Somatic Chromatin Research Articles

Cohesin and CTCF do not assemble TADs in Xenopus sperm and male pronuclei.

Paternal genomes are compacted during spermiogenesis and decompacted following fertilization. These processes are fundamental for inheritance but incompletely understood. We analyzed these processes in the frog Xenopus laevis, whose sperm can be assembled into functional pronuclei in egg extracts in vitro. In such extracts, cohesin extrudes DNA into loops, but in vivo cohesin only assembles topologically associating domains (TADs) at the mid-blastula transition (MBT). Why cohesin assembles TADs only at this stage is unknown. We first analyzed genome architecture in frog sperm and compared it to human and mouse. Our results indicate that sperm genome organization is conserved between frogs and humans and occurs without formation of TADs. TADs can be detected in mouse sperm samples, as reported, but these structures might originate from somatic chromatin contaminations. We therefore discuss the possibility that the absence of TADs might be a general feature of vertebrate sperm. To analyze sperm genome remodeling upon fertilization, we reconstituted male pronuclei in Xenopus egg extracts. In pronuclei, chromatin compartmentalization increases, but cohesin does not accumulate at CTCF sites and assemble TADs. However, if pronuclei are formed in the presence of exogenous CTCF, CTCF binds to its consensus sites, and cohesin accumulates at these and forms short-range chromatin loops, which are preferentially anchored at CTCF's N terminus. These results indicate that TADs are only assembled at MBT because before this stage CTCF sites are not occupied and cohesin only forms short-range chromatin loops.

Impact of genome and epigenome on intratumor heterogeneity in colorectal cancer

Impact of genome and epigenome on intratumor heterogeneity in colorectal cancer

Hydrozoan sperm-specific SPKK motif-containing histone H2B variants stabilise chromatin with limited compaction.

Many animals achieve sperm chromatin compaction and stabilisation by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines during spermatogenesis. Hydrozoan cnidarians and echinoid sea urchins lack protamines and have evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N termini rich in SPK(K/R) motifs. Echinoid sperm packaging is regulated by spH2Bs. Their sperm is negatively buoyant and fertilises on the sea floor. Hydroid cnidarians undertake broadcast spawning but their sperm properties are poorly characterised. We show that Hydractinia echinata and H. symbiolongicarpus sperm chromatin possesses higher stability than somatic chromatin, with reduced accessibility to transposase Tn5 integration and to endonucleases in vitro. In contrast, nuclear dimensions are only moderately reduced in mature Hydractinia sperm. Ectopic expression of spH2B in the background of H2B.1 knockdown results in downregulation of global transcription and cell cycle arrest in embryos, without altering their nuclear density. Taken together, SPKK-containing spH2B variants act to stabilise chromatin and silence transcription in Hydractinia sperm with only limited chromatin compaction. We suggest that spH2Bs could contribute to sperm buoyancy as a reproductive adaptation.

The co-evolution of the genome and epigenome in colorectal cancer

Colorectal malignancies are a leading cause of cancer-related death1 and have undergone extensive genomic study2,3. However, DNA mutations alone do not fully explain malignant transformation4–7. Here we investigate the co-evolution of the genome and epigenome of colorectal tumours at single-clone resolution using spatial multi-omic profiling of individual glands. We collected 1,370 samples from 30 primary cancers and 8 concomitant adenomas and generated 1,207 chromatin accessibility profiles, 527 whole genomes and 297 whole transcriptomes. We found positive selection for DNA mutations in chromatin modifier genes and recurrent somatic chromatin accessibility alterations, including in regulatory regions of cancer driver genes that were otherwise devoid of genetic mutations. Genome-wide alterations in accessibility for transcription factor binding involved CTCF, downregulation of interferon and increased accessibility for SOX and HOX transcription factor families, suggesting the involvement of developmental genes during tumourigenesis. Somatic chromatin accessibility alterations were heritable and distinguished adenomas from cancers. Mutational signature analysis showed that the epigenome in turn influences the accumulation of DNA mutations. This study provides a map of genetic and epigenetic tumour heterogeneity, with fundamental implications for understanding colorectal cancer biology.

Terpyridine Zn(II) Complexes with Azide Units for Visualization of Histone Deacetylation in Living Cells under STED Nanoscopy.

Histones are the alkali proteins in eukaryotic somatic chromatin cells which constitute the nucleosome structure together with DNA. Their abnormality is often associated with multiple tumorigenesis and other human diseases. Nevertheless, a simple and efficient super-resolution method to visualize histone distribution at the subcellular level is still unavailable. Herein, a Zn(II) terpyridine complex with rich-electronic azide units, namely, TpZnA-His, was designed and synthesized. The initial in vitro and in silico studies suggested that this complex is able to detect histones rapidly and selectively via charge-charge interactions with the histone H3 subunit. Its live cell nuclear localization, red-emission tail, and large Stokes shift allowed super-resolution evaluation of histone distributions with a clear distinction against nuclear DNA. We were able to quantitatively conclude three histone morphology alternations in live cells including condensation, aggregation, and cavity during activating histone acetylation. This work offers a better understanding as well as a versatile tool to study histone-involved gene transcription, signal transduction, and differentiation in cells.

Analysis of Genome Architecture during SCNT Reveals a Role of Cohesin in Impeding Minor ZGA

Somatic cell nuclear transfer (SCNT) can reprogram a somatic nucleus to a totipotent state. However, the re-organization of 3D chromatin structure in this process remains poorly understood. Using low-input Hi-C, we revealed that, during SCNT, the transferred nucleus first enters a mitotic-like state (premature chromatin condensation). Unlike fertilized embryos, SCNT embryos show stronger topologically associating domains (TADs) at the 1-cell stage. TADs become weaker at the 2-cell stage, followed by gradual consolidation. Compartments A/B are markedly weak in 1-cell SCNT embryos and become increasingly strengthened afterward. By the 8-cell stage, somatic chromatin architecture is largely reset to embryonic patterns. Unexpectedly, we found cohesin represses minor zygotic genome activation (ZGA) genes (2-cell-specific genes) in pluripotent and differentiated cells, and pre-depleting cohesin in donor cells facilitates minor ZGA and SCNT. These data reveal multi-step reprogramming of 3D chromatin architecture during SCNT and support dual roles of cohesin in TAD formation and minor ZGA repression.

SSRP1-mediated histone H1 eviction promotes replication origin assembly and accelerated development

In several metazoans, the number of active replication origins in embryonic nuclei is higher than in somatic ones, ensuring rapid genome duplication during synchronous embryonic cell divisions. High replication origin density can be restored by somatic nuclear reprogramming. However, mechanisms underlying high replication origin density formation coupled to rapid cell cycles are poorly understood. Here, using Xenopus laevis, we show that SSRP1 stimulates replication origin assembly on somatic chromatin by promoting eviction of histone H1 through its N-terminal domain. Histone H1 removal derepresses ORC and MCM chromatin binding, allowing efficient replication origin assembly. SSRP1 protein decays at mid-blastula transition (MBT) when asynchronous somatic cell cycles start. Increasing levels of SSRP1 delay MBT and, surprisingly, accelerate post-MBT cell cycle speed and embryo development. These findings identify a major epigenetic mechanism regulating DNA replication and directly linking replication origin assembly, cell cycle duration and embryo development in vertebrates.

Heterochromatin formation in Drosophila requires genome-wide histone deacetylation in cleavage chromatin before mid-blastula transition in early embryogenesis

Su(var) mutations define epigenetic factors controlling heterochromatin formation and gene silencing in Drosophila. Here, we identify SU(VAR)2-1 as a novel chromatin regulator that directs global histone deacetylation during the transition of cleavage chromatin into somatic blastoderm chromatin in early embryogenesis. SU(VAR)2-1 is heterochromatin-associated in blastoderm nuclei but not in later stages of development. In larval polytene chromosomes, SU(VAR)2-1 is a band-specific protein. SU(VAR)2-1 directs global histone deacetylation by recruiting the histone deacetylase RPD3. In Su(var)2-1 mutants H3K9, H3K27, H4K8 and H4K16 acetylation shows elevated levels genome-wide and heterochromatin displays aberrant histone hyper-acetylation. Whereas H3K9me2- and HP1a-binding appears unaltered, the heterochromatin-specific H3K9me2S10ph composite mark is impaired in heterochromatic chromocenters of larval salivary polytene chromosomes. SU(VAR)2-1 contains an NRF1/EWG domain and a C2HC zinc-finger motif. Our study identifies SU(VAR)2-1 as a dosage-dependent, heterochromatin-initiating SU(VAR) factor, where the SU(VAR)2-1-mediated control of genome-wide histone deacetylation after cleavage and before mid-blastula transition (pre-MBT) is required to enable heterochromatin formation.

Genetic Instability and Chromatin Remodeling in Spermatids.

The near complete replacement of somatic chromatin in spermatids is, perhaps, the most striking nuclear event known to the eukaryotic domain. The process is far from being fully understood, but research has nevertheless unraveled its complexity as an expression of histone variants and post-translational modifications that must be finely orchestrated to promote the DNA topological change and compaction provided by the deposition of protamines. That this major transition may not be genetically inert came from early observations that transient DNA strand breaks were detected in situ at chromatin remodeling steps. The potential for genetic instability was later emphasized by our demonstration that a significant number of DNA double-strand breaks (DSBs) are formed and then repaired in the haploid context of spermatids. The detection of DNA breaks by 3′OH end labeling in the whole population of spermatids suggests that a reversible enzymatic process is involved, which differs from canonical apoptosis. We have set the stage for a better characterization of the genetic impact of this transition by showing that post-meiotic DNA fragmentation is conserved from human to yeast, and by providing tools for the initial mapping of the genome-wide DSB distribution in the mouse model. Hence, the molecular mechanism of post-meiotic DSB formation and repair in spermatids may prove to be a significant component of the well-known male mutation bias. Based on our recent observations and a survey of the literature, we propose that the chromatin remodeling in spermatids offers a proper context for the induction of de novo polymorphism and structural variations that can be transmitted to the next generation.

18 Characterisation of early embryonic cellular defects after somatic cell nuclear transfer in fish

Cryopreservation of genetic resources is of major interest for the security of biodiversity and the sustainability of the agronomic industry. Cryopreservation of somatic cells is one means of preserving the genome of both parents, but regeneration of functional breeders requires the mastering of somatic cell NT (SCNT). In mammals, SCNT consists of injecting the somatic nuclei into enucleated recipient oocytes to obtain clones that bear the genome of the donor. Cloning in fish presents some advantages over cloning in mammals: oocytes are produced by the hundreds, and development is external. Moreover, embryonic genome activation takes place after 10 mitoses only. These features are favourable to a better understanding of cellular reprogramming of the somatic cell. Nonenucleated oocytes are often used for SCNT, which allows the production of 2n clones bearing only the donor genome when the maternal chromatin is spontaneously removed by an unknown mechanism (25% of cases). This ability can help us understand the fate of the maternal chromatin and the role of its surrounding factors on the cellular behaviour of the somatic cell during the first developmental steps of embryonic development: meiosis resumption (MR) and mitosis. In this study, fin somatic cells and mature oocytes were obtained from a 2-year-old goldfish (Carassius auratus). For SCNT, the whole fin cell was injected into the sperm entry site, and the oocyte was activated by water contact after 30min to begin the development. Somatic and maternal chromatin behaviour during MR and early mitosis were characterised by immunofluorescence (Hoechst/Vybrant labelling for chromatin identification and α-tubulin for spindle organisation). Variability in chromatin condensation was observed after the somatic cell injection before the activation. Clone analysis (n=16) revealed that some of them presented a condensed somatic nuclei (71%) and others presented a decondensed nuclei (29%). After activation, most clones underwent normal polar body extrusion of the maternal chromatin (66%, n=69/95 clones v. 96%, n=96/100 controls oocytes) without extrusion of a somatic polar body. Afterward, clones that presented a first symmetric division (n=14/35) were analysed at the 2-cells stage and compared with fertilized embryos (n=7). The maternal chromatin was observed as fragmented on the cleavage furrow (79%), where it cannot contribute to the development. Spindle defects were observed in 50% of clone cells (v. 0% in controls), such as multicentrosomal spindles, chromosome misalignment, abnormal segregation, and DNA fragmentation. To conclude, those clone (2n or 3n) defects are probably due to the chromatin condensation observed before activation. The fish oocyte volume did not allow the MR of the condensed somatic chromatin, and that may induce an abnormal anaphase and the following clone developmental defects.

Chromatin remodeling in Drosophila preblastodermic embryo extract

Chromatin is known to undergo extensive remodeling during nuclear reprogramming. However, the factors and mechanisms involved in this remodeling are still poorly understood and current experimental approaches to study it are not best suited for molecular and genetic analyses. Here we report on the use of Drosophila preblastodermic embryo extracts (DREX) in chromatin remodeling experiments. Our results show that incubation of somatic nuclei in DREX induces changes in chromatin organization similar to those associated with nuclear reprogramming, such as rapid binding of the germline specific linker histone dBigH1 variant to somatic chromatin, heterochromatin reorganization, changes in the epigenetic state of chromatin, and nuclear lamin disassembly. These results raise the possibility of using the powerful tools of Drosophila genetics for the analysis of chromatin changes associated with this essential process.

Abstract 2354: Candidate non-coding driver mutations in super-enhancers and long-range chromatin interaction networks across 1,800 whole cancer genomes

Abstract A comprehensive understanding of the mutations that drive tumorigenesis and disease progression is essential to understanding tumor biology and designing precision therapies. The landscape of driver mutations in the protein-coding genome has been well-characterized by large exome-sequencing studies. Surprisingly, many tumors do not have mutations in any known protein-coding driver. Non-coding driver mutations are hypothesized to explain many of these cases, but aside from a few regions, like the TERT promoter, our understanding of drivers in the complex regulatory genome remains limited. To fill this gap, we analyzed 150,000 cis-regulatory modules with clustered transcription factor binding sites in 1,844 whole cancer genomes from the ICGC-TCGA PCAWG project. Using a new statistical method, ActiveDriverWGS, we identified dozens of frequently mutated regulatory elements (FMREs) enriched in non-coding SNVs and indels (FDR<0.05) with many structural rearrangements and focal copy number alterations in additional samples. The FMREs were enriched in super-enhancers, long-range chromatin interactions and H3K27ac marks derived from primary tumors, suggesting a gene regulatory role of these mutations through three-dimensional genome organization. The interaction network of chromatin loops and FMREs revealed putative target genes located dozens to hundreds of kilobases away from the mutated regulatory elements. We found known and putative oncogenes and tumor suppressors whose expression significantly correlated with mutations in FMREs, suggesting novel oncogenic mechanisms. Most of the FMREs were also confirmed by additional driver discovery methods, lending confidence to our statistical approach. We also validated ActiveDriverWGS on protein-coding sequence and accurately recovered known driver genes. The non-coding regulatory genome is characterized by diverse mutational processes, regional hypermutations and technically challenging areas with suboptimal sequencing coverage. Thus our findings, most of which are reported for the first time, should be carefully vetted and experimentally validated in future studies. Our integrative analysis of somatic mutations, cis-regulatory regions and long-range chromatin interaction networks is a novel framework for cancer discovery and reveals the currently largest set of potential non-coding drivers in a pan-cancer cohort. Citation Format: Juri Reimand. Candidate non-coding driver mutations in super-enhancers and long-range chromatin interaction networks across 1,800 whole cancer genomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2354.

The Influence of Interspecies Somatic Cell Nuclear Transfer on Epigenetic Enzymes Transcription in Early Embryos

Abstract One of the main reason for the incorrect development of embryos derived from somatic cell nuclear transfer is caused by insufficient demethylation of injected somatic chromatin to a state comparable with an early embryonic nucleus. It is already known that the epigenetic enzymes transcription in oocytes and early embryos of several species including bovine and porcine zygotes is species-dependent process and the incomplete DNA methylation correlates with the nuclear transfer failure rate in mammals. In this study the transcription of DNA methyltransferase 1 and 3a (DNMT1, DNMT3a) genes in early embryonic stages of interspecies (bovine, porcine) nuclear transfer embryos (iSCNT) by RT-PCR were analyzed. Coming out from the diverse timing of embryonic genome activation (EGA) in porcine and bovine preimplantation embryos, the intense effect of ooplasm on transferred somatic cell nucleus was expected. In spite of the detection of ooplasmic DNA methyltransferases, the somatic genes for DNMT1 and DNMT3a enzymes were not expressed and the development of intergeneric embryos stopped at the 4-cell stage. Our results indicate that the epigenetic reprogramming during early mammalian development is strongly influenced by the ooplasmic environment.

Parp1 Deficiency Confers Defects in Chromatin Surveillance and Remodeling During Reprogramming by Nuclear Transfer.

Accumulating evidence suggests that cloned mice production by the injection of a somatic cell nucleus into an enucleated oocyte is inefficient. DNA damage and chromatin remodeling failures that occur during embryogenesis following nuclear transfer (NT) might explain the poor development of cloned embryos. To avoid these problems, it is important to elucidate somatic chromatin remodeling after NT. Because polyADP-ribosylation, which is catalyzed mainly by poly(ADP-ribose) polymerase 1 (Parp1), is a major post-translational modification that facilitates DNA repair and chromatin remodeling, we examined the effects of Parp1 deficiency in developing NT embryos. Parp1 was located within the pseudo-pronuclei (PPN) of NT eggs. We observed that NT eggs, after activation by Sr2+, formed PPN with significantly more efficiency in Parp1-null embryos than in wild-type NT embryos. However, most the Parp1-null embryos stopped developing by the four-cell stage. Immunostaining for γH2AX foci, a marker of DNA double strand breaks, showed longer retention in the PPN of Parp1-/- donor NT embryos than in wild-type NT embryos, suggesting that, in the absence of Parp1, DNA breaks are slowly repaired and consequently, entry into the S phase might be delayed. Furthermore, decreases in histone H3 acetylation, H3 monomethylation at lysine 4, and H3 trimethylation at lysine 27 after the Sr2+ activation step were observed in the PPN of Parp1-/- donor embryos. Taken together, our data suggest that Parp1 is involved in the plastic remodeling of chromatin structure after NT by supporting DNA repair and specific histone code modifications.

Mutations in DNMT3B Modify Epigenetic Repression of the D4Z4 Repeat and the Penetrance of Facioscapulohumeral Dystrophy

Facioscapulohumeral dystrophy (FSHD) is associated with somatic chromatin relaxation of the D4Z4 repeat array and derepression of the D4Z4-encoded DUX4 retrogene coding for a germline transcription factor. Somatic DUX4 derepression is caused either by a 1-10 unit repeat-array contraction (FSHD1) or by mutations in SMCHD1, which encodes a chromatin repressor that binds to D4Z4 (FSHD2). Here, we show that heterozygous mutations in DNA methyltransferase 3B (DNMT3B) are a likely cause of D4Z4 derepression associated with low levels of DUX4 expression from the D4Z4 repeat and increased penetrance of FSHD. Recessive mutations in DNMT3B were previously shown to cause immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome. This study suggests that transcription of DUX4 in somatic cells is modified by variations in its epigenetic state and provides a basis for understanding the reduced penetrance of FSHD within families.

42 CONVERSION OF THE CHROMATIN OF SOMATIC CELLS INTO SPERMATID-LIKE STRUCTURES

The post-meiotic phase of spermatogenesis is characterised by a radical reorganization of the chromatin, leading to its nucleosomal to toroid transition. The replacement of histones with protamine is a gradual process regulated to the hierarchical translation of repressed mRNAs leading to the following events: incorporation of testis-specific histone variants and general histone hyperacetylation, bromodomain proteins, transition proteins, concluded by protamine incorporation on DNA. In this work, we tested whether the induced expression of human protamine 1 (PR1) in sheep somatic cells could induce a protamine/toroid conformation of interphase nuclei. Sheep adult fibroblasts (SAF) were cultured in DMEM with 10% fetal bovine serum from second to eighth passage. Then, SAF at 80% confluence were transfected with 3 μg of pPR1-red fluorescent protein (RFP) and pRFP (CTR) with lipofectamine. At 4 h post-transfection, cells were treated with 5 nM trichostatin A (TSA; histone deacethylase inhibitor) for an additional 16 h. Transfected cells (visualised through the RFP tag) were analysed for nuclear morphology (transmission electron microscopy), PR1-RFP expression (confocal microscope, RT-PCR, Western blot), cytofluorimeter, DNA damage (comet assay, pH2A.X immune-detection), and chromatin immune-precipitation assays (ChIP). Moreover, to visualise the histone/protamine exchange, we transfected with PR1 mouse GFP-H2B fibroblasts. Protaminized cells were used as donors for nuclear transfer (NT) and TH2B (testis/oocyte-specific histone normally present in male pronucleus, a marker of reprogramming) was detected in pronuclear stage of NT zygotes. The χ2 test was used for statistical analyses. We demonstrated that PR1 translocates into the nuclei and gradually compacts them into elongating spermatid-like structures in 48 h; TSA treatment facilitates the process [TSA 83.3% (50/60); without TSA 55.2%; 58/105; P < 0.0003]. A complete histone-to-protamine exchange was also visualised in GFP-H2B nuclei (mouse fibroblasts) 40h after PR1 transfection. Cytofluorimetric analysis demonstrated that protamine incorporation occurs in any cell cycle stage and without DNA breaks. Next, protamine-protamine binding was excluded by ChIP analysis, which confirmed protamine-DNA binding. Finally, protaminized nuclei transplanted into enucleated oocytes incorporated maternal histone TH2B (3/8), whereas no signal was detected in control cells (0/9), suggesting that protaminized nuclei are better remodelled. We conclude that the induced expression of PR1 forces the somatic chromatin to acquire a structure overlapping elongated spermatid/spermatozoa, a conformation that perfectly matches the nuclear reprogramming machinery of the oocyte. Further work will determine whether protaminized cells are better reprogrammed upon nuclear transfer.

Hierarchical Molecular Events Driven by Oocyte-Specific Factors Lead to Rapid and Extensive Reprogramming

SummaryNuclear transfer to oocytes is an efficient way to transcriptionally reprogram somatic nuclei, but its mechanisms remain unclear. Here, we identify a sequence of molecular events that leads to rapid transcriptional reprogramming of somatic nuclei after transplantation to Xenopus oocytes. RNA-seq analyses reveal that reprogramming by oocytes results in a selective switch in transcription toward an oocyte rather than pluripotent type, without requiring new protein synthesis. Time-course analyses at the single-nucleus level show that transcriptional reprogramming is induced in most transplanted nuclei in a highly hierarchical manner. We demonstrate that an extensive exchange of somatic- for oocyte-specific factors mediates reprogramming and leads to robust oocyte RNA polymerase II binding and phosphorylation on transplanted chromatin. Moreover, genome-wide binding of oocyte-specific linker histone B4 supports its role in transcriptional reprogramming. Thus, our study reveals the rapid, abundant, and stepwise loading of oocyte-specific factors onto somatic chromatin as important determinants for successful reprogramming.

Conserved Asf1–importin β physical interaction in growth and sexual development in the ciliate Tetrahymena thermophila

How the eukaryotic cell specifies distinct chromatin domains is a central problem in molecular biology. The ciliate protozoan Tetrahymena thermophila features a separation of structurally and functionally distinct germ-line and somatic chromatin into two distinct nuclei, the micronucleus (MIC) and macronucleus (MAC) respectively. To address questions about how distinct chromatin states are assembled in the MAC and MIC, we have initiated studies to define protein-protein interactions for T. thermophila chromatin-related proteins. Affinity purification followed by mass spectrometry analysis of the conserved Asf1 histone chaperone in T. thermophila revealed that it forms a complex with an importin β, ImpB6. Furthermore, these proteins co-localized to both the MAC and MIC in growth and development. We suggest that newly synthesized histones H3 and H4 in T. thermophila are transported via Asf1-ImpB6 in an evolutionarily conserved pathway to both nuclei where they then enter nucleus-specific chromatin assembly pathways. These studies set the stage for further use of functional proteomics to elucidate details of the characterization and functional analysis of the unique chromatin domains in T. thermophila. Asf1 is an evolutionarily conserved chaperone of H3 and H4 histones that functions in replication dependent and independent chromatin assembly. Although Asf1 has been well studied in humans and yeast (members of the Opisthokonta lineage of eukaryotes), questions remain concerning its mechanism of function. To obtain additional insight into the Asf1 function we have initiated a proteomic analysis in the ciliate protozoan T. thermophila, a member of the Alveolata lineage of eukaryotes. Our results suggest that an evolutionarily conserved function of Asf1 is mediating the nuclear transport of newly synthesized histones H3 and H4.

SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis

SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc1−/− animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at >20 weeks of age) was preceded by a transient increase in the number of undifferentiated Aaligned (Aal) spermatogonia in younger mice (at >10 weeks of age). The number of primary Spoc1−/− gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis was normal, but we noted a significantly elevated level of apoptosis in the Spoc1−/− testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.

SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis

SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc1(-/-) animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at >20 weeks of age) was preceded by a transient increase in the number of undifferentiated A(aligned) (A(al)) spermatogonia in younger mice (at >10 weeks of age). The number of primary Spoc1(-/-) gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis was normal, but we noted a significantly elevated level of apoptosis in the Spoc1(-/-) testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.