatogonia prepared from 8-day-old mice. Transcripts for both Rfx2 and Rfx4 were elevated in mid-late pachytene spermatocytes; however, the dominant Rfx4 transcript present begins at a downstream exon and lacks the DNA binding domain. Transcripts for all four genes were elevated in early haploid cells (round spermatids). In these cells Rfx4 transcripts originate primarily from a newly described promoter with intron 1 but are expected to be translationally compromised due to a poorly situated start codon. Western blotting confirmed that RFX2 is greatly elevated beginning in meiosis and also confirmed that full-length RFX4 protein is not prevalent in mouse testis at any stage. These results imply that RFX2 is the most likely X box binding factor to influence novel gene expression during meiosis, that RFX1-3 may all play roles in haploid cells but that RFX4 is much less prevalent than implied by its high transcript levels.

Expression patterns of SP1 and SP3 during mouse spermatogenesis: SP1 down-regulation correlates with two successive promoter changes and translationally compromised transcripts.

Because of their prominent roles in regulation of gene expression, it is important to understand how levels of Krüpple-like transcription factors SP1 and SP3 change in germ cells during spermatogenesis. Using immunological techniques, we found that both factors decreased sharply during meiosis. SP3 declined during the leptotene-to-pachytene transition, whereas SP1 fell somewhat later, as spermatocytes progressed beyond the early pachytene stage. SP3 reappeared for a period in round spermatids. For Sp1, the transition to the pachytene stage is accompanied by loss of the normal, 8.2-kb mRNA and appearance of a prevalent, 8.8-kb variant, which has not been well characterized. We have now shown that this pachytene-specific transcript contains a long, unspliced sequence from the first intron and that this sequence inhibits expression of a reporter, probably because of its many short open-reading frames. A second testis-specific Sp1 transcript in spermatids of 2.4 kb also has been reported previously. Like the 8.8-kb variant, it is compromised translationally. We have confirmed by Northern blotting that the 8.8-, 8.2-, and 2.4-kb variants account for the major testis Sp1 transcripts. Thus, the unexpected decline of SP1 protein in the face of continuing Sp1 transcription is explained, in large part, by poor translation of both novel testis transcripts. As part of this work, we also identified five additional, minor Sp1 cap sites by 5' rapid amplification of cDNA ends, including a trans-spliced RNA originating from the Glcci1 gene.

RFX2 is a potential transcriptional regulatory factor for histone H1t and other genes expressed during the meiotic phase of spermatogenesis.

H1t is a novel linker histone variant synthesized in mid- to late pachytene spermatocytes. Its regulatory region is of interest because developmentally specific expression has been impressed on an otherwise ubiquitously expressed promoter. Using competitive band-shift assays and specific antisera, we have now shown that the H1t-60 CCTAGG palindrome motif region binds members of the RFX family of transcriptional regulators. The testis-specific binding complex contains RFX2, probably as a homodimer. Other DNA-protein complexes obtained from testis as well as somatic organs contain RFX1, primarily as a heterodimer. Western blots confirmed that RFX2 expression is greatly enhanced in adult testis and that RFX2 is equally prominent in highly enriched populations of late pachytene spermatocytes and round spermatids. Immunohistochemistry carried out on mouse testis showed that RFX2 is strongly expressed in pachytene spermatocytes, remains high in early round spermatids, and declines only in advance of nuclear condensation. Maximum expression correlates well with the appearance of H1t. In contrast, RFX1 immunoreactivity in germ cells was only detected in late round spermatids. RFX-specific band complexes were also identified for both the mouse lamin C2 and Sgy promoters, using either testis nuclear extracts or in vitro-synthesized RFX2. These results call attention to RFX2 as a transcription factor with obvious potential for the regulation of gene expression during meiosis and the early development of spermatids.

BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma.

CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding potential, but with distinct amino and carboxy termini. We named this gene BORIS for Brother of the Regulator of Imprinted Sites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.