Abstract
Anti-silencing function 1 (ASF1) is an evolutionarily conserved histone H3–H4 chaperone involved in the assembly/disassembly of nucleosome and histone modification. Two paralogous genes, Asf1a and Asf1b, exist in the mouse genome. Asf1a is ubiquitously expressed and its loss causes embryonic lethality. Conversely, Asf1b expression is more restricted and has been less studied. To determine the in vivo function of Asf1b, we generated a Asf1b-deficient mouse line (Asf1bGT(ROSA-βgeo)437) in which expression of the lacZ reporter gene is driven by the Asf1b promoter. Analysis of β-galactosidase activity at early embryonic stages indicated a correlation between Asf1b expression and cell differentiation potential. In the gonads of both male and female, Asf1b expression was specifically detected in the germ cell lineage with a peak expression correlated with meiosis. The viability of Asf1b-null mice suggests that Asf1b is dispensable for mouse development. However, these mice showed reduced reproductive capacity compared with wild-type controls. We present evidence that the timing of meiotic entry and the subsequent gonad development are affected more severely in Asf1b-null female mice than in male mice. In female mice, in addition to subfertility related to altered gamete formation, variable defects compromising the development and/or survival of their offspring were also observed. Altogether, our data indicate the importance of Asf1b expression at the time of meiotic entry, suggesting that chromatin modifications may play a central role in this process.
Introduction
Histone chaperones participate in chromatin organization by binding directly to histone proteins and mediating the assembly/disassembly of nucleosomes (Avvakumov et al. 2011, Das et al. 2010, Eitoku et al. 2008, Gurard-Levin et al. 2014). Anti-silencing function 1 (ASF1) is a H3–H4 chaperone that is evolutionarily conserved from yeast to mammals and that plays a role in various chromatin-based processes, such as DNA replication, DNA damage response and repair, DNA recombination, and transcriptional regulation (Mousson et al. 2007).
Asf1 was originally discovered in budding yeast as a gene involved in transcriptional regulation. Indeed, the name of this chaperone (anti-silencing function 1) is derived from its ability to de-repress silent mating-type loci when overexpressed (Le et al. 1997). Among the conserved functions, the contribution of Asf1 homologs to heterochromatin silencing (Meijsing & Ehrenhofer-Murray 2001, Moshkin et al. 2002, Osada et al. 2005, Zhang et al. 2005) and to the transcriptional regulation of euchromatic genes has been reported in various organisms (Adkins et al. 2004, 2007, Akai et al. 2010, Chimura et al. 2002, Schwabish & Struhl 2006, Sutton et al. 2001, Zabaronick & Tyler 2005). In addition, many studies have highlighted the critical role played by ASF1 in DNA replication and cell cycle progression (Grigsby & Finger 2008, Grigsby et al. 2009, Groth et al. 2007, Sanematsu et al. 2006, Schulz & Tyler 2006, Tyler et al. 1999, 2001). ASF1 also promotes acetylation of lysine 56 of histone H3 (Battu et al. 2011, Das et al. 2009, 2014, Li et al. 2008, Recht et al. 2006, Yuan et al. 2009). This histone modification has a crucial role in the assembly of newly formed nucleosomes and in histone eviction or exchange during DNA repair and transcription initiation and elongation (Chen et al. 2008, Masumoto et al. 2005, Ozdemir et al. 2005, Rufiange et al. 2007, Schneider et al. 2006, Tjeertes et al. 2009, Vempati et al. 2010, Xu et al. 2005, Zhou et al. 2006).
In eukaryotes, Asf1 exists either as a single gene (e.g. in yeast) or as two paralogs (Abascal et al. 2013). In mammals, these paralogs are called Asf1a and Asf1b. Due to the high sequence identity of the two ASF1 proteins, their functional redundancy or specialization has been investigated in vitro in human cell lines. Based on these studies, human ASF1A is preferentially involved in DNA repair and cell senescence, while ASF1B primarily contributes to cell proliferation (Groth et al. 2005, 2007). Moreover, Asf1a-knockout mouse embryos die at mid-gestation (E9.5) (Hartford et al. 2011), indicating that Asf1b cannot compensate for the loss of Asf1a in vivo. The lack of redundancy between the Asf1 paralogs might be partially explained by their different expression patterns. In both humans and mice, Asf1a is ubiquitously expressed, whereas Asf1b expression is clearly confined to few tissues, with strong expression in adult testes (Abascal et al. 2013, Umehara & Horikoshi 2003). In testis, Asf1b expression is restricted to pre-meiotic and meiotic germ cells, suggesting a specific function in the germ cell lineage (Umehara & Horikoshi 2003).
Among the differentiated cells of an organism, gametes have a unique cell fate as they ensure, after the fertilization, the accurate transmission of genetic and epigenetic information to the next generation. Germ cells, the gamete precursors, undergo extensive DNA and chromatin modifications during their differentiation (Sasaki & Matsui 2008). Germ cell development is initiated in early fetal life and differs according to sex determination. In mice, primordial germ cells (PGCs) migrate into the genital ridges between embryonic day 10.5 (E10.5) and E11.5. Post-migratory PGCs proliferate actively and are sexually determined only at E11.5. At E13.5, female germ cells enter meiosis and progress through the first stages of the meiotic prophase I during fetal life. Around birth, oocytes are arrested at the diplotene stage and are progressively enclosed into primordial follicles until postnatal day 5 (P5). Meiosis resumption and ovulation will be triggered later in adult life at the end of the folliculogenesis process upon hormonal stimulation. In testes, embryonic germ cells enter mitotic arrest at E14.5 and remain in the G0/G1 phase of the cell cycle. Within the first days after birth, they re-enter the cell cycle and migrate to the basement membrane of the seminiferous tubule. Some of these cells contribute to the spermatogonial stem cell population that supports the continuous production of sperm throughout adult life by initiating successive waves of spermatogenesis. Male meiosis initiates only around P8–P9. Pachytene stage spermatocytes I can be observed starting at P14, round spermatids at P20, and elongating spermatids at P30 (Bellve et al. 1977).
To investigate the specific function(s) of the ASF1B protein, we generated a Asf1b-knockout mouse line. Using this model, we show that, differently from Asf1a, the Asf1b gene is dispensable for early embryonic development. Moreover, its expression is developmentally regulated in the germ cells of both sexes with functional implications on meiosis onset.
Materials and methods
Mice
All animal studies were conducted in accordance with the guidelines on the care and use of laboratory animals of the French Ministry of Agriculture. Animal experiments were supervised by Dr M Vernet (agreement delivered by the French Ministry of Agriculture for animal: n° 92-289) in the mouse facility of the Science Life Division at CEA (agreement n° B 92-032-02). Mice were killed by cervical dislocation. All efforts were made to minimize animal stress and suffering. The different mouse strains (129S2/SvPas, C57BL/6J, FVB, and CD-1) were purchased from Charles River Laboratory, but the NMRI mice from Janvier (Le Genest-Saint-Isle, France). Pou5f1-GFP mice were described previously (Yoshimizu et al. 1999). Mice were housed in controlled 12h light:12h darkness conditions (lights on from 08:00 to 20:00 h) and were supplied with commercial food and tap water ad libitum. For the fertility studies, males were housed in individual cages with two females (3- to 7-month old). The presence of vaginal plugs was checked daily. Plugged females were considered infertile when no birth was observed 3 months after mating. For comparison with the congenic C57BL/6J strain, the presented data were retrieved from the Mouse Phenome Database at the Jackson Laboratory (http://phenome.jax.org). For the continuous breeding assay, all mating pairs of mice (control C57BL/6J and mutants) were housed in the same mouse facility.
ES cell manipulation and derivation of the Asf1bGT(ROSA-βgeo)437 transgenic line
The ES cell line AT1 was established in the laboratory from 129S2/SvPas blastocysts. The pluripotency of AT1 ES cells was confirmed by their successful contribution to the germ line of transmitting chimeras (Buchou et al. 2003). The infection of AT1 cells with the retroviral ROSA-βgeo vector and the selection, isolation amplification, and storage of G418-resistant clones were performed as described previously (Stuhlmann 2003). The overall gene trap strategy, including the differentiation tests, is presented in the supportive information (Supplementary data, see section on supplementary data at the end of the article). The AT1 ES clone 437 that carries the proviral ROSA-βgeo vector inserted in the Asf1b gene was used to generate chimeras following the aggregation technique. Germ line-transmitting chimeras were crossed with outbred CD-1 or inbred C57BL/6 mice. Backcrossing on C57BL/6 was continued for ten successive generations in order to transfer the mutation to this inbred background.
Western blotting
To produce whole cell extracts, cells/tissues were harvested in lysis buffer (50 mM Tris—HCl pH 7.5, 300 mM NaCl, 0.5% NP40, 0.1 mM EDTA, and protease inhibitors) and frozen in dry ice. After thawing and sonication, insoluble material was removed by centrifugation and protein concentration was measured. Protein extracts were resolved by SDS—PAGE (15% gel). For immunoblotting, the following antibodies were used: a rabbit polyclonal antiserum (28026) against human ASF1 (Groth et al. 2005) that recognizes both mouse ASF1A and ASF1B (D Ray-Gallet and G Almouzni, personal communication) and a control anti-b-actin antibody. Protein—antibody complexes were visualized using a chemiluminescence western blotting detection system (Applied Biosystems).
Reverse transcription and quantitative PCR
Total RNA was isolated from cells or tissues using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. For quantitative RT-PCR (RT-qPCR), total RNA was reverse transcribed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) or the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). An aliquot of cDNA was amplified in a 7900HT Sequence Detection System apparatus (Applied Biosystems) using the Power SYBR Green PCR Master Mix (Applied Biosystems). The comparative Ct method was used to determine the relative quantities of mRNA. The target gene expression was normalized to the expression of the Actb gene. The localization and sequence of the oligonucleotides used are given in Fig. 1 and Supplementary Table 1 (see section on supplementary data given at the end of this article) respectively. The software Primer3 was used to design the primers (Koressaar & Remm 2007, Untergasser et al. 2012).
Generation of Asf1b-deficient mice by gene trapping. (A) Schematic representation of the wild type (Asf1b) and gene trap (Asf1bGT(ROSA-βgeo)437) alleles. Open and black boxes indicate non-coding and coding exons, respectively. The proviral ROSA-βgeo vector is inserted in the first exon of Asf1b. The proviral vector contains LTR (Long Terminal Repeats) at both ends (dotted boxes), a splice acceptor (SA), a fusion gene (βgeo), and an SV40 polyadenylation site (pA). The cryptic splice donor (SD) site within the LTR sequence is indicated. (B) Western blot analysis of testis tissue extracts (30 mg) from adult Asf1b+/+ and Asf1b−/− mice using a polyclonal antibody against ASF1. b-actin was used as a loading control. (C) Comparison of Asf1a, Asf1b, and Asf1b-βgeo mRNA expression levels, measured by RT-qPCR, in adult testes from mutants (Asf1b+/− and Asf1b−/−) and wild type C57BL/6 mice (C57). The position of the primers is indicated by black arrows in (A). Primers (8-9) and (5-10) were used, respectively, to assess Asf1b and Asf1b-βgeo expression. Results are presented as the percentage of the highest expression (defined as 100%) normalized to the expression of the Actb gene. The error bars represent the s.e.m. of three independent experiments.
Citation: Reproduction 151, 5; 10.1530/REP-15-0327
β-Galactosidase activity
The procedure for in situ detection of β-galactosidase expression using X-Gal staining was described previously (Bonnerot & Nicolas 1993, Vernet et al. 1993). Pre-implantation embryos were obtained from CD-1 females mated with hybrid (129S2/CD-1) heterozygous or homozygous mutant males. β-Galactosidase expression was analyzed in freshly recovered fertilized eggs, two-cell and morula embryos. Expression was assessed in blastocysts after in vitro overnight culture of morula embryos in M16 medium at 37°C with 5% CO2 in air. Post-implantation embryos were obtained from heterozygous females mated with hybrid (129S2/CD-1) homozygous males and expression was assessed immediately after recovery at E7.0. After whole-mount X-Gal staining, fetal and post-natal gonads (E13.5 to P2) were fixed in 2% paraformaldehyde (PFA) overnight and then dehydrated and embedded in paraffin wax. Sections of 5 μm size were cut and counterstained with nuclear fast red (Vector). P12 ovaries were fixed and transferred in 1X PBS with 30% (w/v) sucrose for 48 h, then embedded in OCT embedding matrix (CellPath, Newtown, Powys, UK) and snap-frozen in liquid nitrogen. Cryostat sections of 10–12 μm size were mounted on glass slides and X-Gal stained overnight. After washing with 1X PBS, sections were counterstained with nuclear fast red.
Histology and immunochemistry
Fetal, postnatal, and adult gonads were fixed in 10% neutral formalin (Carlo Erba Reagents, Peypin, France) at 4°C overnight. Then, they were dehydrated and embedded in paraffin wax. Adult ovaries (5 μm sections) were stained with hematoxylin and eosin, while fetal and postnatal gonads (5 μm sections) were used for immunostaining with the appropriate antibodies as described previously (Messiaen et al. 2013). When different genotypes were compared, tissues from the corresponding animals were mounted on the same slide. Primary antibodies are listed in supplementary Table 2.
Sperm counts
For sperm counts, cauda epididymides were isolated from 16- to 18-week-old mice (C57Bl/6 and mutant Asf1-/-) (four or five mice housed per cage). The two cauda epididymides (right and left) of each male were placed in 1 mL of pre-warmed M2 medium (Sigma). Then, an incision was made in each epididymis and the sperm suspension was allowed to swim in the medium for 15 min. A dilution of this sperm suspension (1:10 in water) was used for counting the sperm concentration using a hemocytometer (each sample was counted twice).
Germ cell counting
Postnatal oocytes were counted in one per every five sections equidistantly distributed along the gonad, using Histolab software (Microvision Instruments, Evry, France). The oocyte nuclei were stained with p63 (a marker of late pachytene/diplotene oocytes) and the stage of follicular development was determined, as described previously (Guigon et al. 2003). Briefly, follicles were classified according to the shape and number of layers of somatic cells surrounding the oocyte: primordial follicles (flattened cells) and growing follicles (one or more complete layers of cuboidal cells), which included both primary and secondary follicles. The nucleus diameter was measured to correct for any double counting resulting from the appearance of a single cell in two successive sections using the Abercrombie formula (Abercrombie 1946). For quantifying the percentage of positive germ cells (OCT4, STRA8, SYCP3, or gH2AX) in female and male gonads, three sections were randomly chosen from each gonad. At least 200 germ cells (VASA/DDX4-positive) in each section were scored.
Flow cytometry
Cells from adult testes were isolated from 2- to 3-month-old mice using a two-step enzymatic digestion procedure to remove interstitial cells. One million cells were stained with 5 mg/mL Hoechst 33342 (Sigma, France) at 32°C for 1 h, as described previously (Bastos et al. 2005). Then, cells were labeled with the biotinylated anti-c-KIT (2B8) and the PE-conjugated anti-integrin-α-6 (GoH3) monoclonal antibodies (BD Pharmingen, Le Pont de Claix, France). Before analysis, 2 mg/mL propidium iodide (Sigma, France) was added to exclude dead cells. Analysis and cell sorting were performed on a LSRII and an ARIA flow cytometer respectively (Becton Dickinson, Le Pont de Claix, France). Embryonic germ cells from E13.5 gonads of (Pou5f1-GFP) mice crossed with NRMI mice were purified by flow cytometry (MoFlo; Beckman Coulter, Brea, CA, USA) (Ohbo et al. 2003).
Data analysis
Data were analyzed using GraphPad Prism, version 6.0, for Mac OS X, GraphPad Software, La Jolla, CA, USA; www.graphpad.com.
Results
Generation of Asf1b mutant mice
Originally, we identified Asf1b during a functional genetic screen designed to identify new potential pluripotency markers (details in supplementary data file). Our strategy was based on a gene trap approach in the ES cell line AT1 using the retroviral ROSA-βgeo vector that encodes the β-galactosidase/neomycin phosphotransferase (βgeo) fusion protein. In one of the selected clones, the retroviral insertion disrupted the first exon of the Asf1b gene, 35 nucleotides downstream of the ATG codon (Fig. 1A).
To assess the in vivo role of Asf1b, we derived a transgenic mouse line from this mutated clone. The location and transmission of the gene trap allele were confirmed by genomic DNA analysis of heterozygous mice (see Supplementary Figure 1, see section on supplementary data given at the end of this article). Then, homozygous mutants were generated by crossing heterozygous mice. Homozygous mice developed normally and reached adulthood. To confirm the complete loss of Asf1b gene expression, its expression was assessed in adult testes, the major Asf1b-expressing tissue in adult mice. Neither the full-length transcript (Supplementary Figure 1D) nor the protein (Fig. 1B) could be detected in adult testes from homozygous mice, thus validating the Asf1bGT(ROSA-βgeo)437 mouse line as a new model of Asf1b loss-of-function mutation (Asf1b−/− thereafter). This finding also implies that Asf1b function is dispensable for embryo development and basic physiology. Quantification of the mRNA expression level of Asf1b and Asf1a by RT-qPCR showed that in heterozygous mice, Asf1b was reduced nearly by half compared with wild-type littermates. In homozygous mice, Asf1b was not expressed in most of the tissues tested; however, using primers chosen in exons 2 and 3, a residual Asf1b expression was specifically detected in testis, despite the absence of a full-length transcript. However, Asf1a gene expression in heterozygous and homozygous mice was similar to that of wild-type C57BL/6 (Figs 1C and Supplementary Figure 2).
We conclude that Asf1b gene expression is biallelic and that Asf1b gene disruption has no impact on the transcriptional expression of its paralog Asf1a.
Impaired reproductive output of Asf1b−/− females
For further phenotypic characterization, the Asf1bGT(ROSA-βgeo)437 gene trap mutation was transferred to the C57BL/6 inbred background. When congenic heterozygous mice were interbred, the distribution of the three expected genotypes was consistent with Mendelian inheritance. From 20 litters derived from ten independent crossings, the cumulative number of mice per genotype was 62, 23, and 23 for heterozygous, wild-type, and homozygous mice respectively (χ2 = 2.370 and P = 0.305). Thus, viable homozygous mutant adult mice can be efficiently generated from heterozygous inter-crosses.
Conversely, the generation of Asf1b−/− pups by mating homozygous animals was less consistent. First, 23% of Asf1b−/− females (three of 13 females from three independent litters) failed to reproduce with known fertile C57BL/6 males. In addition, the number of weaned Asf1b−/− pups per litter was highly variable and generally less. Specifically, the mean number of pups obtained from Asf1b−/− homozygous matings was significantly lower compared with the inter-crosses between heterozygous Asf1b+/− or C57BL/6 reference mice (http://phenome.jax.org) (Fig. 2A). These observations prompted us to further evaluate the fertility of mutant mice. Homozygous mice were crossed with C57BL/6 animals. Comparison of female and male mutants showed that the number of weaned pups per litter was significantly lower compared with control matings (C57BL/6 × C57BL/6), only when Asf1b−/− females were mated with C57BL/6 males and not the contrary (Fig. 2B). The subfertility of Asf1b−/− females was also illustrated by the weak cumulative number of progeny per female obtained in continuous breeding assays (Fig. 2C). These data indicate that the defective generation of mutant offspring is clearly linked to the maternal genotype.
Reduced reproductive output of Asf1b−/− mice. By convention, the results shown here concern pups obtained by female x male crosses. (A) Mean number of weaned pups per litter obtained from crosses between heterozygous and homozygous mice and in the inbred strain C57BL/6. The total litter number is shown in each column. (B) Mean number of weaned pups per litter obtained from backcrosses of Asf1b−/− males or females with C57BL/6 mice and from C57BL/6 crosses. The total litter number appears in each column. For (A) and (B) the Kruskal–Wallis test was used for statistical analysis and P-values were corrected using the Dunn’s multiple comparison test (P < 0.05). The litter number from Asf1b−/− inter-crosses and from Asf1b−/− females (backcrosses) is significantly lower (****P < 0.0001 and *P < 0.05) than that from C57BL/6 crosses. (C) Fertility assessment by continuous breeding. During nine weeks, 3 to 4-month-old mice were housed as breeding pairs and litters were discarded to allow continuous mating. The number of pups obtained for each breeding pair was recorded and presented as the cumulative number of pups per female. (D) The percentage of litters in which a defined number of pups (0; 1-3; 4-6; >7) survived from birth until weaning was calculated (crosses between heterozygous and homozygous mice and in the inbred strain C57BL/6). (E) The percentage of litters in which a defined number of pups (0; 1-3; 4-6; >7) survived from birth until weaning was calculated (backcrosses of Asf1b−/− males or females with C57BL/6 mice and C57BL/6 crosses). (F) Comparison of the development of Asf1b−/− embryos from homozygous parents and of wild type C57BL/6 embryos during the second half of pregnancy (from E9.5 to E18.5). The abnormal class includes resorbed or dead embryos and embryos with morphological defects. The number of abnormal embryos is significantly higher among Asf1b−/− than control embryos (Mann–Whitney test; *P < 0.05). Error bars represent the s.e.m.; (+/−), heterozygous; (−/−), homozygous; C57, C57BL/6.
Citation: Reproduction 151, 5; 10.1530/REP-15-0327
Besides the decreased female reproductive performance, perinatal lethality was also increased in the offspring of Asf1b−/− females. Pup lethality during the first 2 d after birth accounted in part for the low number of Asf1b−/− pups that survived until weaning in homozygous crosses. Indeed, in more than 20% of homozygous litters, no new born survived (Fig. 2D). Similarly, the number of live pups at weaning was significantly lower when Asf1b was invalidated specifically in the mother (Fig. 2E). The development of embryos derived from homozygous parents was also impaired. Analyses performed during the second half of pregnancy revealed a significantly higher number of abnormal embryos (resorbed embryos, developmentally retarded or dead embryos, and embryos with morphological defects) in homozygous crossings than in C57BL/6 crossings (Fig. 2F). These results suggest that Asf1b−/− females produce gametes that are less compatible with normal embryo development.
Asf1b is expressed in early embryos and germ cells
Due to the impaired reproductive performance of Asf1b−/− animals, we focused our analysis on the expression pattern of Asf1b in embryos and gonads. As in Asf1bGT(ROSA-βgeo)437 mice β-galactosidase expression is under the control of the Asf1b promoter (Fig. 1C, right panel), the Asf1b expression pattern can be easily determined by X-Gal staining. The expression was first analyzed in heterozygous embryos obtained from wild-type females mated with transgenic males. No expression was detected in the transcriptionally silent one-cell embryos (Supplementary Figure 3A), while in two-cell embryos, blastomeres were homogeneously stained (Supplementary Figure 3B) concomitantly with zygotic genome activation (Bensaude et al. 1983). X-Gal staining in eight-cell embryos was heterogeneous (Supplementary Figure 3C), and a faint staining was still observed at the blastocyst stage (Supplementary Figure 3D). In post-implantation E7.0 embryos, X-Gal staining was absent in extra-embryonic tissues and was strictly located in the epiblast within the embryonic region (Supplementary Figure 3E). These data indicate that the Asf1b transgene expression is associated with cell pluripotency during early embryogenesis.
Next, as the dynamics of germ cell differentiation differ between sexes, in particular meiosis, we investigated gametogenesis in both sexes. The relative transcript levels of Asf1a and Asf1b were compared using RT-qPCR in mouse gonads of outbred NMRI mice during development and post-natal life. Asf1a gene expression was comparable in both sexes, particularly during development (Fig. 3A). Conversely, Asf1b transcription seemed to be finely regulated (Fig. 3B). During fetal development, Asf1b was stably expressed at low levels in male gonads, whereas its expression increased dramatically in female gonads at E13.5, peaked at E14.5, and then rapidly decreased to levels comparable to those observed in male samples by E16.5. In contrast, after birth, Asf1b expression progressively increased only in male gonads, peaking at P15 and then remained stable in adults. These increases in Asf1b transcription matched the timing of meiotic onset observed in female (E13.5) and male germ cells (P8/9).
Asf1a and Asf1b genes expression in mouse gonads. (A, B) The expression of Asf1a and Asf1b was quantified by RT-qPCR using mRNA isolated from whole mouse gonads harvested at the indicated developmental stages. (C) The transgenic line Pou5f1-GFP(ΔPE) was used to measure Asf1b gene expression in FACS-sorted germ (GFP+) and somatic (GFP-) cells from E13.5 gonads. The relative expression of the Asf1b and Asf1a genes was calculated using the Actb gene as a reference and the F9 mouse cell line as a calibrator (A, B and C). The error bars represent the s.e.m. of three independent experiments; E, embryonic day; P post-natal day.
Citation: Reproduction 151, 5; 10.1530/REP-15-0327
To investigate more accurately the germ cell specificity of Asf1b expression in developing gonads, we performed additional experiments using the Pou5f1-GFP (ΔPE) mouse transgenic line in which GFP is specifically expressed in embryonic germ cells (Yoshimizu et al. 1999). Asf1b gene expression analysis by RT-qPCR of sorted GFP-positive and GFP-negative cells from gonads of both sexes at E13.5 clearly demonstrated the germ cell specificity of Asf1b gene expression (Fig. 3C).
Finally, X-Gal staining of Asf1b−/− gonads (hybrid genetic background 129S2/CD-1) accurately illustrated the differences observed between male and female gonads, confirming the RT-qPCR data. X-Gal-stained cells (blue) were exclusively detected in the gonads of E13.5 female embryos with more pronounced staining in the anterior part (Fig. 4A). By E15.5, the whole female gonad was robustly stained, while in male gonads only a few blue cells, localized in the testis cords, were detected (Fig. 4A). At E16.5, the increased staining observed in arrested male germ cells was probably due to accumulation of β-galactosidase (Fig. 4B) in the cytoplasm.
Expression pattern of the lacZ reporter gene under the control of the Asf1b promoter in Asf1b−/− fetal gonads. Gonads were isolated from homozygous male and female embryos at E13.5, E15.5, and E16.5 and stained with X-Gal. (A) At E13.5, female gonads are X-gal positive and male gonads X-gal negative. At E15.5, few blue cells are present in male gonads. Lower panels: tissue sections of male gonads at E15.5 (scale bar, 20 μm). (B) At E16.5, the number of blue cells in testis cords are increased (left panel, scale bar, 20 μm) and their localization is suggestive of germ cells (right panel, scale bar, 25 μm).
Citation: Reproduction 151, 5; 10.1530/REP-15-0327
After birth, X-Gal staining in Asf1b−/− ovaries was confined to oocytes at P2 and P12 (Fig. 5A). For unknown reasons, the X-Gal staining in oocytes appeared as a single dot in the cytoplasmic compartment, as previously reported in other transgenic models (Gallardo et al. 2007). In testes, a few blue cells (possibly spermatogonia) at the periphery of the seminiferous tubules were observed at P6. After meiotic entry, testis cross sections clearly showed strong staining of meiotic cells (pachytene stage) in the center of tubules at P18 (Fig. 5A). The staining observed in spermatogonia at this stage was less intense than that observed in spermatocytes (Fig. 5A, image on right side). As previously reported (Umehara & Horikoshi 2003), no staining was detected at post-meiotic stages (e.g. round spermatids: data not shown). In adult testes, X-Gal staining of whole-mount seminiferous tubules recapitulated the stage-specific expression during the spermatogenic cycle, as indicated by the regular blue cell clumps that could correspond to spermatocytes (Fig. 5B).
Expression pattern of the lacZ reporter gene under the control of the Asf1b promoter in Asf1b−/− postnatal gonads. (A) Histological cross-sections of X-Gal stained gonads retrieved from homozygous animals. In P2 ovaries, X-Gal staining is confined to oocytes in primordial (arrow) and primary follicles (arrowhead) and maintained at P12. In P6 testes, X-Gal staining is observed in germ cells near the basement membrane. At P18, staining is present in spermatocytes (pachytene) in the center of the seminiferous tubules and also in few cells near the basement membrane. (B) In Asf1b−/− adult testes, whole-mount X-gal staining shows a dotted staining along the seminiferous tubules regularly interspersed with clumps of blue cells that probably correspond to spermatogonia and spermatocytes, respectively. (C) Quantification of Asf1a and Asf1b genes expression in the two spermatogonial subpopulations (SPa-6+c-kit-; undifferentiated) and (SPa-6+c-kit+; differentiated) by RT-qPCR using the Actb gene as a reference and cells from adult testis as a calibrator. The error bars represent the s.e.m. of two independent experiments. Scale bars, 20 μm except for P12 ovary: scale bar, 50 μm.
Citation: Reproduction 151, 5; 10.1530/REP-15-0327
To investigate more specifically the expression of Asf1b and Asf1a in spermatogonial cells from adult testes, sorted spermatogonial populations from FVB inbred mice were analyzed by RT-qPCR. Using the a-6 integrin-positive side population (SPa-6+) and the c-KIT marker, two spermatogonial populations were purified (SPa-6+c-Kit- and SPa-6+c-Kit+) that corresponded, respectively, to “undifferentiated” and “differentiated” spermatogonia (Barroca et al. 2009, Schrans-Stassen et al. 1999). As already noted, Asf1a was similarly expressed in the two subpopulations. In contrast, Asf1b was strongly expressed in the differentiated (SPa-6+c-Kit+) subpopulation and only moderately in the “undifferentiated” (SPa-6+c-Kit-) fraction (Fig. 5C). From these results, we conclude that the Asf1b gene is specifically expressed in the germ cell lineage and correlates with meiosis in both sexes. Similarly, in adult testes, Asf1b was expressed in the spermatogonial cell population, preferentially in the more differentiated and proliferative compartment. Altogether, Asf1b expression appears to be finely regulated in germ cells. Moreover, the specific Asf1b expression in germ and embryonic pluripotent cells could explain the reproductive and developmental defects observed in Asf1b−/− animals.
Gonad morphological defects in both sexes of adult Asf1b−/− mice
Due to the expression of Asf1b in germ cells and the reduced fertility of Asf1b−/− crosses, we accurately analyzed the reproductive systems of both sexes. Although the male reproductive apparatus appeared morphologically normal, testis size was smaller in Asf1b−/− than in wild-type C57BL/6 mice (Fig. 6A). To quantify this effect, the testis/body weight ratios of Asf1b+/−, Asf1b−/−, and wild-type mice were compared. No significant difference was observed between Asf1b+/− and wild-type animals; conversely, the testis/body weight ratio of Asf1b−/− mice was significantly lower than in control mice (Supplementary Figure 4). Body weight was comparable in the three groups. Histological examination of testis sections revealed a normal abundance of cells at all spermatogenic stages; however, epididymal sperm counts were significantly lower in Asf1b−/− mice than in control mice (Fig. 6B). Therefore, while no major defect in cell differentiation can be observed in adult testes, the moderate testis hypoplasia and hypospermia observed in Asf1b−/− males is likely insufficient to impair their fertility.
Testis hypospermy and decreased ovarian follicle number in adult Asf1b−/− mice. (A) Whole adult testes from wild type (left) and Asf1b−/− (right) mice. (B) Comparison of the testis weight/body weight ratio (mg/g) in C57BL/6 and Asf1b−/− (n = 5) mice reveals hypoplasia in Asf1b−/− males. Comparison of the corresponding sperm counts shows a slight hypospermy in mutant mice. Significant differences (Mann–Whitney tests) are indicated by asterisks: **P < 0.01). (C) Hematoxylin and eosin staining of histological sections of ovaries from three 31–37 week/old female mice: one C57BL/6 female control, one Asf1b−/− female after successful pregnancy, and one Asf1b−/− female from an infertile couple. Scale bars, 500 μm.
Citation: Reproduction 151, 5; 10.1530/REP-15-0327
To analyze potential defects in Asf1b−/− ovaries, histological sections from ovaries of control C57Bl/6 females and from fertile and infertile Asf1b−/− females were compared. Ovarian histology was similar in fertile Asf1b−/− and control females with several types of follicles easily identified in ovarian sections (Fig. 6C). In contrast, ovaries of infertile Asf1b−/− females were sometimes completely devoid of follicles (example in Fig. 6C). Analysis of multiple sections per ovary allowed us to exclude a localized defect of follicles. In this case, infertility could obviously be directly correlated with the lack of germ cells.
These results underline the variability of the phenotype observed in Asf1b−/− females, possibly due to incomplete penetrance of the mutation or variable expressivity.
Asf1b gene loss impairs meiosis initiation
In female gonads, Asf1b gene expression peaks at E14.5 during fetal development and coincides with meiotic entry (Fig. 3B). Therefore, to evaluate the putative consequences of Asf1b deficiency on meiosis initiation, the expression of OCT4, a marker of undifferentiated germ cells encoded by the Pou5f1 gene, and of SYCP3 (synaptonemal complex protein 3, involved in meiotic prophase) was assessed in E15.5 female gonads. In C57BL/6 control mice, SYCP3 staining pattern indicated that, at E15.5, germ cells were predominantly engaged in meiosis, while only a few cells continued to express OCT4 (Fig. 7A). Conversely, fewer meiotic cells were observed in Asf1b−/− gonads compared with wild-type C57BL/6 and a high proportion of cells still expressed OCT4 (Fig. 7A and B). Thus, meiotic entry appears to be delayed in Asf1b−/− germ cells. Notably, no global delay of embryonic development was observed in the analyzed embryos. Moreover, germ cell proliferation before meiotic entry was not affected by Asf1b invalidation, as indicated by BrdU incorporation (34.0 ± 2.5% vs 38.8 ± 3.4% of BrdU-positive germ cells in C57Bl/6 and Asf1b−/− E13.5 ovaries respectively, n = 4–6) (Supplementary Figure 5). We thus hypothesized that Asf1b expression in fetal oocytes is required to regulate either the entry into meiosis or its progression.
Asf1b gene knockout affects germ cell meiotic entry. Gonads were obtained from E15.5 female embryos and P9 females. (A) Expression of OCT4, a marker of undifferentiated germ cells, and of SYCP3, a meiotic marker, was assessed by immunohistochemistry in C57BL/6 and Asf1b−/− E15.5 ovaries. (B) The cumulative number of OCT4- and SYCP3-positive cells was calculated in three embryos for each genotype. (C) P9 ovaries were stained with an anti-p63 antibody (brown nuclei). (D) The cumulative number of p63-positive oocytes in ovaries was calculated per animal from respectively three and six C57BL/6 and Asf1b−/− females (upper panel). Among the p63-positive oocytes, the number of primordial and growing oocytes per ovary was also calculated. (B, D) Significant differences (Mann–Whitney test) are indicated by asterisks: ***P < 0.001; ****P < 0.0001). The error bars represent the s.e.m. (A) scale bar, 20 mm; (E) scale bar, 100 μm.
Citation: Reproduction 151, 5; 10.1530/REP-15-0327
To evaluate progression through the meiosis of germ cells after birth, P9 female gonads were examined using the p63 protein, a marker of the late pachytene/diplotene stage of meiosis (Nakamuta & Kobayashi 2007). Compared with C57BL/6 control ovaries, the size of P9 Asf1b−/− ovaries widely varied. In some Asf1b−/− animals, ovary size was similar, whereas in others, ovaries were smaller than in control animals (Fig. 7C). The total number of p63-positive oocytes in Asf1b−/− females was significantly reduced by almost 50% compared with controls. The number of growing oocytes per ovary was only slightly decreased in Asf1b−/− animals compared with C57BL/6 controls. Conversely, the number of primordial oocytes in Asf1b−/− ovaries ranged from nearly normal to significantly decreased (Fig. 7C and D), and its decrease was correlated with the reduction in the ovary size. As the reduction of primordial oocytes suggests a perinatal loss of oocytes, we investigated apoptosis at P0 by measuring the percentage of cleaved caspase 3-positive cells (Supplementary Figure 5). At this stage, the apoptosis rate was higher in Asf1b−/− than in wild-type ovaries (9.1 ± 2.9% vs 1.8 ± 0.2%, respectively, n = 4, P < 0.05, Mann—Whitney test). Altogether, these results suggest that during female gametogenesis, loss of Asf1b mostly affects meiosis initiation rather than progression. Furthermore, examination of ovulated oocytes at the metaphase 2 stage did not identify any additional late meiotic or maturation defect, based on chromosome alignment and measurement of the volume of metaphase plates (Supplementary Figure 6).
As Asf1b expression increases at meiotic entry in both sexes, we also investigated male meiosis initiation during the first round of spermatogenesis at P9 (pre-pubertal testes). The number of meiotic cells, based on SYCP3 expression, was decreased in seminiferous tubules of Asf1b−/− testes compared with Asf1b+/+ littermates (see Supplementary Figure 7). This observation was reinforced by the decreased number of cells expressing the phosphorylated histone variants gH2AX, a DNA double-strand break marker that is considered as a hallmark of meiosis (Hunter et al. 2001), and STRA8, a key factor for meiotic entry (Koubova et al. 2006). We then examined in adult testes the various spermatogenic subpopulations. Fluorescence-activated cell sorting analysis (Fig. 5C) indicated that the frequency of undifferentiated (SPa-6+c-Kit-) and differentiated (SPa-6+c-Kit+) spermatogonial subpopulations was not significantly different in Asf1b−/− and wild-type testes (data not shown). Characterization of the germ cell populations based on their ploidy also failed to reveal any obvious changes in the partition of the different cell populations (data not shown).
In conclusion, meiosis was delayed in both male and female germ cells; although in males, this delay could only be detected at the time of first spermatogenetic wave. Conversely, gametogenesis was severely impaired only in females.
Discussion
The generation and characterization of Asf1b-null mice presented in this study, together with the previously published Asf1a inactivation model, indicates that the two mouse Asf1 genes have distinct functions in vivo, despite their high-sequence similarity. Indeed, while Asf1a gene knockout leads to an embryonic lethal phenotype, Asf1b−/− mice are viable, but their breeding efficiency is reduced and females are subfertile. The correlation between Asf1b-specific expression in germ cells and the reproductive phenotype of Asf1b−/− mice suggests that Asf1b has a specialized role during gametogenesis.
Asf1b is preferentially expressed in pluripotent and progenitor cells
In this study, we refined Asf1b spatial and temporal expression specifically in early embryos and in developing gonads. Consistent with the previously published gene expression data (Umehara 2003, Abascal 2013), we confirmed the ubiquitous expression of Asf1a in mouse tissues and the restricted expression of Asf1b. The cell type-specific expression of Asf1b in gonads during development and in early embryos suggests that Asf1b is preferentially expressed in cells that retain pluripotent characteristics and differentiation potentialities. This is in agreement with our initial identification of Asf1b as a potential marker of undifferentiated ES cells. Similarly, in adult testis, Asf1b transcripts are detected in spermatogonia, particularly in the proliferative progenitor compartment. Therefore, it is tempting to speculate that similarly to testis, Asf1b expression in the few other adult tissues where it is detected (i.e. thymus, colon, and spleen; this study and (Abascal et al. 2013)) could be primarily linked to the presence of progenitor cells undergoing differentiation. However, our findings on the germ line-specific Asf1b expression suggest that Asf1b function is not strictly correlated with cell proliferation. In agreement, we did not observe any specific impairment of proliferation in the germ cells of Asf1-/- female embryos. This finding apparently goes against the putatively conserved proliferative function of ASF1B in human cells and the positive correlation established between the level of ASF1B expression and breast cancer aggressiveness (Corpet et al. 2011). Nevertheless, these differences could be reconciled if ASF1B up-regulation in human tumors reflected their undifferentiated state, related to the tumor grade, rather than their proliferative activity per se.
Asf1b expression correlates with the control of meiotic entry
We then showed that Asf1b upregulation in both male and female germ cells coincides with meiotic entry. In addition, a correlation between Asf1b expression and upregulation of Stra8, which is currently considered as the gatekeeper of the mitotic/meiotic shift, was documented during the synchronized neonatal wave of mouse spermatogenesis (Evans et al. 2014) and can be retrieved from transcriptomic data on human fetal gonads (Houmard et al. 2009). Therefore, we hypothesize that the loss of Asf1b function directly or indirectly affects the transcriptional activation of Stra8, leading to delayed meiotic entry. However, due to the reported implication of ASF1B in human cancer cell replication (Corpet et al. 2011), a specific role of ASF1B during the pre-meiotic S-phase cannot be excluded. Interestingly, in C. elegans, double mutants for the two Asf1 orthologs are sterile due to DNA replication blockage in the germ line (Grigsby et al. 2009). Therefore, and as proposed for the worm germ line, we hypothesize that partially overlapping Asf1a and Asf1b functions in the mouse germ line might explain the partial penetrance of the reproductive defects observed in Asf1b−/− mice.
Finally, our findings indicate that overall gonad morphology and fertility are more severely affected in Asf1b−/− females (at least in some) than in males, where only a moderate hypospermia is observed. This may be explained by the fact that different from spermatogenesis, oogenesis is not a continuous process throughout the reproductive life span. Specifically, in females, germ cell meiotic initiation occurs only once in the embryonic gonads and defines the pool of primordial follicles that will be available during the entire reproductive life. Therefore, any alteration in the process of meiotic entry could directly and definitively affect the pool of oocytes and, thereby, reduce fertility. Conversely, in males, germ cell meiotic entry occurs continuously throughout adult life and a moderate defect in this process may be less visible.
In conclusion, the generation of this first Asf1b-knockout model already allowed us to demonstrate the specific role of Asf1b in reproduction and germ cell differentiation. Of interest, similarly in C. elegans, the Asf1b ortholog (Asfl-1) is also upregulated specifically in germ cells, while the Asf1a ortholog (unc85) is expressed throughout development, suggesting its conserved roles throughout evolution. Besides the defective oogenesis caused by the deregulation of germ cell meiotic entry, Asf1b loss affects embryonic development more globally. This transgenic model thus represents a useful tool to decipher the potential role of Asf1b in cell differentiation. In regard to the role of ASF1B as histone chaperone, further studies are now required to investigate whether epigenetic mechanisms are affected in the mutant mice.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/REP-15-0327.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the Association pour la Recherche sur le Cancer (ARC grant number 9041); the Life Sciences Division of CEA on Epigenetic parameters (Programme Incitatif et Collaboratif between Institut Curie and CEA); and the Region Rhônes-Alpes (Stem cells Program). C. Aigueperse and I. Fliniaux were supported by Life Sciences Division of CEA postdoctoral fellowship.
Author contribution
All the authors contributed equally to this work.
Acknowledgements
The authors are grateful to P Soriano (Mount Sinai Hospital, New York, USA) for the gift of the packaging cell line to produce the retrovirus vector ROSA-βgeo and for his advices and encouragement all along our genetic screen. They thank G Almouzni and D Ray-Gallet (Institut Curie, Paris, France) for their help and the gift of antibodies against ASF1, and D Moison and C Duquenne for their technical help. They thank the iRCM and iMETI Flow Cytomery Platforms. They are grateful to M-H Verlhac for her precious advices and interesting exchanges, L Irbah for her help with oocyte imaging at the iRCM microscopy facility and to T Kortulewski for his advices. They also thank the teams of the mouse transgenic facilities at the iRCM and at the iRTSV. M V thanks P-H Roméo for his continued support and encouragement, D Gay for critically reading the manuscript, and E Andermarcher for English language editing.
References
Abascal F, Corpet A, Gurard-Levin ZA, Juan D, Ochsenbein F, Rico D, Valencia A & Almouzni G 2013 Subfunctionalization via adaptive evolution influenced by genomic context: the case of histone chaperones ASF1a and ASF1b. Molecular Biology & Evolution 30 1853–1866. (doi:10.1093/molbev/mst086)
Abercrombie M 1946 Estimation of nuclear population from microtome sections. The Anatomical Record 94 239–247. (doi:10.1002/(ISSN)1097-0185)
Adkins MW, Howar SR & Tyler JK 2004 Chromatin disassembly mediated by the histone chaperone Asf1 is essential for transcriptional activation of the yeast PHO5 and PHO8 genes. Molecular Cell 14 657–666. (doi:10.1016/j.molcel.2004.05.016)
Adkins MW, Williams SK, Linger J & Tyler JK 2007 Chromatin disassembly from the PHO5 promoter is essential for the recruitment of the general transcription machinery and coactivators. Molecular Cell Biology 27 6372–6382. (doi:10.1128/MCB.00981-07)
Akai Y, Adachi N, Hayashi Y, Eitoku M, Sano N, Natsume R, Kudo N, Tanokura M, Senda T & Horikoshi M 2010 Structure of the histone chaperone CIA/ASF1-double bromodomain complex linking histone modifications and site-specific histone eviction. Proceedings of the National Academy of Sciences U S A 107 8153–8158. (doi:10.1073/pnas.0912509107)
Avvakumov N, Nourani A & Cote J 2011 Histone chaperones: modulators of chromatin marks. Molecular Cell 41 502–514.
Barroca V, Lassalle B, Coureuil M, Louis JP, Le Page F, Testart J, Allemand I, Riou L & Fouchet P 2009 Mouse differentiating spermatogonia can generate germinal stem cells in vivo. Nature Cell Biology 11 190–196. (doi:10.1038/ncb1826)
Bastos H, Lassalle B, Chicheportiche A, Riou L, Testart J, Allemand I & Fouchet P 2005 Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis. Cytometry A 65 40–49. (doi:10.1002/cyto.a.20129)
Battu A, Ray A & Wani AA 2011 ASF1A and ATM regulate H3K56-mediated cell-cycle checkpoint recovery in response to UV irradiation. Nucleic Acids Research 39 7931–7945. (doi:10.1016/j.mrfmmm.2015.01.005)
Bellve AR, Cavicchia JC, Millette CF, O’Brien DA, Bhatnagar YM & Dym M 1977 Spermatogenic cells of the prepuberal mouse. Isolation and morphological characterization. Journal of Cell Biology 74 68–85. (doi:10.1083/jcb.74.1.68)
Bensaude O, Babinet C, Morange M & Jacob F 1983 Heat shock proteins, first major products of zygotic gene activity in mouse embryo. Nature 305 331–333. (doi:10.1007/978-3-540-46712-0)
Bonnerot C & Nicolas JF 1993 Application of LacZ gene fusions to postimplantation development. Methods Enzymol 225 451–469. (doi:10.1091/mbc.E06-12-1114)
Buchou T, Vernet M, Blond O, Jensen HH, Pointu H, Olsen BB, Cochet C, Issinger OG & Boldyreff B 2003 Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality. Molecular Cell Biol 23 908–915.
Chen CC, Carson JJ, Feser J, Tamburini B, Zabaronick S, Linger J & Tyler JK 2008 Acetylated lysine 56 on histone H3 drives chromatin assembly after repair and signals for the completion of repair. Cell 134 231–243. (doi:10.1016/j.cell.2008.06.035)
Chimura T, Kuzuhara T & Horikoshi M 2002 Identification and characterization of CIA/ASF1 as an interactor of bromodomains associated with TFIID. Proceedings of the National Academy of Sciences U S A 99 9334–9339. (doi:10.1073/pnas.142627899)
Corpet A, De Koning L, Toedling J, Savignoni A, Berger F, Lemaitre C, O’Sullivan RJ, Karlseder J, Barillot E, Asselain B, Sastre-Garau X & Almouzni G 2011 Asf1b, the necessary Asf1 isoform for proliferation, is predictive of outcome in breast cancer. The EMBO Journal 30 480–493.
Das C, Lucia MS, Hansen KC & Tyler JK 2009 CBP/p300-mediated acetylation of histone H3 on lysine 56. Nature 459 113–117. (doi:10.1038/nature07861)
Das C, Roy S, Namjoshi S, Malarkey CS, Jones DN, Kutateladze TG, Churchill ME & Tyler JK 2014 Binding of the histone chaperone ASF1 to the CBP bromodomain promotes histone acetylation. Proceedings of the National Academy of Sciences U S A 111 E1072–1081. (doi:10.1073/pnas.1319122111)
Das C, Tyler JK & Churchill ME 2010 The histone shuffle: histone chaperones in an energetic dance. Trends in Biochemical Sciences 35 476–489. (doi:10.1073/pnas.1319122111)
Eitoku M, Sato L, Senda T & Horikoshi M 2008 Histone chaperones: 30 years from isolation to elucidation of the mechanisms of nucleosome assembly and disassembly. Cellular & molecular life sciences 65 414–444. (doi:10.1007/s00018-007-7305-6)
Evans E, Hogarth C, Mitchell D & Griswold M 2014 Riding the spermatogenic wave: profiling gene expression within neonatal germ and sertoli cells during a synchronized initial wave of spermatogenesis in mice. Biology of Reproduction 90 108. (doi:10.1095/biolreprod.115.129718)
Gallardo T, Shirley L, John GB & Castrillon DH 2007 Generation of a germ cell-specific mouse transgenic Cre line, Vasa-Cre. Genesis 45 413–417. (doi:10.1016/j.ydbio.2008.06.017)
Grigsby IF & Finger FP 2008 UNC-85, a C. elegans homolog of the histone chaperone Asf1, functions in post-embryonic neuroblast replication. Journal of Developmental Biology 319 100–109. (doi:10.1128/JVI.02358-14)
Grigsby IF, Rutledge EM, Morton CA & Finger FP 2009 Functional redundancy of two C. elegans homologs of the histone chaperone Asf1 in germline DNA replication. Journal of Developmental Biology 329 64–79. (doi:10.1016/j.ydbio.2009.02.015)
Groth A, Corpet A, Cook AJ, Roche D, Bartek J, Lukas J & Almouzni G 2007 Regulation of replication fork progression through histone supply and demand. Science 318 1928–1931. (doi:10.1126/science.1148992)
Groth A, Ray-Gallet D, Quivy JP, Lukas J, Bartek J & Almouzni G 2005 Human Asf1 regulates the flow of S phase histones during replicational stress. Molecular Cell 17 301–311. (doi:10.1016/j.molcel.2004.12.018)
Guigon CJ, Mazaud S, Forest MG, Brailly-Tabard S, Coudouel N & Magre S 2003 Unaltered development of the initial follicular waves and normal pubertal onset in female rats after neonatal deletion of the follicular reserve. Endocrinology 144 3651–3662. (doi:10.1210/en.2003-0072)
Gurard-Levin ZA, Quivy JP & Almouzni G 2014 Histone chaperones: assisting histone traffic and nucleosome dynamics. Annual Review of Biochemistry 83 487–517. (doi:10.1146/annurev-biochem-060713-035536)
Hartford SA, Luo Y, Southard TL, Min IM, Lis JT & Schimenti JC 2011 Minichromosome maintenance helicase paralog MCM9 is dispensible for DNA replication but functions in germ-line stem cells and tumor suppression. PNAS 108 17702–17707. (doi:10.1073/pnas.1113524108)
Houmard B, Small C, Yang L, Naluai-Cecchini T, Cheng E, Hassold T & Griswold M 2009 Global gene expression in the human fetal testis and ovary. Biology of Reproduction 81 438–443. (doi:10.1095/biolreprod.108.075747)
Hunter N, Borner GV, Lichten M & Kleckner N 2001 Gamma-H2AX illuminates meiosis. Nature Genetics 27 236–238. (doi:10.1038/85781)
Koressaar T & Remm M 2007 Enhancements and modifications of primer design program Primer3. Bioinformatics 23 1289–1291. (doi:10.1016/j.mimet.2014.07.034)
Koubova J, Menke DB, Zhou Q, Capel B, Griswold MD & Page DC 2006 Retinoic acid regulates sex-specific timing of meiotic initiation in mice. Proceedings of the National Academy of Sciences USA 103 2474–2479. (doi:10.1073/pnas.0510813103)
Le S, Davis C, Konopka JB & Sternglanz R 1997 Two new S-phase-specific genes from Saccharomyces cerevisiae. Yeast 13 1029–1042. (doi:10.1002/(ISSN)1097-0061)
Li Q, Zhou H, Wurtele H, Davies B, Horazdovsky B, Verreault A & Zhang Z 2008 Acetylation of histone H3 lysine 56 regulates replication-coupled nucleosome assembly. Cell 134 244–255. (doi:10.1016/j.cell.2008.06.018)
Masumoto H, Hawke D, Kobayashi R & Verreault A 2005 A role for cell-cycle-regulated histone H3 lysine 56 acetylation in the DNA damage response. Nature 436 294–298. (doi:10.1038/nature03714)
Meijsing SH & Ehrenhofer-Murray AE 2001 The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae. Genetics & Development 15 3169–3182. (doi:10.1007/978-1-4939-2895-8)
Messiaen S, Le Bras A, Duquenne C, Barroca V, Moison D, Dechamps N, Doussau M, Bauchet AL, Guerquin MJ, Livera G, Essers J, Kanaar R, Habert R & Bernardino-Sgherri J 2013 Rad54 is required for the normal development of male and female germ cells and contributes to the maintainance of their genome integrity after genotoxic stress. Cell Death & Diseases 4 e774.
Moshkin YM, Armstrong JA, Maeda RK, Tamkun JW, Verrijzer P, Kennison JA & Karch F 2002 Histone chaperone ASF1 cooperates with the Brahma chromatin-remodelling machinery. Genetics & Development 16 2621–2626. (doi:10.1101/gad.231202)
Mousson F, Ochsenbein F & Mann C 2007 The histone chaperone Asf1 at the crossroads of chromatin and DNA checkpoint pathways. Chromosoma 116 79–93. (doi:10.1073/pnas.1106023109)
Nakamuta N & Kobayashi S 2007 Expression of p63 in the mouse ovary. Journal of Reproduction & Development 53 691–697.
Ohbo K, Yoshida S, Ohmura M, Ohneda O, Ogawa T, Tsuchiya H, Kuwana T, Kehler J, Abe K, Scholer HR & Suda T 2003 Identification and characterization of stem cells in prepubertal spermatogenesis in mice. Journal of Developmental Biology 258 209–225.
Osada S, Kurita M, Nishikawa J & Nishihara T 2005 Chromatin assembly factor Asf1p-dependent occupancy of the SAS histone acetyltransferase complex at the silent mating-type locus HMLalpha. Nucleic Acids Research 33 2742–2750. (doi:10.1093/nar/gki560)
Ozdemir A, Spicuglia S, Lasonder E, Vermeulen M, Campsteijn C, Stunnenberg HG & Logie C 2005 Characterization of lysine 56 of histone H3 as an acetylation site in Saccharomyces cerevisiae. Journal of Biological Chemistry 280 25949–25952. (doi:10.1074/jbc.C500181200)
Recht J, Tsubota T, Tanny JC, Diaz RL, Berger MJ, Zhang X, Garcia BA, Shabanowitz J, Burlingame AL, Hunt DF, Kaufman PD & Allis CD 2006 Histone chaperone Asf1 is required for histone H3 lysine 56 acetylation, a modification associated with S phase in mitosis and meiosis. Proceedings of the National Academy of Sciences U S A 103 6988–6993. (doi:10.1073/pnas.0601676103)
Rufiange A, Jacques PE, Bhat W, Robert F & Nourani A 2007 Genome-wide replication-independent histone H3 exchange occurs predominantly at promoters and implicates H3 K56 acetylation and Asf1. Molecular Cell 27 393–405. (doi:10.1016/j.molcel.2007.07.011)
Sanematsu F, Takami Y, Barman HK, Fukagawa T, Ono T, Shibahara K & Nakayama T 2006 Asf1 is required for viability and chromatin assembly during DNA replication in vertebrate cells. Journal of Biological Chemistry 281 13817–13827. (doi:10.1074/jbc.M511590200)
Sasaki H & Matsui Y 2008 Epigenetic events in mammalian germ-cell development: reprogramming and beyond. Nature Reviews Genetics 9 129–140.
Schneider J, Bajwa P, Johnson FC, Bhaumik SR & Shilatifard A 2006 Rtt109 is required for proper H3K56 acetylation: a chromatin mark associated with the elongating RNA polymerase II. Journal of Biological Chemistry 281 37270–37274. (doi:10.1074/jbc.C600265200)
Schrans-Stassen BH, van de Kant HJ, de Rooij DG & van Pelt AM 1999 Differential expression of c-kit in mouse undifferentiated and differentiating type A spermatogonia. Endocrinology 140 5894–5900. (doi:10.1210/endo.140.12.7172)
Schulz LL & Tyler JK 2006 The histone chaperone ASF1 localizes to active DNA replication forks to mediate efficient DNA replication. FASEB Journal 20 488–490. (doi:10.2527/jas.2015-9136)
Schwabish MA & Struhl K 2006 Asf1 mediates histone eviction and deposition during elongation by RNA polymerase II. Molecular Cell 22 415–422. (doi:10.1128/MCB.00717-07)
Stuhlmann H 2003 Gene trap vector screen for developmental genes in differentiating ES cells. Methods in Enzymology 365 386–406. (doi:10.1016/S0076-6879(03)65027-5)
Sutton A, Bucaria J, Osley MA & Sternglanz R 2001 Yeast ASF1 protein is required for cell cycle regulation of histone gene transcription. Genetics 158 587–596.
Tjeertes JV, Miller KM & Jackson SP 2009 Screen for DNA-damage-responsive histone modifications identifies H3K9Ac and H3K56Ac in human cells. EMBO Journal 28 1878–1889.
Tyler JK, Adams CR, Chen SR, Kobayashi R, Kamakaka RT & Kadonaga JT 1999 The RCAF complex mediates chromatin assembly during DNA replication and repair. Nature 402 555–560.
Tyler JK, Collins KA, Prasad-Sinha J, Amiott E, Bulger M, Harte PJ, Kobayashi R & Kadonaga JT 2001 Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors. Molecular Cell Biology 21 6574–6584. (doi:10.1128/MCB.21.19.6574-6584.2001)
Umehara T & Horikoshi M 2003 Transcription initiation factor IID-interactive histone chaperone CIA-II implicated in mammalian spermatogenesis. Journal of Biological Chemistry 278 35660–35667.
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M & Rozen SG 2012 Primer3–new capabilities and interfaces. Nucleic Acids Research 40 e115.
Vempati RK, Jayani RS, Notani D, Sengupta A, Galande S & Haldar D 2010 p300-mediated acetylation of histone H3 lysine 56 functions in DNA damage response in mammals. Joirnal of Biological Chemistry 285 28553–28564. (doi:10.1074/jbc.M110.149393)
Vernet M, Bonnerot C, Briand P & Nicolas JF 1993 Application of LacZ gene fusions to preimplantation development. Methods in Enzymology 225 434–451. (doi:10.1016/0076-6879(93)25030-6)
Xu F, Zhang K & Grunstein M 2005 Acetylation in histone H3 globular domain regulates gene expression in yeast. Cell 121 375–385.
Yoshimizu T, Sugiyama N, De Felice M, Yeom YI, Ohbo K, Masuko K, Obinata M, Abe K, Scholer HR & Matsui Y 1999 Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Development Growth & Differentiation 41 675–684.
Yuan J, Pu M, Zhang Z & Lou Z 2009 Histone H3-K56 acetylation is important for genomic stability in mammals. Cell Cycle 8 1747–1753. (doi:10.4161/cc.8.11.8620)
Zabaronick SR & Tyler JK 2005 The histone chaperone anti-silencing function 1 is a global regulator of transcription independent of passage through S phase. Molecular Cell Biology 25 652–660. (doi:10.1128/MCB.25.2.652-660.2005)
Zhang R, Poustovoitov MV, Ye X, Santos HA, Chen W, Daganzo SM, Erzberger JP, Serebriiskii IG, Canutescu AA, Dunbrack RL, Pehrson JR, Berger JM, Kaufman PD & Adams PD 2005 Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA. Development Cell 8 19–30.
Zhou H, Madden BJ, Muddiman DC & Zhang Z 2006 Chromatin assembly factor 1 interacts with histone H3 methylated at lysine 79 in the processes of epigenetic silencing and DNA repair. Biochemistry 45 2852–2861. (doi:10.1021/bi0521083)
