Epidermal growth factor represses differentiation of mouse trophoblast stem cells into spongiotrophoblast cells via epidermal growth factor receptor.

The mouse placenta is composed of three different trophoblast layers that are occupied by particular trophoblast subtypes to maintain placental function and pregnancy. Accurate control of trophoblast differentiation is required for proper placental function; however, the molecular mechanisms underlying cell fate decisions in trophoblast stem cells remain poorly understood. Epidermal growth factor (EGF) signaling is involved in multiple biological processes including cell survival, proliferation, and differentiation. The effect of EGF on trophoblast function has been reported in various species; however, the role of EGF signaling in mouse trophoblast specification remains unclear. In this study, we aimed to elucidate the role of EGF signaling in mouse trophoblast differentiation using mouse trophoblast stem cells (mTSCs) in an in vitro culture system. EGF stimulation at the early stage of differentiation repressed mTSC differentiation into spongiotrophoblast cells (SpT). Gene deletion and inhibitor experiments showed that the effect of EGF exposure went through epidermal growth factor receptor (Egfr) activity in mTSCs. EGF stimuli induced acute downstream activation of MAPK/ERK, PI3K/AKT, and JNK pathways, and inhibition of the MAPK/ERK pathway, but not others, alleviated EGF-mediated repression of SpT differentiation. Moreover, expression of Mash2, a master regulator of SpT differentiation, was repressed by EGF stimulation, and MAPK/ERK inhibition counteracted this repression. The Mash2 overexpression recovered SpT marker expression, indicating that the decrease in Mash2 expression was due to abnormal SpT differentiation in EGF-treated mTSCs. Our findings suggest that the EGF-Egfr-MAPK/ERK-Mash2 axis is a core regulatory mechanism for the EGF-mediated repression of SpT differentiation.

N-Oleoyldopamine promotes the differentiation of mouse trophoblast stem cells into parietal trophoblast giant cells.

The placenta plays various roles in a healthy pregnancy, and abnormalities in the placenta result in adverse outcomes. Adequate differentiation of trophoblast subtypes is necessary for placental function, but the molecular mechanisms that determine trophoblast cell fate remain unclear. Here, we screened small molecular compound (SMC) libraries (1904 SMCs) to identify particular SMCs which regulate trophoblast differentiation in mouse trophoblast stem cells (mTSCs) to understand the molecular mechanisms underlying cell fate decision in trophoblast cells. The two-step screening revealed a novel effect of N-oleoyldopamine (OLDA), an endogenic vanilloid, to promote differentiation into parietal trophoblast giant cells (P-TGCs) and repress them into spongiotrophoblast cells in mTSCs. Analyses by gene deletion and inhibitor treatments indicated that transient receptor potential cation channel subfamily V member 3 (Trpv3), one of the candidates for targeting by OLDA, was involved in maintaining stem status and P-TGC differentiation in mTSCs. Finally, transcriptome analysis revealed that Fosl1, a key regulatory factor in differentiation into P-TGCs, was upregulated by OLDA treatment, suggesting that OLDA promoted the differentiation of mTSCs into P-TGCs via regulation of Fosl1 expression.

Oocyte-specific linker histone H1foo interacts with Esrrb to induce chromatin decondensation at specific gene loci.

Linker histone H1 is mainly localized in the linker DNA region, between two nucleosome cores, and regulates chromatin structures linking gene expression. Mammalian oocytes contain the histone H1foo, a distinct member with low sequence similarity to other members in the H1 histone family. Although, from various previous studies, evidence related to H1foo function in chromatin structures is being accumulated, the distribution of H1foo at the target gene loci in a genome-wide manner and the molecular mechanism of H1foo-dependent chromatin architecture remain unclear. In this study, we aimed to identify the target loci and the physiological factor bound to H1foo at the loci. Chromatin immunoprecipitation sequencing analysis of H1foo-overexpressing mouse embryonic stem cells showed that H1foo is enriched around the transcriptional start sites of genes such as oocyte-specific genes and that the chromatin structures at these regions were relaxed. We demonstrated that H1foo was physiologically bound to the nuclear receptor estrogen-related receptor beta (Esrrb), and Esrrb was necessary for H1foo activity of chromatin decondensation at the target loci. The specific localization and interaction with Esrrb were validated in endogenous H1foo of oocytes. Overall, H1foo induces chromatin decondensation in a locus-specific manner and this function is achieved by interacting with Esrrb.

Leptin production capacity determines food intake and susceptibility to obesity-induced diabetes in Oikawa-Nagao Diabetes-Prone and Diabetes-Resistant mice.

AIMS/HYPOTHESIS: Obesity caused by overeating plays a pivotal role in the development of type 2 diabetes. However, it remains poorly understood how individual meal size differences are determined before the development of obesity. Here, we investigated the underlying mechanisms in determining spontaneous food intake in newly established Oikawa-Nagao Diabetes-Prone (ON-DP) and Diabetes-Resistant (ON-DR) mice. METHODS: Food intake and metabolic phenotypes of ON-DP and ON-DR mice under high-fat-diet feeding were compared from 5 weeks to 10 weeks of age. Differences in leptin status at 5 weeks of age were assessed between the two mouse lines. Adipose tissue explant culture was also performed to evaluate leptin production capacity in vitro. RESULTS: ON-DP mice showed spontaneous overfeeding compared with ON-DR mice. Excessive body weight gain and fat accumulation in ON-DP mice were completely suppressed to the levels seen in ON-DR mice by pair-feeding with ON-DR mice. Deterioration of glucose tolerance in ON-DP mice was also ameliorated under the pair-feeding conditions. While no differences were seen in body weight and adipose tissue mass when comparing the two mouse lines at 5 weeks of age, the ON-DP mice had lower plasma leptin concentrations and adipose tissue leptin gene expression levels. In accordance with peripheral leptin status, ON-DP mice displayed lower anorexigenic leptin signalling in the hypothalamic arcuate nucleus when compared with ON-DR mice without apparent leptin resistance. Explant culture studies revealed that ON-DP mice had lower leptin production capacity in adipose tissue. ON-DP mice also displayed higher DNA methylation levels in the leptin gene promoter region of adipocytes when compared with ON-DR mice. CONCLUSIONS/INTERPRETATION: The results suggest that heritable lower leptin production capacity plays a critical role in overfeeding-induced obesity and subsequent deterioration of glucose tolerance in ON-DP mice. Leptin production capacity in adipocytes, especially before the development of obesity, may have diagnostic potential for predicting individual risk of obesity caused by overeating and future onset of type 2 diabetes. Graphical abstract.

Linker histone variant H1T functions as a chromatin de-condenser on genic regions.

Linker histone H1 is mainly localized in the linker DNA region, between two nucleosome cores, and regulates chromatin structures linking gene expression. There are 11 variants in histone H1, and each variant has unique functions. Our previous study demonstrates that one of the H1 variants, H1T is mainly localized in the nucleolus and targets the rDNA repeat region. Moreover, H1T condenses the chromatin structures on rDNA to repress pre-rRNA expression. Although H1T is partially localized in the nucleoplasm area, the functions of H1T in the non-repeat genic region are unclear. In this study, we aimed to identify the target loci and the role of H1T in the genic region. Chromatin immunoprecipitation sequencing analysis showed that H1T is localized around the transcriptional start site and the chromatin structures of the region were relaxed. H1T knockdown and overexpression experiments revealed that H1T induced chromatin de-condensation and was associated with the increased expression of target genes. Moreover, we observed H1T co-localization with transcriptional factor SPZ1 on the genic region. Collectively, H1T has opposing roles in the genic region and in rDNA repeats; H1T functions to facilitate chromatin relaxation linked gene activation.

Extracellular glucose levels in cultures of undifferentiated mouse trophoblast stem cells affect gene expression during subsequent differentiation with replicable cell line-dependent variation.

Mouse trophoblast stem cells (TSCs) have been established and maintained using hyperglycemic conditions (11 mM glucose) for no apparent good reason. Because glucose metabolites are used as resources for cellular energy production, biosynthesis, and epigenetic modifications, differences in extracellular glucose levels may widely affect cellular function. Since the hyperglycemic culture conditions used for TSC culture have not been fully validated, the effect of extracellular glucose levels on the properties of TSCs remains unclear. To address this issue, we investigated the gene expression of stemness-related transcription factors in TSCs cultured in the undifferentiated state under various glucose concentrations. We also examined the expression of trophoblast subtype markers during differentiation, after returning the glucose concentration to the conventional culture concentration (11 mM). As a result, it appeared that the extracellular glucose conditions in the stem state not only affected the gene expression of stemness-related transcription factors before differentiation but also affected the expression of marker genes after differentiation, with some line-to-line variation. In the TS4 cell line, which showed the largest glucose concentration-dependent fluctuations in gene expression among all the lines examined, low glucose (1 mM glucose, LG) augmented H3K27me3 levels. An Ezh2 inhibitor prevented these LG-induced changes in gene expression, suggesting the possible involvement of H3K27me3 in the changes in gene expression seen in LG. These results collectively indicate that the response of the TSCs to the change in the extracellular glucose concentration is cell line-dependent and a part of which may be epigenetically memorized.

Epigenetic switching by the metabolism-sensing factors in the generation of orexin neurons from mouse embryonic stem cells.

The orexin system plays a central role in the integration of sleep/wake and feeding behaviors in a broad spectrum of neural-metabolic physiology. Orexin-A and orexin-B are produced by the cleavage of prepro-orexin, which is encoded on the Hcrt gene. To date, methods for generating other peptide neurons could not induce orexin neurons from pluripotent stem cells. Considering that the metabolic status affects orexin expression, we supplemented the culture medium with a nutrient factor, ManNAc, and succeeded in generating functional orexin neurons from mouse ES cells. Because DNA methylation inhibitors and histone deacetylase inhibitors could induce Hcrt expression in mouse ES cells, the epigenetic mechanism may be involved in this orexin neurogenesis. DNA methylation analysis showed the presence of a tissue-dependent differentially methylated region (T-DMR) around the transcription start site of the Hcrt gene. In the orexin neurons induced by supplementation of ManNAc, the T-DMR of the Hcrt gene was hypomethylated in association with higher H3/H4 acetylation. Concomitantly, the histone acetyltransferases p300, CREB-binding protein (CBP), and Mgea5 (also called O-GlcNAcase) were localized to the T-DMR in the orexin neurons. In non-orexin-expressing cells, H3/H4 hypoacetylation and hyper-O-GlcNAc modification were observed at the T-DMRs occupied by O-GlcNAc transferase and Sirt1. Therefore, the results of the present study suggest that the glucose metabolite, ManNAc, induces switching from the inactive state by Ogt-Sirt1 to the active state by Mgea5, p300, and CBP at the Hcrt gene locus.

An Anatomical Dissection Method for Observation of Fibrous Facial Structures.

BACKGROUND: In recent years, structures including the superficial musculoaponeurotic system and retaining ligaments that support the facial soft tissue have been clarified. However, these structures are very difficult to observe in their entirety by the standard gross anatomical procedure (ie, dissection from superficial to deep layers). Furthermore, accurate descriptions of these structures are rare in both anatomical and plastic surgery textbooks. The aim of this study was to clarify the facial fibrous structures in a gross anatomical view. METHODS: The authors' novel method used soft facial tissue and bone. The tissue was fixed in gelatin and sectioned at a thickness of 5 to 10 mm. Each section was placed on a wooden board; the bone was then pinned, and the skin was pulled outward with sutures to hyperextend the soft tissue. Subsequently, the loose connective tissue was torn and fat tissue was removed under a surgical microscope. After the removal of fat tissue, the fibrous facial structures (eg, the superficial musculoaponeurotic system and retaining ligaments) could be observed clearly. RESULTS: The thickness of the sections allowed three-dimensional observation, such that a structure located deep within a section could be clearly observed. The expansion of soft tissue facilitated observation of the facial layer and fibrous structures, and the locations of nerves and vessels. Therefore, the facial layer structure was readily discerned. CONCLUSION: This method is likely to be very useful in the field of plastic surgery because it enabled intuitive identification of facial layers and their characteristics. CLINICAL RELEVANCE STATEMENT: The dissection method developed by the authors reveals the connected morphology of each tissue of the face, thus providing basic data for analyzing soft tissue changes due to aging and gravity. This will be useful for the development of anti-aging medicine.

Bridging sequence diversity and tissue-specific expression by DNA methylation in genes of the mouse prolactin superfamily.

Much of the DNA in genomes is organized within gene families and hierarchies of gene superfamilies. DNA methylation is the main epigenetic event involved in gene silencing and genome stability. In the present study, we analyzed the DNA methylation status of the prolactin (PRL) superfamily to obtain insight into its tissue-specific expression and the evolution of its sequence diversity. The PRL superfamily in mice consists of two dozen members, which are expressed in a tissue-specific manner. The genes in this family have CpG-less sequences, and they are located within a 1-Mb region as a gene cluster on chromosome 13. We tentatively grouped the family into several gene clusters, depending on location and gene orientation. We found that all the members had tissue-dependent differentially methylated regions (T-DMRs) around the transcription start site. The T-DMRs are hypermethylated in nonexpressing tissues and hypomethylated in expressing cells, supporting the idea that the expression of the PRL superfamily genes is subject to epigenetic regulation. Interestingly, the DNA methylation patterns of T-DMRs are shared within a cluster, while the patterns are different among the clusters. Finally, we reconstituted the nucleotide sequences of T-DMRs by converting TpG to CpG based on the consideration of a possible conversion of 5-methylcytosine to thymine by spontaneous deamination during the evolutionary process. On the phylogenic tree, the reconstituted sequences were well matched with the DNA methylation pattern of T-DMR and orientation. Our study suggests that DNA methylation is involved in tissue-specific expression and sequence diversity during evolution.

Protein phosphatase 6 promotes neurite outgrowth by promoting mTORC2 activity in N2a cells.

Understanding the molecular mechanism of neuronal differentiation is important to overcome the incurable diseases caused by nervous system damage. Neurite outgrowth is prerequisite for neuronal differentiation and regeneration, and cAMP response element-binding protein (CREB) is one of the major transcriptional factors positively regulating this process. Neuronal differentiation stimuli activate mammalian target of rapamycin (mTOR) complex 2 (mTORC2)/Akt signalling to phosphorylate CREB; however, the precise molecular mechanism of this event has not been fully understood. In this manuscript, we show that neuronal differentiation stimuli increased a protein level of protein phosphatase 6 (PP6), a member of type 2A Ser/Thr protein phosphatases. PP6 knockdown suppressed mTORC2/Akt/CREB signalling and results in failure of neurite outgrowth. SIN1 is a unique component of mTORC2 that enhances mTORC2 activity towards Akt when it is in dephosphorylated form. We found PP6 knockdown increased SIN1 phosphorylation. These data suggest that PP6 may positively regulate neurite outgrowth by dephosphorylating SIN1 to activate mTORC2/Akt/CREB signalling.

Transcriptome analysis using patient iPSC-derived skeletal myocytes: Bet1L as a new molecule possibly linked to neuromuscular junction degeneration in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease in which patients gradually become paralyzed due to loss of motor function. Many genetically inheritable mutations have been linked to ALS; however, the majority of ALS patients are considered sporadic. Therefore, there is a need for a common therapy that is effective for all ALS patients. Although there is evidence of the disease beginning in the periphery at the neuromuscular junction (NMJ), the specific processes involved in skeletal muscle and at the NMJ are still largely unknown. To study common disease mechanisms in ALS skeletal muscle, we performed RNA sequencing of skeletal myocytes differentiated from induced pluripotent stem cells (iPSCs) derived from familial ALS (with C9ORF72, SOD1, or TARDBP mutations) and sporadic ALS patients. Compared to healthy control lines, the myocytes from all ALS lines showed downregulation of four genes: BET1L, DCX, GPC3, and HNRNPK. We next measured the expression levels of these four genes in hind limb muscle samples from a rat model of familial ALS (SOD1G93A transgenic) and found that only the Bet1L gene, which encodes Bet1 Golgi Vesicular Membrane Trafficking Protein Like, was commonly downregulated. Bet1L protein appeared to be localized to the basal lamina of the NMJ, with decreased expression over time in SOD1G93A transgenic rats. Importantly, the expression levels began to decrease early in the disease process. Our results indicate that loss of Bet1L at the NMJ could be of interest for better understanding ALS disease progression.

Kynurenine, 3-OH-kynurenine, and anthranilate are nutrient metabolites that alter H3K4 trimethylation and H2AS40 O-GlcNAcylation at hypothalamus-related loci.

Epigenetic mechanisms can establish and maintain mitotically stable patterns of gene expression while retaining the DNA sequence. These mechanisms can be affected by environmental factors such as nutrients. The importance of intracellular dosages of nutrient metabolites such as acetyl coenzyme A and S-adenosylmethionine, which are utilized as donors for post-translational modifications, is well-known in epigenetic regulation; however, the significance of indirect metabolites in epigenetic regulation is not clear. In this study, we screened for metabolites that function as epigenetic modulators. Because the expression of genes related to hypothalamic function is reportedly affected by nutritional conditions, we used a neural cell culture system and evaluated hypothalamic-linked loci. We supplemented the culture medium with 129 metabolites separately during induction of human-iPS-derived neural cells and used high-throughput ChIP-qPCR to determine the epigenetic status at 37 hypothalamus-linked loci. We found three metabolites (kynurenine, 3-OH-kynurenine, and anthranilate) from tryptophan pathways that increased H3K4 trimethylation and H2AS40 O-GlcNAcylation, resulting in upregulated gene expression at most loci, except those encoding pan-neural markers. Dietary supplementation of these three metabolites and the resulting epigenetic modification were important for stability in gene expression. In conclusion, our findings provide a better understanding of how nutrients play a role in epigenetic mechanisms.

C9ORF72-related cellular pathology in skeletal myocytes derived from ALS-patient induced pluripotent stem cells.

Amyotrophic lateral sclerosis (ALS) is a late-onset neuromuscular disease with no cure and limited treatment options. Patients experience a gradual paralysis leading to death from respiratory complications on average only 2-5 years after diagnosis. There is increasing evidence that skeletal muscle is affected early in the disease process, yet the pathological processes occurring in the skeletal muscle of ALS patients are still mostly unknown. Specifically, the most common genetic cause of ALS, a hexanucleotide repeat expansion in the C9ORF72 gene, has yet to be fully characterized in the context of skeletal muscle. In this study, we used the protocol previously developed in our lab to differentiate skeletal myocytes from induced pluripotent stem cells (iPSCs) of C9ORF72 ALS (C9-ALS) patients in order to create an in vitro disease model of C9-ALS skeletal muscle pathology. Of the three C9ORF72 mutation hallmarks, we did not see any evidence of haploinsufficiency, but we did detect RNA foci and dipeptide repeat (DPR) proteins. Additional abnormalities included changes in the expression of mitochondrial genes and a susceptibility to oxidative stress, indicating that mitochondrial dysfunction may be a critical feature of C9-ALS skeletal muscle pathology. Finally, the C9-ALS myocytes had increased expression and aggregation of TDP-43. Together, these data show that skeletal muscle cells experience pathological changes due to the C9ORF72 mutation. Our in vitro model could facilitate further study of cellular and molecular pathology in ALS skeletal muscle in order to discover new therapeutic targets against this devastating disease.This article has an associated First Person interview with the first author of the paper.

Improvement in the in vitro maturation rate of porcine oocytes vitrified at the germinal vesicle stage by treatment with a mitochondrial permeability transition inhibitor.

In this study, we examined the effects of inhibitors of mitochondrial permeability transition (MPT), caspase activity, intracellular Ca(2+) chelator and mitochondrial Ca(2+) uniporter on survival assessed by morphological observation and in vitro maturation (IVM) of porcine vitrified germinal vesicle (GV) oocytes. When vitrified GV oocytes were matured only present in the IVM medium with an MPT inhibitor, cyclosporin A (CsA), the survival and IVM rates (36.1% and 26.8%, respectively) were significantly higher (P<0.05) than those in the other vitrified groups (10.3-12.3% and 6.2-10.3%, respectively). However, Z-VAD-fmk (Z-VAD), a caspase inhibitor, did not improve the survival and IVM rates (11.7-21.6% and 8.5-155%, respectively). When BAPTA-AM, an intracellular Ca(2+) chelator, was present in the IVM medium, the survival and IVM rates of vitrified GV oocytes (34.5-36.2% and 25.0-26.9%, respectively) were significantly higher (P<0.05) than those in the absent vitrified groups (17.2-24.2% and 12.9-19.3%, respectively). When ruthenium red (RR), an inhibitor of mitochondrial Ca(2+) uniporter, was present only in the IVM medium, the survival and IVM rates (54.5% and 39.4%, respectively) were significantly higher than those in the other vitrified groups (25.8-38.4% and 14.4-24.2%, respectively). Furthermore, blastocysts were successfully produced using porcine vitrified GV oocytes matured in the IVM medium with RR after in vitro fertilization. These results suggested that CsA, BAPTA-AM and RR but not Z-VAD have improved the survival and IVM rates of porcine vitrified GV oocytes.

Nucleosomes of polyploid trophoblast giant cells mostly consist of histone variants and form a loose chromatin structure.

Trophoblast giant cells (TGCs) are one of the cell types that form the placenta and play multiple essential roles in maintaining pregnancy in rodents. TGCs have large, polyploid nuclei resulting from endoreduplication. While previous studies have shown distinct gene expression profiles of TGCs, their chromatin structure remains largely unknown. An appropriate combination of canonical and non-canonical histones, also known as histone variants, allows each cell to exert its cell type-specific functions. Here, we aimed to reveal the dynamics of histone usage and chromatin structure during the differentiation of trophoblast stem cells (TSCs) into TGCs. Although the expression of most genes encoding canonical histones was downregulated, the expression of a few genes encoding histone variants such as H2AX, H2AZ, and H3.3 was maintained at a relatively high level in TGCs. Both the micrococcal nuclease digestion assay and nucleosome stability assay using a microfluidic device indicated that chromatin became increasingly loose as TSCs differentiated. Combinatorial experiments involving H3.3-knockdown and -overexpression demonstrated that variant H3.3 resulted in the formation of loose nucleosomes in TGCs. In conclusion, our study revealed that TGCs possessed loose nucleosomes owing to alterations in their histone composition during differentiation.

H2A O-GlcNAcylation at serine 40 functions genomic protection in association with acetylated H2AZ or γH2AX.

BACKGROUND: We have previously reported a novel O-GlcNAc modification at serine 40 (S40) of H2A (H2AS40Gc). S40-type H2A isoforms susceptible to O-GlcNAcylation are evolutionarily new and restricted to the viviparous animals; however, the biological function of H2AS40Gc is largely unknown. H2A isoforms are consisted of S40 and alanine 40 (A40) type and this residue on H2A is located in the L1 of the globular domain, which is also known as a variable portion that distinguishes between the canonical and non-canonical H2A variants. In this study, by considering the similarity between the S40-type H2A and histone H2A variants, we explored the function of H2AS40Gc in mouse embryonic stem cells (mESCs). RESULTS: We found several similarities between the S40-type H2A isoforms and histone H2A variants such H2AZ and H2AX. mRNA of S40-type H2A isoforms (H2A1 N and H2A3) had a poly(A) tail and was produced throughout the cell cycle in contrast to that of A40-type. Importantly, H2AS40Gc level increased owing to chemical-induced DNA damage, similar to phosphorylated H2AX (γH2AX) and acetylated H2AZ (AcH2AZ). H2AS40Gc was accumulated at the restricted area (± 1.5 kb) of DNA damage sites induced by CRISPR/CAS9 system in contrast to accumulation of γH2AX, which was widely scattered. Overexpression of the wild-type (WT) H2A3, but not the S40 to A40 mutation (S40A-mutant), protected the mESC genome against chemical-induced DNA damage. Furthermore, 3 h after the DNA damage treatment, the genome was almost recovered in WT mESCs, whereas the damage advanced further in the S40A-mutant mESCs, suggesting functions of H2AS40Gc in the DNA repair mechanism. Furthermore, the S40A mutant prevented the accumulation of the DNA repair apparatus such as DNA-PKcs and Rad51 at the damage site. Co-immunoprecipitation experiment in WT and S40A-mutant mESCs revealed that H2AS40Gc physiologically bound to AcH2AZ at the initial phase upon DNA damage, followed by binding with γH2AX during the DNA damage repair process. CONCLUSIONS: These data suggest that H2AS40Gc functions to maintain genome integrity through the DNA repair mechanism in association with AcH2AZ and γH2AX.

Reactivation of hyperglycemia-induced hypocretin (HCRT) gene silencing by N-acetyl-d-mannosamine in the orexin neurons derived from human iPS cells.

Orexin neurons regulate critical brain activities for controlling sleep, eating, emotions, and metabolism, and impaired orexin neuron function results in several neurologic disorders. Therefore, restoring normal orexin function and understanding the mechanisms of loss or impairment of orexin neurons represent important goals. As a step toward that end, we generated human orexin neurons from induced pluripotent stem cells (hiPSCs) by treatment with N-acetyl-d-mannosamine (ManNAc) and its derivatives. The generation of orexin neurons was associated with DNA hypomethylation, histone H3/H4 hyperacetylation, and hypo-O-GlcNAcylation on the HCRT gene locus, and, thereby, the treatment of inhibitors of SIRT1 and OGT were effective at inducing orexin neurons from hiPSCs. The prolonged exposure of orexin neurons to high glucose in culture caused irreversible silencing of the HCRT gene, which was characterized by H3/H4 hypoacetylation and hyper-O-GlcNAcylation. The DNA hypomethylation status, once established in orexin neurogenesis, was maintained in the HCRT-silenced orexin neurons, indicating that histone modifications, but not DNA methylation, were responsible for the HCRT silencing. Thus, the epigenetic status of the HCRT gene is unique to the hyperglycemia-induced silencing. Intriguingly, treatment of ManNAc and its derivatives reactivated HCRT gene expression, while inhibitors SIRT1 and the OGT did not. The present study revealed that the HCRT gene was silenced by the hyperglycemia condition, and ManNAc and its derivatives were useful for restoring the orexin neurons.

Linker histone variant H1T targets rDNA repeats.

H1T is a linker histone H1 variant that is highly expressed at the primary spermatocyte stage through to the early spermatid stage of spermatogenesis. While the functions of the somatic types of H1 have been extensively investigated, the intracellular role of H1T is unclear. H1 variants specifically expressed in germ cells show low amino acid sequence homology to somatic H1s, which suggests that the functions or target loci of germ cell-specific H1T differ from those of somatic H1s. Here, we describe the target loci and function of H1T. H1T was expressed not only in the testis but also in tumor cell lines, mouse embryonic stem cells (mESCs), and some normal somatic cells. To elucidate the intracellular localization and target loci of H1T, fluorescent immunostaining and ChIP-seq were performed in tumor cells and mESCs. We found that H1T accumulated in nucleoli and predominantly targeted rDNA repeats, which differ from somatic H1 targets. Furthermore, by nuclease sensitivity assay and RT-qPCR, we showed that H1T repressed rDNA transcription by condensing chromatin structure. Imaging analysis indicated that H1T expression affected nucleolar formation. We concluded that H1T plays a role in rDNA transcription, by distinctively targeting rDNA repeats.

Novel O-GlcNAcylation on Ser(40) of canonical H2A isoforms specific to viviparity.

We report here newly discovered O-linked-N-acetylglucosamine (O-GlcNAc) modification of histone H2A at Ser(40) (H2AS40Gc). The mouse genome contains 18 H2A isoforms, of which 13 have Ser(40) and the other five have Ala(40). The combination of production of monoclonal antibody and mass spectrometric analyses with reverse-phase (RP)-high performance liquid chromatography (HPLC) fractionation indicated that the O-GlcNAcylation is specific to the Ser(40) isoforms. The H2AS40Gc site is in the L1 loop structure where two H2A molecules interact in the nucleosome. Targets of H2AS40Gc are distributed genome-wide and are dramatically changed during the process of differentiation in mouse trophoblast stem cells. In addition to the mouse, H2AS40Gc was also detected in humans, macaques and cows, whereas non-mammalian species possessing only the Ala(40) isoforms, such as silkworms, zebrafish and Xenopus showed no signal. Genome database surveys revealed that Ser(40) isoforms of H2A emerged in Marsupialia and persisted thereafter in mammals. We propose that the emergence of H2A Ser(40) and its O-GlcNAcylation linked a genetic event to genome-wide epigenetic events that correlate with the evolution of placental animals.