Maternal epigenetic factors in embryonic and postnatal development.

Maternal factors present in oocytes and surrounding granulosa cells influence early development of embryos. In this study, we searched for epigenetic regulators that are expressed in oocytes and/or granulosa cells. Some of the 120 epigenetic regulators examined were expressed specifically in oocytes and/or granulosa cells. When their expression was examined in young versus aged oocytes or granulosa cells, many were significantly up- or downregulated in aged cells. The maternal role of six genes in development was investigated by generating oocyte-specific knock-out (MKO) mice. Two genes (Mllt10, Kdm2b) did not show maternal effects on later development, whereas maternal effects were evident for Kdm6a, Kdm4a, Prdm3, and Prdm16 for MKO female mice. Offspring from Kdm6a MKO mice underwent perinatal lethality at a higher rate. Pups derived from Prdm3;Prdm16 double MKO showed a higher incidence of postnatal death. Finally, embryos derived from Kdm4a MKO mice showed early developmental defects as early as the peri-implantation stage. These results suggest that many of maternal epigenetic regulators undergo differential expression upon aging. Some, such as Kdm4a, Kdm6a, Prdm3, and Prdm16, have maternal role in later embryonic or postnatal development.

Enhanced transcriptional heterogeneity mediated by NF-κB super-enhancers.

The transcription factor NF-κB, which plays an important role in cell fate determination, is involved in the activation of super-enhancers (SEs). However, the biological functions of the NF-κB SEs in gene control are not fully elucidated. We investigated the characteristics of NF-κB-mediated SE activity using fluorescence imaging of RelA, single-cell transcriptome and chromatin accessibility analyses in anti-IgM-stimulated B cells. The formation of cell stimulation-induced nuclear RelA foci was abolished in the presence of hexanediol, suggesting an underlying process of liquid-liquid phase separation. The gained SEs induced a switch-like expression and enhanced cell-to-cell variability in transcriptional response. These properties were correlated with the number of gained cis-regulatory interactions, while switch-like gene induction was associated with the number of NF-κB binding sites in SE. Our study suggests that NF-κB SEs have an important role in the transcriptional regulation of B cells possibly through liquid condensate formation consisting of macromolecular interactions.

Erratum: Derepression of inflammation-related genes link to microglia activation and neural maturation defect in a mouse model of Kleefstra syndrome.

[This corrects the article DOI: 10.1016/j.isci.2021.102741.].

Local states of chromatin compaction at transcription start sites control transcription levels.

The 'open' and 'compact' regions of chromatin are considered to be regions of active and silent transcription, respectively. However, individual genes produce transcripts at different levels, suggesting that transcription output does not depend on the simple open-compact conversion of chromatin, but on structural variations in chromatin itself, which so far have remained elusive. In this study, weakly crosslinked chromatin was subjected to sedimentation velocity centrifugation, which fractionated the chromatin according to its degree of compaction. Open chromatin remained in upper fractions, while compact chromatin sedimented to lower fractions depending on the level of nucleosome assembly. Although nucleosomes were evenly detected in all fractions, histone H1 was more highly enriched in the lower fractions. H1 was found to self-associate and crosslinked to histone H3, suggesting that H1 bound to H3 interacts with another H1 in an adjacent nucleosome to form compact chromatin. Genome-wide analyses revealed that nearly the entire genome consists of compact chromatin without differences in compaction between repeat and non-repeat sequences; however, active transcription start sites (TSSs) were rarely found in compact chromatin. Considering the inverse correlation between chromatin compaction and RNA polymerase binding at TSSs, it appears that local states of chromatin compaction determine transcription levels.

Derepression of inflammation-related genes link to microglia activation and neural maturation defect in a mouse model of Kleefstra syndrome.

Haploinsufficiency of EHMT1, which encodes histone H3 lysine 9 (H3K9) methyltransferase G9a-like protein (GLP), causes Kleefstra syndrome (KS), a complex disorder of developmental delay and intellectual disability. Here, we examined whether postnatal supply of GLP can reverse the neurological phenotypes seen in Ehmt1 Δ/+ mice as a KS model. Ubiquitous GLP supply from the juvenile stage ameliorated behavioral abnormalities in Ehmt1 Δ/+ mice. Postnatal neuron-specific GLP supply was not sufficient for the improvement of abnormal behaviors but still reversed the reduction of H3K9me2 and spine number in Ehmt1 Δ/+ mice. Interestingly, some inflammatory genes, including IL-1ß (Il1b), were upregulated and activated microglial cells increased in the Ehmt1 Δ/+ brain, and such phenotypes were also reversed by neuron-specific postnatal GLP supply. Il1b inactivation canceled the microglial and spine number phenotypes in the Ehmt1 Δ/+ mice. Thus, H3K9me2 and some neurological phenotypes are reversible, but behavioral abnormalities are more difficult to improve depending on the timing of GLP supply.

Single-oocyte transcriptome analysis reveals aging-associated effects influenced by life stage and calorie restriction.

Chromosome segregation errors in oocytes lead to the production of aneuploid eggs, which are the leading cause of pregnancy loss and of several congenital diseases such as Down syndrome. The frequency of chromosome segregation errors in oocytes increases with maternal age, especially at a late stage of reproductive life. How aging at various life stages affects oocytes differently remains poorly understood. In this study, we describe aging-associated changes in the transcriptome profile of mouse oocytes throughout reproductive life. Our single-oocyte comprehensive RNA sequencing using RamDA-seq revealed that oocytes undergo transcriptome changes at a late reproductive stage, whereas their surrounding cumulus cells exhibit transcriptome changes at an earlier stage. Calorie restriction, a paradigm that reportedly prevents aging-associated egg aneuploidy, promotes a transcriptome shift in oocytes with the up-regulation of genes involved in chromosome segregation. This shift is accompanied by the improved maintenance of chromosomal cohesin, the loss of which is a hallmark of oocyte aging and causes chromosome segregation errors. These findings have implications for understanding how oocytes undergo aging-associated functional decline throughout their reproductive life in a context-dependent manner.

Tracing the origin of hair follicle stem cells.

Tissue stem cells are generated from a population of embryonic progenitors through organ-specific morphogenetic events1,2. Although tissue stem cells are central to organ homeostasis and regeneration, it remains unclear how they are induced during development, mainly because of the lack of markers that exclusively label prospective stem cells. Here we combine marker-independent long-term 3D live imaging and single-cell transcriptomics to capture a dynamic lineage progression and transcriptome changes in the entire epithelium of the mouse hair follicle as it develops. We found that the precursors of different epithelial lineages were aligned in a 2D concentric manner in the basal layer of the hair placode. Each concentric ring acquired unique transcriptomes and extended to form longitudinally aligned, 3D cylindrical compartments. Prospective bulge stem cells were derived from the peripheral ring of the placode basal layer, but not from suprabasal cells (as was previously suggested3). The fate of placode cells is determined by the cell position, rather than by the orientation of cell division. We also identified 13 gene clusters: the ensemble expression dynamics of these clusters drew the entire transcriptional landscape of epithelial lineage diversification, consistent with cell lineage data. Combining these findings with previous work on the development of appendages in insects4,5, we describe the 'telescope model', a generalized model for the development of ectodermal organs in which 2D concentric zones in the placode telescope out to form 3D longitudinally aligned cylindrical compartments.

The Number of Transcription Factors at an Enhancer Determines Switch-like Gene Expression.

NF-κB is a transcription factor that activates super enhancers (SEs) and typical enhancers (TEs) and triggers threshold and graded gene expression, respectively. However, the mechanisms by which NF-κB selectively participates in these enhancers remain unclear. Here we show using mouse primary B lymphocytes that SE activity simultaneously associates with chromatin opening and enriched NF-κB binding, resulting in a higher fold change and threshold expression upon B cell receptor (BCR) activation. The higher fold change results from longer DNA, whereas the threshold response is explained by synergy in DNA-NF-κB binding and is supported by the coexistence of PU.1 and NF-κB in a SE before cell stimulation. This model indicates that the pre-existing NF-κB functions as a seed and triggers its processive binding upon BCR activation. Our mathematical modeling of the single-cell transcriptome reveals an additional role for SEs in divergent clonal responses in B cells.

Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells.

Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

Millefy: visualizing cell-to-cell heterogeneity in read coverage of single-cell RNA sequencing datasets.

BACKGROUND: Read coverage of RNA sequencing data reflects gene expression and RNA processing events. Single-cell RNA sequencing (scRNA-seq) methods, particularly "full-length" ones, provide read coverage of many individual cells and have the potential to reveal cellular heterogeneity in RNA transcription and processing. However, visualization tools suited to highlighting cell-to-cell heterogeneity in read coverage are still lacking. RESULTS: Here, we have developed Millefy, a tool for visualizing read coverage of scRNA-seq data in genomic contexts. Millefy is designed to show read coverage of all individual cells at once in genomic contexts and to highlight cell-to-cell heterogeneity in read coverage. By visualizing read coverage of all cells as a heat map and dynamically reordering cells based on diffusion maps, Millefy facilitates discovery of "local" region-specific, cell-to-cell heterogeneity in read coverage. We applied Millefy to scRNA-seq data sets of mouse embryonic stem cells and triple-negative breast cancers and showed variability of transcribed regions including antisense RNAs, 3 ' UTR lengths, and enhancer RNA transcription. CONCLUSIONS: Millefy simplifies the examination of cellular heterogeneity in RNA transcription and processing events using scRNA-seq data. Millefy is available as an R package (https://github.com/yuifu/millefy) and as a Docker image for use with Jupyter Notebook (https://hub.docker.com/r/yuifu/datascience-notebook-millefy).

An NMF-based approach to discover overlooked differentially expressed gene regions from single-cell RNA-seq data.

Single-cell RNA sequencing has enabled researchers to quantify the transcriptomes of individual cells, infer cell types and investigate differential expression among cell types, which will lead to a better understanding of the regulatory mechanisms of cell states. Transcript diversity caused by phenomena such as aberrant splicing events have been revealed, and differential expression of previously unannotated transcripts might be overlooked by annotation-based analyses. Accordingly, we have developed an approach to discover overlooked differentially expressed (DE) gene regions that complements annotation-based methods. Our algorithm decomposes mapped count data matrix for a gene region using non-negative matrix factorization, quantifies the differential expression level based on the decomposed matrix, and compares the differential expression level based on annotation-based approach to discover previously unannotated DE transcripts. We performed single-cell RNA sequencing for human neural stem cells and applied our algorithm to the dataset. We also applied our algorithm to two public single-cell RNA sequencing datasets correspond to mouse ES and primitive endoderm cells, and human preimplantation embryos. As a result, we discovered several intriguing DE transcripts, including a transcript related to the modulation of neural stem/progenitor cell differentiation.

Polysaccharide hydrolase of the hadal zone amphipods Hirondellea gigas.

Hirondellea species are common inhabitants in the hadal region deeper than 7,000 m. We found that Hirondellea gigas thrived in the Challenger Deep possessed polysaccharide hydrolases as digestive enzymes. To obtain various enzymes of other H. gigas, we captured amphipods from the Japan Trench, and Izu-Ogasawara (Bonin) Trench. A phylogenetic analysis based on the cytochrome oxidase I gene showed close relationships among amphipods, despite the geographic distance between the localities. However, several differences in enzymatic properties were observed in these H. gigas specimens. We also carried out RNA sequencing of H. gigas from the Izu-Ogasawara Trench. The cellulase gene of H. gigas was highly homologous to cellobiohydrolase of Glucosyl Hydrolase family 7 (GH7). On the other hand, enzymatic properties of H. gigas's cellulase were different from those of typical GH7 cellobiohydrolase. Thus, these results indicate that hadal-zone amphipod can be good candidates as the new enzyme resource.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.

Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.

The novel heme-dependent inducible protein, SRRD regulates heme biosynthesis and circadian rhythms.

Heme plays a role in the regulation of the expression of genes related to circadian rhythms and heme metabolism. In order to identify new heme-regulated proteins, an RNA sequence analysis using mouse NIH3T3 cells treated without or with 5-aminolevulinic acid (ALA) was performed. Among the changes observed in the levels of various mRNAs including heme oxygenase-1 (HO-1) and ALA synthase-1 (ALAS1), a mouse homologue of the plant circadian-regulating protein SRR1, SRR1 domain containing (SRRD) was induced by the ALA treatment. The expression of SRRD was dependent on heme biosynthesis, and increased the production of heme. SRRD was expressed under circadian rhythms, and influenced the expression of clock genes including PER2, BMAL1, and CLOCK. The knockout of SRRD arrested the growth of cells, indicating that SRRD plays roles in heme-regulated circadian rhythms and cell proliferation.

The p53-dependent expression of frataxin controls 5-aminolevulinic acid-induced accumulation of protoporphyrin IX and photo-damage in cancerous cells.

Mitochondrial frataxin is involved in various functions such as iron homeostasis, iron-sulfur cluster biogenesis, the protection from oxidative stress and apoptosis and acts as a tumor suppressor protein. We now show that the expression of frataxin is stimulated in a p53-dependent manner and prove that frataxin is a direct p53 target gene by showing that the p53-responsive element in the promoter of the mouse frataxin gene is bound by p53. The bacterial expression of human frataxin stimulated maturation of human ferrochelatase, which catalyzes the insertion of iron into protoporphyrin at the last step of heme biosynthesis. Overexpression of frataxin in human cancer A431 and HeLa cells lowered 5-aminolevulinic acid(ALA)-induced accumulation of protoporphyrin and induced resistance to ALA-induced photo-damage, whereas p53 silencing with siRNA in non tumor HEK293T cells down-regulated the expression of frataxin and increased the accumulation of protoporphyrin. Thus, the decrease of the expression of frataxin unregulated by p53 in tumor cells enhances ALA-induced photo-damage, by down-regulation of mitochondrial functions.