Search

Cilia assembly is under strict transcriptional control during animal development. In vertebrates, a hierarchy of transcription factors (TFs) are involved in controlling the specification, differentiation and function of multiciliated epithelia. RFX TFs play key functions in the control of ciliogenesis in animals. Whereas only one RFX factor regulates ciliogenesis in C. elegans, several distinct RFX factors have been implicated in this process in vertebrates. However, a clear understanding of the specific and redundant functions of different RFX factors in ciliated cells remains lacking. Using RNA-seq and ChIP-seq approaches we identified genes regulated directly and indirectly by RFX1, RFX2 and RFX3 in mouse ependymal cells. We show that these three TFs have both redundant and specific functions in ependymal cells. Whereas RFX1, RFX2 and RFX3 occupy many shared genomic loci, only RFX2 and RFX3 play a prominent and redundant function in the control of motile ciliogenesis in mice. Our results provide a valuable list of candidate ciliary genes. They also reveal stunning differences between compensatory processes operating in vivo and ex vivo.

H1 histones bind to linker DNA. H1t (H1f6), a testis-specific linker histone variant, is present in pachytene spermatocytes and spermatids. The expression of H1t histone coincides with the acquisition of metaphase I competence in pachytene spermatocytes. Here we report the generation of H1t-GFP transgenic mice. The H1t-GFP (H1 histone testis-green fluorescence protein) fusion protein expression recapitulates the endogenous H1t expression pattern. This protein appears first in mid pachytene spermatocytes in stage V seminiferous tubules, persists in round spermatids and elongating spermatids, but is absent in elongated spermatids. The strong green fluorescence signal, due to the high abundance of H1t-GFP, is maintained in spermatocytes after induction towards metaphase I through treatment with okadaic acid. Therefore, H1t-GFP can be used as a visual marker for monitoring the progression of meiosis in vitro and in vivo, as well as fluorescence-activated cell sorting (FACS) sorting of germ cells.

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.

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. gene regulation, meiosis, spermatogenesis, testis, transcription factor

H1t is a testis-specific variant histone 1 gene transcribed in pachytene spermatocytes. As part of a program to understand its transcriptional control, we have investigated the effect of the cap-proximal, GC-rich silencer element in the context of various lengths of upstream sequence. By transient transfection of NIH 3T3 cells, we showed that a targeted mutation in the silencer has a large (>10-fold) effect on reporter gene expression, regardless of the length of upstream sequence present. No other discrete silencing activity was observed in the upstream region extending to nucleotide -1842. Similarly, when the silencer mutation was introduced into the natural gene, H1t expression was readily detected in permanently transfected cells by both RNase protection and Western blot analysis, regardless of the extent of 5' or 3' flanking genomic DNA. In constructs with the mutated silencer, we showed interdependence of the characteristic H1 AC and TG box regulatory elements. Promoter up-regulation occurred only when both were intact, and possibly identical binding factors were demonstrated for each by electrophoretic mobility shift assays. In view of its precisely regulated but limited expression, it is interesting that H1t retains all the promoter elements known to activate standard H1 genes, including the TG/AC unit, SP1 site, and CCAAT element. Their presence emphasizes the apparent dominance of the silencer element in most cells.

H1t is an H1 histone variant unique to late spermatocytes and early spermatids. Using gene targeting and embryonic stem cell technologies, we have produced mice with a disrupted H1t gene. Homozygous H1t-null mice have normal fertility and show no obvious phenotypic consequence due to the lack of this histone. Biochemical and immunohistochemical approaches were used to show that normal changes in chromosomal proteins occurred during spermatid development, including the appearance and disappearance of transition proteins 1 and 2. Both protamines 1 and 2 are present in normal amounts in sonication-resistant spermatid nuclei from H1t-null mice. Analysis of H1 histones by quantitative gel electrophoresis in enriched populations of pachytene spermatocytes and round spermatids showed that the lack of H1t is only partially compensated for by somatic H1s, so that the chromatin of these cells is H1 deficient. Because H1t is thought to create a less tightly compacted chromatin environment, it may be that H1...

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.

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.

H1t is a testis-specific histone 1 variant restricted to the male germ line and expressed only in pachytene spermatocytes. Understanding the regulation of the H1t gene is an interesting challenge as its promoter shares all of the recognized control elements of standard somatic H1 genes, yet H1t is not expressed in somatic or in early spermatogenic cells. To investigate the mechanism of this apparent repression, we exchanged three promoter subregions between H1t and a major somatic H1 gene (H1d) by introduction of suitable restriction sites just 5' of the TATA box and 3' of the conserved H1 AC box. Hybrid promoters were joined to a lacZ reporter gene and assayed by transient transfection in NIH3T3 fibroblasts. In this system the wild type H1d promoter was 20-fold stronger than the H1t promoter. Much of this difference in activity was traced to inhibitory sequences immediately downstream of the TATA box in H1t, although sequences upstream of the H1t AC box and within the H1t 5'-untranslated region played some role as well. A series of deletions and short oligonucleotide mutations scanned across the region between the TATA box and cap site identified two tracts of C (GC box 2) as the inhibitory sequences. While both Sp1 and Sp3 bind to this region weakly in vitro, they are unlikely to be responsible for the inhibitory effect of GC box 2, and additional binding proteins (CTB-4 and CTB-5) were identified by electrophoretic mobility shift assays as better candidates for mediating the repressive effect. When repression of the H1t promoter was relieved by mutation of GC box 2, additional mutations introduced into GC box 1 upstream of the CAAT box led to a large decrease in activity, indicating that these two G/C-rich elements have opposite effects on promoter activity.

Spermatogenesis demonstrates a regulated program of genetic expression driving a complex progression of cellular differentiation. A current challenge is to understand the factors responsible for the many examples of differential gene expression documented during spermatogenesis (1,2). An aspect of male germ-cell differentiation that is fairly well described at the molecular level involves changes in DNA packaging by basic chromosomal proteins. Histones are responsible for the fundamental packaging unit of chromatin, the nucleosome, and early male germ cells contain the standard somatic forms of histones. However, beginning in late spermatogonia, novel histone variants that are unique to the male germ line start to appear. These include the core histone variants TH2A, TH2B, and TH3, as well as the linker histone variant H1t. The variants replace some, though not all, of their somatic-type counterparts, so that by the last third of meiotic prophase the spermatocyte nucleus is substantially enriched for testis-specific histones. This chromatin state persists into round spermatids until the spermatid nuclei elongate and condense (3, 4).

BackgroundMammalian 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 forRfx2.ResultsSearches of pachytene up-regulated genes identified high affinity RFX binding sites (X boxes) in promoter regions of several new genes:Adam5,Pdcl2, andSpag6. We confirmed a strong promoter-region X-box forAlf, a germ cell-specific variant of general transcription factor TFIIA. UsingAlfas 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 theRfx2promoter, we identified a cluster of three binding sites (MBS) for the MYB family of transcription factors. Because testis is one of the few sites ofA-mybexpression, and because spermatogenesis arrests in pachytene inA-mybknockout mice, the MBS cluster implicatesRfx2as anA-mybtarget. Electrophoretic gel-shift, ChIP, and co-transfection assays all support a role for these MYB sites inRfx2expression. Further,Rfx2expression was virtually eliminated inA-mybknockout 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.ConclusionWith an expanding list of likely target genes, RFX2 is potentially an important transcriptional regulator in pachytene spermatocytes.Rfx2itself is a good candidate to be regulated by A-MYB, which is essential for meiotic progression. IfAlfis a genuine RFX2 target, thenA-myb,Rfx2, andAlfmay form part of a transcriptional network that is vital for completion of meiosis and preparation for post-meiotic differentiation.

H1t is a testis-specific H1 histone variant that appears in germ cells during the meiotic prophase of mammalian spermatogenesis. Using a tritiated antisense RNA probe, H1t mRNA was identified by in situ hybridization in the mid and late pachytene spermatocytes found in seminiferous tubules of approximately stages VII to XIII.

Double stranded cDNA was made to partially purified mRNA for the small seminal vesicle secretory proteins IV and V. This ds cDNA was then inserted into the Pst 1 site of pBR322 by the (G-C) homopolymer tailing technique. Bacterial transformants harboring plasmids with specific inserts were identified by translation of mRNA that was hybridized to plasmid DNA immobilized on nitrocellulose. Separate plasmids were obtained with cDNA inserts for both SVS IV and V. Neither hybridization results nor preliminary restriction analysis gave any indication for homology between them.

Summary Nuclear transition protein 2 (TP2) along with TP1 are major basic chromosomal proteins of rat spermatids during the period of transition from histone-associated to protamine-associated DNA. TP2 isolated by reversed phase high pressure liquid chromatography was cleaved with S. aureus V8 protease to yield two fragments. The complete amino acid sequence of the 27 residue peptide assigned to the carboxyl terminus was established. It contains most of the basic residues of the protein and is likely to be a major site of DNA binding. Thus, TP2 is differentiated from core histones in having its basic domain at the carboxyl rather than amino terminal end.

Cloned gene sequences have been isolated for two testis-specific chromosomal proteins, one of which, histone (H1t), appears during meiosis, whereas the other, transition protein 1 (TP1), appears only during the later steps of spermatid development. Aspects of the regulation of each gene have been examined. In the case of H1t, analysis of its promoter region shows that it contains excellent matches to each of the four sequence homologies identified for the usual somatic H1 variants, so that the factor(s) that restrict H1t expression to spermatocytes remain a mystery. In the case of TP1, a cDNA clone allowed identification of its message by Northern blots as well as by in situ hybridization. The message appears postmeiotically in late round spermatids but is translationally repressed until the spermatid nucleus begins to condense.

H1t is a testis-specific H1 histone variant recently isolated from the rat [13]. We have now identified H1t in testicular extracts from mice, rabbits, hamsters, bulls, and boars, thus establishing its widespread presence in animals. H1t from all species could be differentiated from standard somatic H1 forms by a variety of criteria. It was poorly extracted by 5% trichloroacetic acid (TCA), migrated more rapidly than standard H1 forms during electrophoresis in the presence of sodium dodecyl sulfate (SDS), and eluted more slowly than standard H1 species during chromatography over cross-linked polyacrylamide beads (Bio-Gel P100). Using an improved purification procedure, H1t was isolated from two new species, mouse and rabbit. In these species as with the rat, H1t is readily identified by its low lysine (ca 19 mol%) and high arginine (6–7 mol%) content as compared with that of standard somatic H1 forms (ca 25% lysine and 3% arginine).

Hlt is a testis-specific Hl histone variant associated with meiosis and post-meiotic stages of male germ cell development. We have now made tentative identification of Hlt in humans by a variety of criteria including electrophoretic and extractive properties. While Hlt was readily identified in extracts from normal testes, it was not detectable in extracts from aspermatogenic testes or from placenta. Identification of Hlt in humans confirms the widespread association of this unusual Hl variant with spermatogenesis among mammals ranging from rodents to primates.

The genes for seminal vesicle secretory protein IV and prostate steroid binding protein component 3 of the rat share upstream homologies of which the most striking is a 30 nucleotide sequence located between position −190 and −330 relative to the major transcriptional initiation sites. This sequence does not appear to be a common repetitive element and deserves consideration as a potential site involved in the androgen regulated expression of these genes.

A unique acid-soluble basic protein was detected in the testes of sexually mature rats and a number of other mammalian species. This protein was not found in comparable acid extracts prepared from many other rat organs or from the testes of juvenile rats. It makes its appearance only after spermatids are evident in normally developing testes and is absent from the gonads of male rats rendered artificially cryptorchid for periods of 10 days or longer. This protein was purified extensively and was found to be devoid of phenylalanine, tryptophan, glutamic acid, glutamine, isoleucine, and cyst(e)ine. The specific basic protein from rat testes could not be extracted from epididymal sperm by a variety of procedures. In contrast, certain techniques reproducibly extracted from both epididymal spermatozoa and whole testis a highly basic protein fraction that is strikingly rich both in arginine and cyst(e)ine.

The entire coding region for rat seminal vesicle secretory protein IV was obtained on a 3.5 kb Eco RI fragment isolated from a genomic library in lambda Charon 4A. The coding sequence for SVS IV message is interrupted twice by introns. The first lies just downstream from the juncture of the 21 amino acid secretory signal peptide with the start of the mature protein, and the second lies in the 3'-nontranslated region. The major transcriptional start site was mapped by primer extention and is 22 nucleotides upstream from the translational initiation codon. S1 protection experiments indicated additional minor transcriptional starts about 27 and 50 nucleotides further upstream from the major cap site. The entire transcriptional unit comprises about 1740 nucleotides. The SVS IV gene does not belong to an obvious gene family, and it is conserved in mice and guinea pigs.

An investigation of the primary structure of the basic chromosomal protein of rat spermatozoa by automated Edman degradation and by carboxypeptidase digestion has provided a general structural outline of the entire molecule. The exact or approximate location of virtually all residues other than arginine or cysteine is reported. Of particular interest, because of the occurrence of phosphorylated derivatives of this protein, is the location of three of the four serine residues.

H1 histones were examined in testes and somatic organs from mice and rabbits. They were extracted from washed chromatin by aqueous 5% (w/v) trichloroacetic acid and analyzed by several electrophoretic systems as well as column chromatography employing Bio-Rex 70. In each species the testicular H1 population contains at least two components that are scarce or absent in the somatic organs examined. The unusual testicular species do not appear to be phosphorylated derivatives. The studies in this and the accompanying report [Seyedin, S.M., & Kistler, W.S. (1979) biochemistry 18 (preceding paper in this issue) confirm that marked changes from the isomatic type H1 population are associated with spermatogenesis in mice, rabbits, and rats. However, in terms of electrophoretic and chromatographic behavior, the pattern of change in species specific.