Hira-dependent histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells.

Polycomb repressive complex 2 (PRC2) regulates gene expression during lineage specification through trimethylation of lysine 27 on histone H3 (H3K27me3). In Drosophila, polycomb binding sites are dynamic chromatin regions enriched with the histone variant H3.3. Here, we show that, in mouse embryonic stem cells (ESCs), H3.3 is required for proper establishment of H3K27me3 at the promoters of developmentally regulated genes. Upon H3.3 depletion, these promoters show reduced nucleosome turnover measured by deposition of de novo synthesized histones and reduced PRC2 occupancy. Further, we show H3.3-dependent interaction of PRC2 with the histone chaperone, Hira, and that Hira localization to chromatin requires H3.3. Our data demonstrate the importance of H3.3 in maintaining a chromatin landscape in ESCs that is important for proper gene regulation during differentiation. Moreover, our findings support the emerging notion that H3.3 has multiple functions in distinct genomic locations that are not always correlated with an "active" chromatin state.

Distinct factors control histone variant H3.3 localization at specific genomic regions.

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.

The role of lipids in genome integrity and pluripotency.

Pluripotent stem cells (PSCs), comprising embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), offer immense potential for regenerative medicine due to their ability to differentiate into all cell types of the adult body. A critical aspect of harnessing this potential is understanding their metabolic requirements during derivation, maintenance, and differentiation in vitro. Traditional culture methods using fetal bovine serum often lead to issues such as heterogeneous cell populations and diminished pluripotency. Although the chemically-defined 2i/LIF medium has provided solutions to some of these challenges, prolonged culturing of these cells, especially female ESCs, raises concerns related to genome integrity. This review discusses the pivotal role of lipids in genome stability and pluripotency of stem cells. Notably, the introduction of lipid-rich albumin, AlbuMAX, into the 2i/LIF culture medium offers a promising avenue for enhancing the genomic stability and pluripotency of cultured ESCs. We further explore the unique characteristics of lipid-induced pluripotent stem cells (LIP-ESCs), emphasizing their potential in regenerative medicine and pluripotency research.

Sphingosine kinases protect murine embryonic stem cells from sphingosine-induced cell cycle arrest.

Sphingosine-1-phosphate (S1P) is a bioactive lipid molecule regulating organogenesis, angiogenesis, cell proliferation, and apoptosis. S1P is generated by sphingosine kinases (SPHK1 and SPHK2) through the phosphorylation of ceramide-derived sphingosine. Phenotypes caused by manipulating S1P metabolic enzymes and receptors suggested several possible functions for S1P in embryonic stem cells (ESCs), yet the mechanisms by which S1P and related sphingolipids act in ESCs are controversial. We designed a rigorous test to evaluate the requirement of S1P in murine ESCs by knocking out both Sphk1 and Sphk2 to create cells incapable of generating S1P. To accomplish this, we created lines mutant for Sphk2 and conditionally mutant (floxed) for Sphk1, allowing evaluation of ESCs that transition to double-null state. The Sphk1/2-null ESCs lack S1P and accumulate the precursor sphingosine. The double-mutant cells fail to grow due to a marked cell cycle arrest at G2/M. Mutant cells activate expression of telomere elongation factor genes Zscan4, Tcstv1, and Tcstv3 and display longer telomeric repeats. Adding exogenous S1P to the medium had no impact, but the cell cycle arrest is partially alleviated by the expression of a ceramide synthase 2, which converts excess sphingosine into ceramide. The results indicate that sphingosine kinase activity is essential in mouse ESCs for limiting the accumulation of sphingosine that otherwise drives cell cycle arrest.

Long noncoding RNAs sustain high expression levels of exogenous octamer-binding protein 4 by sponging regulatory microRNAs during cellular reprogramming.

Long noncoding RNAs (lncRNAs) modulate gene expression as competing endogenous RNAs (ceRNAs) that sponge regulatory microRNAs (miRNAs). During cellular reprogramming, genes associated with pluripotency establishment need to be up-regulated, and developmental genes need to be silenced. However, how ceRNAs control cellular reprogramming still awaits full elucidation. Here, we used doxycycline-inducible expression of the four transcription factors octamer-binding protein 4 (OCT4), SRY-box 2 (SOX2), Krüppel-like factor 4 (KLF4), and proto-oncogene c-Myc (c-Myc) to generate induced pluripotent stem cells (iPSCs) from mouse embryonic fibroblasts (MEFs). Using RNA-Seq and bioinformatics approaches, we found that the expression levels of miRNAs from MEFs remain high from day 0 to 6 after the doxycycline induction. Many genes targeted by these miRNAs were up-regulated, and long intergenic noncoding RNAs (lincRNAs) and circular RNAs (circRNAs), which have complementary binding sites to these miRNAs, were highly expressed, indicating lincRNAs and circRNAs may function as ceRNAs. Intriguingly, knockdown of the linc/circRNAs that sponge the miRNAs, which target OCT4 down-regulated exogenous OCT4, decreased reprogramming efficiency, and resulted in low-grade iPSCs. Our results suggest that the ceRNA network plays an important role in cellular reprogramming.

Histone variant H3.3-mediated chromatin remodeling is essential for paternal genome activation in mouse preimplantation embryos.

Derepression of chromatin-mediated transcriptional repression of paternal and maternal genomes is considered the first major step that initiates zygotic gene expression after fertilization. The histone variant H3.3 is present in both male and female gametes and is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA-tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different sources of H3.3 protein in embryos, we show here that sperm-derived H3.3 (sH3.3) protein is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PBII) during embryogenesis. We also found that the maternal H3.3 (mH3.3) protein is incorporated into the paternal genome as early as 2 h postfertilization and is detectable in the paternal genome until the morula stage. Knockdown of maternal H3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis.

Histone variant H3.3 is an essential maternal factor for oocyte reprogramming.

Mature oocyte cytoplasm can reprogram somatic cell nuclei to the pluripotent state through a series of sequential events including protein exchange between the donor nucleus and ooplasm, chromatin remodeling, and pluripotency gene reactivation. Maternal factors that are responsible for this reprogramming process remain largely unidentified. Here, we demonstrate that knockdown of histone variant H3.3 in mouse oocytes results in compromised reprogramming and down-regulation of key pluripotency genes; and this compromised reprogramming for developmental potentials and transcription of pluripotency genes can be rescued by injecting exogenous H3.3 mRNA, but not H3.2 mRNA, into oocytes in somatic cell nuclear transfer embryos. We show that maternal H3.3, and not H3.3 in the donor nucleus, is essential for successful reprogramming of somatic cell nucleus into the pluripotent state. Furthermore, H3.3 is involved in this reprogramming process by remodeling the donor nuclear chromatin through replacement of donor nucleus-derived H3 with de novo synthesized maternal H3.3 protein. Our study shows that H3.3 is a crucial maternal factor for oocyte reprogramming and provides a practical model to directly dissect the oocyte for its reprogramming capacity.

Genome editing a mouse locus encoding a variant histone, H3.3B, to report on its expression in live animals.

Chromatin remodeling via incorporation of histone variants plays a key role in the regulation of embryonic development. The histone variant H3.3 has been associated with a number of early events including formation of the paternal pronucleus upon fertilization. The small number of amino acid differences between H3.3 and its canonical counterparts (H3.1 and H3.2) has limited studies of the developmental significance of H3.3 deposition into chromatin due to difficulties in distinguishing the H3 isoforms. To this end, we used zinc-finger nuclease (ZFN) mediated gene editing to introduce a small C-terminal hemagglutinin (HA) tag to the endogenous H3.3B locus in mouse embryonic stem cells (ESCs), along with an internal ribosome entry site (IRES) and a separately translated fluorescent reporter of expression. This system will allow detection of expression driven by the reporter in cells, animals, and embryos, and will facilitate investigation of differential roles of paternal and maternal H3.3 protein during embryogenesis that would not be possible using variant-specific antibodies. Further, the ability to monitor endogenous H3.3 protein in various cell lineages will enhance our understanding of the dynamics of this histone variant over the course of development.

Dual role of lipids for genome stability and pluripotency facilitates full potency of mouse embryonic stem cells.

While Mek1/2 and Gsk3ß inhibition ("2i") supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of DNMT3s that prevent DNA hypomethylation. Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female "all-ESC" mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.

Altered chromatin occupancy of patient-associated H4 mutants misregulate neuronal differentiation.

Chromatin is a crucial regulator of gene expression and tightly controls development across species. Mutations in only one copy of multiple histone genes were identified in children with developmental disorders characterized by microcephaly, but their mechanistic roles in development remain unclear. Here we focus on dominant mutations affecting histone H4 lysine 91. These H4K91 mutants form aberrant nuclear puncta at specific heterochromatin regions. Mechanistically, H4K91 mutants demonstrate enhanced binding to the histone variant H3.3, and ablation of H3.3 or the H3.3-specific chaperone DAXX diminishes the mutant localization to chromatin. Our functional studies demonstrate that H4K91 mutant expression increases chromatin accessibility, alters developmental gene expression through accelerating pro-neural differentiation, and causes reduced mouse brain size in vivo, reminiscent of the microcephaly phenotypes of patients. Together, our studies unveil a distinct molecular pathogenic mechanism from other known histone mutants, where H4K91 mutants misregulate cell fate during development through abnormal genomic localization.

Generation of sex-reversed female clonal mice via CRISPR/Cas9-mediated Y chromosome deletion in male embryonic stem cells.

Mice derived entirely from embryonic stem (ES) cells can be generated through tetraploid complementation. Although XY male ES cell lines are commonly used in this system, occasionally, monosomic XO female mice are produced through spontaneous Y chromosome loss. Here, we describe an efficient method to obtain monosomic XO ES cells by CRISPR/Cas9-mediated deletion of the Y chromosome allowing generation of female clonal mice by tetraploid complementation. The monosomic XO female mice are viable and able to produce normal male and female offspring. Direct generation of clonal mice in both sexes can significantly accelerate the production of complex genetically modified mouse models.

Histone variant H3.3 maintains adult haematopoietic stem cell homeostasis by enforcing chromatin adaptability.

Histone variants and the associated post-translational modifications that govern the stemness of haematopoietic stem cells (HSCs) and differentiation thereof into progenitors (HSPCs) have not been well defined. H3.3 is a replication-independent H3 histone variant in mammalian systems that is enriched at both H3K4me3- and H3K27me3-marked bivalent genes as well as H3K9me3-marked endogenous retroviral repeats. Here we show that H3.3, but not its chaperone Hira, prevents premature HSC exhaustion and differentiation into granulocyte-macrophage progenitors. H3.3-null HSPCs display reduced expression of stemness and lineage-specific genes with a predominant gain of H3K27me3 marks at their promoter regions. Concomitantly, loss of H3.3 leads to a reduction of H3K9me3 marks at endogenous retroviral repeats, opening up binding sites for the interferon regulatory factor family of transcription factors, allowing the survival of rare, persisting H3.3-null HSCs. We propose a model whereby H3.3 maintains adult HSC stemness by safeguarding the delicate interplay between H3K27me3 and H3K9me3 marks, enforcing chromatin adaptability.

Breakthrough of interspecific chimerism: blocking apoptosis.

On January 28 in Nature online, Zheng et al. (Nature, 2021 (Online ahead of print)) reported that they developed an ingenious method of interspecies PSC co-culture system in vitro which unfolded interspecific cell competition. This study paves the way for discovering the mechanism of interspecific chimera and for further interspecific organogenesis between evolutionarily distant species.

Generation of Sex-Reversed Female Clonal Mice via CRISPR-Cas9-Mediated Y Chromosome Deletion in Male Embryonic Stem Cells.

Mice derived entirely from embryonic stem (ES) cells can be generated through tetraploid complementation. Although XY male ES cell lines are commonly used in this system, occasionally, monosomic XO female mice are produced through spontaneous Y chromosome loss. Here, we describe an efficient method to obtain monosomic XO ES cells by CRISPR-Cas9-mediated deletion of the Y chromosome, allowing generation of female clonal mice by tetraploid complementation. The monosomic XO female mice are viable and able to produce normal male and female offspring. Direct generation of clonal mice in both sexes can significantly accelerate the production of complex genetically modified mouse models.

Exogenous insulin-like growth factor 1 accelerates growth and maturation of follicles in human cortical xenografts and increases ovarian output in mice.

OBJECTIVE: To measure the influence of exogenous insulin-like growth factor 1 (IGF1) on follicle growth and maturation in human ovarian cortical xenografts. DESIGN: Xenotransplantation model. SETTING: University-based research laboratory. PATIENTS/ANIMALS: Ovarian tissue was donated with consent and institutional review board approval by brain-dead organ donors or patients undergoing ovarian tissue cryopreservation for fertility preservation. Cortical fragments were transplanted into immunocompromised mice. INTERVENTIONS: Cryopreserved ovarian cortical fragments from four women (aged 19, 25, 33, and 46 years) were transplanted into the gluteus muscle of immunocompromised mice in a fibrin matrix containing endothelial cells that were transduced with lentiviral particles encoding secreted IGF1. Xenografts were recovered after 3, 8, and 14 weeks. In addition, C57/Bl6 mice underwent intraovarian injection of saline or recombinant IGF1 (60 µg), followed by superovulation, analysis of ethynyl-deoxyuridine incorporation, and ribonucleic acid sequencing of the whole ovaries. MAIN OUTCOME MEASURES: For xenografts: follicle count and distribution; antral follicle count; and corpora lutea/albicans count. For mice: follicle count and distribution; oocyte yield, ethynyl-deoxyuridine incorporation (granulosa cell proliferation); and ovarian transcriptomic signature. RESULTS: At 3 weeks, xenografts in the IGF1 condition revealed a decreased percentage of primary follicles and increased percentage of secondary follicles that were concentrated in the preantral subtype; at 8 weeks, an increase in secondary follicles was concentrated in the simple subtype; after 14 weeks, primordial follicles were reduced, and while the number of advanced follicles did not power the experiment to demonstrate significance, antral follicles reduced and corpora lutea increased. Supporting experiments in mice revealed an increase in normal oocytes following intraovarian injection of recombinant IGF1 (60 µg) as well as increased proliferative index among follicles of secondary and preantral stages. Ribonucleic acid sequencing analysis of the whole ovaries following injection of recombinant IGF1 (25 µg) revealed an acute (24 hours) upregulation of transcripts related to steroidogenesis and luteinization. CONCLUSIONS: Exogenous IGF1 advances the pace of growth among primordial, primary, and secondary stage follicles but results in near absence of antral stage follicles in long-term (14 weeks) xenografts. In mice, acute administration of IGF1 promotes follicle advance and increased oocyte yield. The results suggest that while superphysiological IGF1 alone advances the pace of growth among early/preantral follicles, a sustained and/or later-stage influence undermines antral follicle growth/survival or promotes premature luteinization. These findings provide a temporal framework for interpreting follicle growth/mobilization and may be useful in understanding the clinical application of human growth hormone in the context of assisted reproduction.

CD34+ testicular stromal cells support long-term expansion of embryonic and adult stem and progenitor cells.

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multipotent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. Disclosure of potential conflicts of interest is found at the end of this article.

Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity.

A robust network of transcription factors and an open chromatin landscape are hallmarks of the naive pluripotent state. Recently, the acetyllysine reader Brd4 has been implicated in stem cell maintenance, but the relative contribution of Brd4 to pluripotency remains unclear. Here, we show that Brd4 is dispensable for self-renewal and pluripotency of embryonic stem cells (ESCs). When maintained in their ground state, ESCs retain transcription factor binding and chromatin accessibility independent of Brd4 function or expression. In metastable ESCs, Brd4 independence can be achieved by increased expression of pluripotency transcription factors, including STAT3, Nanog or Klf4, so long as the DNA methylcytosine oxidases Tet1 and Tet2 are present. These data reveal that Brd4 is not essential for ESC self-renewal. Rather, the levels of pluripotency transcription factor abundance and Tet1/2 function determine the extent to which bromodomain recognition of protein acetylation contributes to the maintenance of gene expression and cell identity.

Activated ovarian endothelial cells promote early follicular development and survival.

BACKGROUND: New data suggests that endothelial cells (ECs) elaborate essential "angiocrine factors". The aim of this study is to investigate the role of activated ovarian endothelial cells in early in-vitro follicular development. METHODS: Mouse ovarian ECs were isolated using magnetic cell sorting or by FACS and cultured in serum free media. After a constitutive activation of the Akt pathway was initiated, early follicles (50-150 um) were mechanically isolated from 8-day-old mice and co-cultured with these activated ovarian endothelial cells (AOEC) (n = 32), gel (n = 24) or within matrigel (n = 27) in serum free media for 14 days. Follicular growth, survival and function were assessed. RESULTS: After 6 passages, flow cytometry showed 93% of cells grown in serum-free culture were VE-cadherin positive, CD-31 positive and CD 45 negative, matching the known EC profile. Beginning on day 4 of culture, we observed significantly higher follicular and oocyte growth rates in follicles co-cultured with AOECs compared with follicles on gel or matrigel. After 14 days of culture, 73% of primary follicles and 83% of secondary follicles co-cultured with AOEC survived, whereas the majority of follicles cultured on gel or matrigel underwent atresia. CONCLUSIONS: This is the first report of successful isolation and culture of ovarian ECs. We suggest that co-culture with activated ovarian ECs promotes early follicular development and survival. This model is a novel platform for the in vitro maturation of early follicles and for the future exploration of endothelial-follicular communication. CAPSULE: In vitro development of early follicles necessitates a complex interplay of growth factors and signals required for development. Endothelial cells (ECs) may elaborate essential "angiocrine factors" involved in organ regeneration. We demonstrate that co-culture with ovarian ECs enables culture of primary and early secondary mouse ovarian follicles.

Epigenetic profiles signify cell fate plasticity in unipotent spermatogonial stem and progenitor cells.

Spermatogonial stem and progenitor cells (SSCs) generate adult male gametes. During in vitro expansion, these unipotent murine cells spontaneously convert to multipotent adult spermatogonial-derived stem cells (MASCs). Here we investigate this conversion process through integrative transcriptomic and epigenomic analyses. We find in SSCs that promoters essential to maintenance and differentiation of embryonic stem cells (ESCs) are enriched with histone H3-lysine4 and -lysine 27 trimethylations. These bivalent modifications are maintained at most somatic promoters after conversion, bestowing MASCs an ESC-like promoter chromatin. At enhancers, the core pluripotency circuitry is activated partially in SSCs and completely in MASCs, concomitant with loss of germ cell-specific gene expression and initiation of embryonic-like programs. Furthermore, SSCs in vitro maintain the epigenomic characteristics of germ cells in vivo. Our observations suggest that SSCs encode innate plasticity through the epigenome and that both conversion of promoter chromatin states and activation of cell type-specific enhancers are prominent features of reprogramming.