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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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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.

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-deficient chromatin is functionally similar to chromatin with H1t present, at least with respect to permitting spermatogenesis to proceed.

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.

Histone H1t is synthesized only in male germ cells during the late pachytene stage of meiosis and is retained in spermatids until the nucleus elongates. Transgenic experiments suggest that spermatocyte-directing sequences lie within 140 base pairs of the cap site. To study the mechanism of this specificity we compared the DNase I footprints made on the immediate promoter regions of H1t and H1d (a typical somatic H1) by testis and liver extracts and observed both common and differentially protected regions. The common footprints of H1t included an Sp1 consensus (GC box 1) and a CCAAT motif. Electrophoretic mobility shift assays (EMSA) identified ubiquitous binding factors for GC box 1 and a binding factor for the CCAAT element that we identified immunologically as H1TF2. H1t-specific footprints occurred over the palindrome CCTAGG and a GC-rich sequence downstream of the TATA box (GC box 2). EMSA analysis of the palindrome identified testis-specific as well as ubiquitous binding factors. UV irradiation of a palindrome-binding reaction generated a cross-linked doublet of about 50 kDa from both testis and liver. Protein factors that bound to the GC box 2 sequence were similar from testis and liver, and GC box 1 and an Sp1 consensus competed for them. In vitro transcription directed by H1t occurred at comparable levels in testis and liver extracts. The importance of both GC box 1 and CCAAT elements was demonstrated by deletion analysis and by oligonucleotide competition. No dependence on the H1t palindrome was observed for in vitro transcription.

Transition proteins replace testis-specific histones and are finally replaced by protamines in the nucleus of germ cells during spermiogenesis. In this study, immunoperoxidase and immunogold localization were used to determine both qualitatively and quantitatively the intracellular distribution of testis-specific histone (H1t), transition protein 1(TP1), and transition protein 2 (TP2) during rat spermatogenesis. H1t labeling was concentrated over heterochromatin in the nucleus of late-pachytene spermatocytes and spermatids up to mid-steps 10. In step 9 spermatids, H1t was confined to the caudal end of the nucleus where heterochromatin was still present, while in early step 10 spermatids, only a few of the nuclei remained caudally labeled. In late step 10 spermatids, a fibrillar chromatin network was distributed throughout the nucleus coincident with the loss of H1t. A statistically significant rise in TP1 and TP2 labeling density over control values was first encountered in the nucleus of step 11 spermatids coincident with the initiation of condensation of the fibrillar chromatin. The TP1 and TP2 labeling density progressively increased in nucleus of step 11-13 spermatids with the apical to caudal condensation of the fibrillar chromatin, In step 13 spermatids, the chromatin was homogeneously condensed throughout the nucleus. In the case of TP1, the nuclear labeling density gradually declined after step 13 and disappeared by step 17. In the case of TP2, the nuclear labeling density disappeared by step 16. This study shows that, coincident with the loss of H1t, the chromatin of the spermatid is reorganized into a fibrillar network, whereas, coincident with the appearance and progressive increase of TP1 and TP2, the fibrillar chromatin condenses in an apical to caudal direction in the nucleus of the spermatid. Thus the remodeling of chromatin structure during spermiogenesis appears to be a two-step process that is sequentially influenced by the loss of spermatid-specific histones and the appearance of transition proteins.

Transition protein 1 (TP1) is a small basic chromosomal protein that appears in mammalian spermatids during the period of chromatin condensation. The gene for TP1 from several species contains an apparent cAMP response element (CRE) in the immediate 5'-flanking region. The recent identification of high expression of the novel CRE-activating protein (CREM tau) in advanced testicular germ cells provided a stimulus to ask whether or not the TP1 CRE is functional. To this end we show both by gel retardation and by footprint assays that TP1 CRE forms specific bound complexes with proteins in whole testis nuclear extracts and that these complexes involve CREM as evidenced by recognition by a specific antibody. In addition, the TP1 CRE forms specific bound complexes with bacterially expressed CREM tau. Finally, the TPI CRE conveys protein kinase A-dependent induction to a linked chloramphenicol acetyl transferase gene when transfected into JEG-3 cells. Accordingly, TP1 is a good candidate for a testis-specific gene subject to CREM tau regulation.

Transition protein 2 (TP2) of the rat was isolated by differential precipitation with trichloroacetic acid, chromatography over Bio-Rex 70, and preparative gel electrophoresis. A polyclonal rabbit antiserum was raised that did not cross-react with unrelated acid-soluble proteins from liver or testes. The antiserum was used to identify TP2-related proteins obtained from testes of mice, hamsters, guinea pigs, rabbits, and boars by Western blotting. Immunohistochemical techniques were used to localize TP2 in paraffin-embedded testis sections from mice and rats. In both species, TP2 was first detected in spermatids that had essentially completed the morphological change from a round to an elongate nucleus and that were undergoing chromosomal condensation (spermatids of step 13 in rat and step 12 in mouse). TP2 was retained in spermatid nuclei until early step 16 in the rat and step 14 in the mouse. Serial sections of rat testis exposed separately to antisera to TP1 and TP2 showed that the great majority of labeled tubules were reactive to both antisera. However, in occasional tubules, TP1 reactivity was retained in relatively late spermatids that were negative for TP2. Thus both TP1 and TP2 appear in the nucleus essentially simultaneously, in association with the beginning of chromatin condensation and at a point well after much of the nuclear shaping has occurred.

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.

Rat seminal vesicle secretion (SVS) contains a variety of protein complexes that seem to be linked by interchain disulfide bonds. Upon reduction and analysis by sodium dodecyl sulfate (SDS) gel electrophoresis, this pattern resolves to 3 major high molecular weight (SVS I-100,000, SVS II-50,000, SVS III-37,000) and 3 major low molecular weight protein bands (SVS IV, V, and VI). A two-dimensional SDS gel (1st dimension unreduced, 2nd dimension reduced) permitted identification of the components of the cross-linked species. In the native secretion, SVS I forms a series of oligomers that include both SVS II and III. Essentially all of SVS III is involved in these complexes, while the bulk of SVS II occurs instead as an apparent homodimer. The smaller proteins (SVS IV-VI) are not involved in covalently crosslinked complexes. The reduced forms of the larger polypeptides were isolated by a variety of procedures involving agarose gel filtration in 6M guanidine hydrochloride, reversed-phase high pressure liquid chromatography, ammonium sulfate fractionation, and preparative polyacrylamide gel electrophoresis. Based on its size, solubility, and amino acid composition, SVS II was identified as the major clottable protein of the secretion.