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Endogenous retroviruses (ERVs) occupy a significant part of the human genome, with some encoding proteins that influence the immune system or regulate cell-cell fusion in early extra-embryonic development. However, whether ERV-derived proteins regulate somatic development is unknown. Here, we report a somatic developmental function for the primate-specific ERVH48-1 (SUPYN/Suppressyn). ERVH48-1 encodes a fragment of a viral envelope that is expressed during early embryonic development. Loss of ERVH48-1 led to impaired mesoderm and cardiomyocyte commitment and diverted cells to an ectoderm-like fate. Mechanistically, ERVH48-1 is localized to sub-cellular membrane compartments through a functional N-terminal signal peptide and binds to the WNT antagonist SFRP2 to promote its polyubiquitination and degradation, thus limiting SFRP2 secretion and blocking repression of WNT/β-catenin signaling. Knockdown of SFRP2 or expression of a chimeric SFRP2 with the ERVH48-1 signal peptide rescued cardiomyocyte differentiation. This study demonstrates how ERVH48-1 modulates WNT/β-catenin signaling and cell type commitment in somatic development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The complicated process of neuronal development is initiated early in life, with the genetic mechanisms governing this process yet to be fully elucidated. Single-cell RNA sequencing (scRNA-seq) is a potent instrument for pinpointing biomarkers that exhibit differential expression across various cell types and developmental stages. By employing scRNA-seq on human embryonic stem cells, we aim to identify differentially expressed genes (DEGs) crucial for early-stage neuronal development. Our focus extends beyond simply identifying DEGs. We strive to investigate the functional roles of these genes through enrichment analysis and construct gene regulatory networks to understand their interactions. Ultimately, this comprehensive approach aspires to illuminate the molecular mechanisms and transcriptional dynamics governing early human brain development. By uncovering potential links between these DEGs and intelligence, mental disorders, and neurodevelopmental disorders, we hope to shed light on human neurological health and disease. In this study, we have used scRNA-seq to identify DEGs involved in early-stage neuronal development in hESCs. The scRNA-seq data, collected on days 26 (D26) and 54 (D54), of the in vitro differentiation of hESCs to neurons were analyzed. Our analysis identified 539 DEGs between D26 and D54. Functional enrichment of those DEG biomarkers indicated that the up-regulated DEGs participated in neurogenesis, while the down-regulated DEGs were linked to synapse regulation. The Reactome pathway analysis revealed that down-regulated DEGs were involved in the interactions between proteins located in synapse pathways. We also discovered interactions between DEGs and miRNA, transcriptional factors (TFs) and DEGs, and between TF and miRNA. Our study identified 20 significant transcription factors, shedding light on early brain development genetics. The identified DEGs and gene regulatory networks are valuable resources for future research into human brain development and neurodevelopmental disorders., (© The Author(s) 2024. Published by Oxford University Press.)

Somatic cell reprogramming and oncogenic transformation share surprisingly similar features, yet transformed cells are resistant to reprogramming. Epigenetic barriers must block transformed cells from reprogramming, but the nature of those barriers is unclear. In this study, we generated a systematic panel of transformed mouse embryonic fibroblasts (MEFs) using oncogenic transgenes and discovered transformed cell lines compatible with reprogramming when transfected with Oct4/Sox2/Klf4/Myc. By comparing the reprogramming-capable and incapable transformed lines we identified multiple stages of failure in the reprogramming process. Some transformed lines failed at an early stage, whilst other lines seemed to progress through a conventional reprogramming process. Finally, we show that MEK inhibition overcomes one critical reprogramming barrier by indirectly suppressing a hyperacetylated active epigenetic state. This study reveals that diverse epigenetic barriers underly resistance to reprogramming of transformed cells., (© 2023. The Author(s).)

Grasscutter ( Thryonomys swinderianus ) is a large-body old world rodent found in sub-Saharan Africa. The body size and the unique taste of the meat of this major crop pest have made it a target of intense hunting and a potential consideration as a micro-livestock. However, there is insufficient knowledge on the genetic diversity of its populations across African Guinean forests. Herein, we investigated the genetic diversity, population structures and evolutionary history of seven Nigerian wild grasscutter populations together with individuals from Cameroon, Republic of Benin, and Ghana, using five mitochondrial fragments, including D-loop and cytochrome b ( CYTB ). D-loop haplotype diversity ranged from 0.571 (± 0.149) in Republic of Benin to 0.921 (± 0.013) in Ghana. Within Nigeria, the haplotype diversity ranged from 0.659 (± 0.059) in Cross River to 0.837 (± 0.075) in Ondo subpopulation. The fixation index (F ST ), haplotype frequency distribution and analysis of molecular variance revealed varying levels of population structures across populations. No significant signature of population contraction was detected in the grasscutter populations. Evolutionary analyses of CYTB suggests that South African population might have diverged from other populations about 6.1 (2.6-10.18, 95% CI) MYA. Taken together, this study reveals the population status and evolutionary history of grasscutter populations in the region., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LHD declared a shared affiliation with the author CAMSD to the handling editor at the time of review., (Copyright © 2023 Babarinde, Adeola, Djagoun, Nneji, Okeyoyin, Niba, Wanzie, Oladipo, Adebambo, Bello, Ng’ang’a, Olaniyi, Okoro, Adedeji, Olatunde, Ayoola, Matouke, Wang, Sanke, Oseni, Nwani and Murphy.)

To satisfy both the safety and rapidity of glycemic control, muscles' insulin response must be bistable, as theoretically predicted. Here, we test the bistability hypothesis by combining cellular experiments (to measure the threshold values in vitro ) with mathematical modeling (to test the relevance of bistability in vivo ). We examine bistability in C2C12 myotubes by both single-cell analysis (Fӧrster resonance energy transfer) and cultured cells analysis (immunoblot). These technologies demonstrate bistable insulin response, with typical switch-on and switch-off thresholds of approximately 300 and 100 pM, respectively. Our mathematical model demonstrates the indispensability of bistability in interpreting experimental data, reveals fine details of plasma glucose-insulin dynamics, and explains unclear phenomena. These results suggest that the body's ability to simultaneously avoid both hypoglycemia and hyperglycemia is mediated by bistability. The switch-on threshold is a promising biomarker for metabolic complications due to its deep quantitative connection with body composition, which is easy to measure., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Investigating the functions and activities of genes requires proper annotation of the transcribed units. However, transcript assembly efforts have produced a surprisingly large variation in the number of transcripts, and especially so for noncoding transcripts. This heterogeneity in assembled transcript sets might be partially explained by sequencing depth. Here, we used real and simulated short-read sequencing data as well as long-read data to systematically investigate the impact of sequencing depths on the accuracy of assembled transcripts. We assembled and analyzed transcripts from 671 human short-read data sets and four long-read data sets. At the first level, there is a positive correlation between the number of reads and the number of recovered transcripts. However, the effect of the sequencing depth varied based on cell or tissue type, the type of read and the nature and expression levels of the transcripts. The detection of coding transcripts saturated rapidly with both short and long-reads, however, there was no sign of early saturation for noncoding transcripts at any sequencing depth. Increasing long-read sequencing depth specifically benefited transcripts containing transposable elements. Finally, we show how single-cell RNA-seq can be guided by transcripts assembled from bulk long-read samples, and demonstrate that noncoding transcripts are expressed at similar levels to coding transcripts but are expressed in fewer cells. This study highlights the impact of sequencing depth on transcript assembly., (© 2022. The Author(s).)

Around 60% of in vitro fertilized (IVF) human embryos irreversibly arrest before compaction between the 3- to 8-cell stage, posing a significant clinical problem. The mechanisms behind this arrest are unclear. Here, we show that the arrested embryos enter a senescent-like state, marked by cell cycle arrest, the down-regulation of ribosomes and histones and down-regulation of MYC and p53 activity. The arrested embryos can be divided into 3 types. Type I embryos fail to complete the maternal-zygotic transition, and Type II/III embryos have low levels of glycolysis and either high (Type II) or low (Type III) levels of oxidative phosphorylation. Treatment with the SIRT agonist resveratrol or nicotinamide riboside (NR) can partially rescue the arrested phenotype, which is accompanied by changes in metabolic activity. Overall, our data suggests metabolic and epigenetic dysfunctions underlie the arrest of human embryos., Competing Interests: The authors have declared that no competing interests exist.

Transposable elements (TEs) are mobile genetic elements that can randomly integrate into other genomic sites. They have successfully replicated and now occupy around 40% of the total DNA sequence in humans. TEs in the genome have a complex relationship with the host cell, being both potentially deleterious and advantageous at the same time. Only a tiny minority of TEs are still capable of transposition, yet their fossilized sequence fragments are thought to be involved in various molecular processes, such as gene transcriptional activity, RNA stability and subcellular localization, and chromosomal architecture. TEs have also been implicated in biological processes, although it is often hard to reveal cause from correlation due to formidable technical issues in analyzing TEs. In this review, we compare and contrast two views of TE activity: one in the pluripotent state, where TEs are broadly beneficial, or at least mechanistically useful, and a second state in human disease, where TEs are uniformly considered harmful., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ma, Babarinde, Zhou and Hutchins.)

CTCF mediates chromatin insulation and long-distance enhancer-promoter (EP) interactions; however, little is known about how these regulatory functions are partitioned among target genes in key biological processes. Here, we show that Ctcf expression is progressively increased during induced pluripotency. In this process, CTCF first functions as a chromatin insulator responsible for direct silencing of the somatic gene expression program and, interestingly, elevated Ctcf expression next ensures chromatin accessibility and contributes to increased EP interactions for a fraction of pluripotency-associated genes. Therefore, CTCF functions in a context-specific manner to modulate the 3D genome to enable cellular reprogramming. We further discover that these context-specific CTCF functions also enlist SMARCA5, an imitation switch (ISWI) chromatin remodeler, together rewiring the epigenome to facilitate cell-fate switch. These findings reveal the dual functions of CTCF in conjunction with a key chromatin remodeler to drive reprogramming toward pluripotency., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Transposable elements (TEs) occupy nearly 40% of mammalian genomes and, whilst most are fragmentary and no longer capable of transposition, they can nevertheless contribute to cell function. TEs within genes transcribed by RNA polymerase II can be copied as parts of primary transcripts; however, their full contribution to mature transcript sequences remains unresolved. Here, using long and short read (LR and SR) RNA sequencing data, we show that 26% of coding and 65% of noncoding transcripts in human pluripotent stem cells (hPSCs) contain TE-derived sequences. Different TE families are incorporated into RNAs in unique patterns, with consequences to transcript structure and function. The presence of TE sequences within a transcript is correlated with TE-type specific changes in its subcellular distribution, alterations in steady-state levels and half-life, and differential association with RNA Binding Proteins (RBPs). We identify hPSC-specific incorporation of endogenous retroviruses (ERVs) and LINE:L1 into protein-coding mRNAs, which generate TE sequence-derived peptides. Finally, single cell RNA-seq reveals that hPSCs express ERV-containing transcripts, whilst differentiating subpopulations lack ERVs and express SINE and LINE-containing transcripts. Overall, our comprehensive analysis demonstrates that the incorporation of TE sequences into the RNAs of hPSCs is more widespread and has a greater impact than previously appreciated., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Transposable elements (TEs) make up a majority of a typical eukaryote's genome, and contribute to cell heterogeneity in unclear ways. Single-cell sequencing technologies are powerful tools to explore cells, however analysis is typically gene-centric and TE expression has not been addressed. Here, we develop a single-cell TE processing pipeline, scTE, and report the expression of TEs in single cells in a range of biological contexts. Specific TE types are expressed in subpopulations of embryonic stem cells and are dynamically regulated during pluripotency reprogramming, differentiation, and embryogenesis. Unexpectedly, TEs are expressed in somatic cells, including human disease-specific TEs that are undetectable in bulk analyses. Finally, we apply scTE to single-cell ATAC-seq data, and demonstrate that scTE can discriminate cell type using chromatin accessibly of TEs alone. Overall, our results classify the dynamic patterns of TEs in single cells and their contributions to cell heterogeneity.

High-throughput sequencing technologies are increasingly used in molecular cell biology to assess genome-wide chromatin dynamics of proteins bound to DNA, through techniques such as chromatin immunoprecipitation sequencing (ChIP-seq). These techniques often rely on an analysis strategy based on identifying genomic regions with increased sequencing signal to infer the binding location or chemical modifications of proteins bound to DNA. Peak calling within individual samples has been well described, however relatively little attention has been devoted to the merging of replicate samples, and the cross-comparison of many samples. Here, we present a generalized strategy to enable the unification of ChIP-seq datasets, enabling enhanced cross-comparison of binding patterns. The strategy works by merging peak data between different (even unrelated) samples, and then using a local background to recalculate enrichment. This strategy redefines the peaks within each experiment, allowing for more accurate cross-comparison of datasets.

The interplay between the Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) and transcriptional/epigenetic co-regulators in somatic cell reprogramming is incompletely understood. Here, we demonstrate that the histone H3 lysine 27 trimethylation (H3K27me3) demethylase JMJD3 plays conflicting roles in mouse reprogramming. On one side, JMJD3 induces the pro-senescence factor Ink4a and degrades the pluripotency regulator PHF20 in a reprogramming factor-independent manner. On the other side, JMJD3 is specifically recruited by KLF4 to reduce H3K27me3 at both enhancers and promoters of epithelial and pluripotency genes. JMJD3 also promotes enhancer-promoter looping through the cohesin loading factor NIPBL and ultimately transcriptional elongation. This competition of forces can be shifted towards improved reprogramming by using early passage fibroblasts or boosting JMJD3's catalytic activity with vitamin C. Our work, thus, establishes a multifaceted role for JMJD3, placing it as a key partner of KLF4 and a scaffold that assists chromatin interactions and activates gene transcription.

Capybara (Hydrochoerus hydrochaeri) is the largest species among the extant rodents. The draft genome of capybara was sequenced with the estimated genome size of 2.6 Gb. Although capybara is about 60 times larger than guinea pig, comparative analyses revealed that the neutral evolutionary rates of the two species were not substantially different. However, analyses of 39 mammalian genomes revealed very heterogeneous evolutionary rates. The highest evolutionary rate, 8.5 times higher than the human rate, was found in the Cricetidae-Muridae common ancestor after the divergence of Spalacidae. Muridae, the family with the highest number of species among mammals, emerged after the rate acceleration. Factors responsible for the evolutionary rate heterogeneity were investigated through correlations between the evolutionary rate and longevity, gestation length, litter frequency, litter size, body weight, generation interval, age at maturity, and taxonomic order. The regression analysis of these factors showed that the model with three factors (taxonomic order, generation interval, and litter size) had the highest predictive power (R2 = 0.74). These three factors determine the number of meiosis per unit time. We also conducted transcriptome analysis and found that the evolutionary rate dynamics affects the evolution of gene expression patterns., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that β-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Summary: Cells are generally resistant to cell type conversions, but can be converted by the application of growth factors, chemical inhibitors and ectopic expression of genes. However, it remains difficult to accurately identify the destination cell type or differentiation bias when these techniques are used to alter cell type. Consequently, there is demand for computational techniques that can help researchers understand both the cell type and differentiation bias. While advanced tools identifying cell types exist for single cell data and the deconvolution of mixed cell populations, the problem of exploring partially differentiated cells of indeterminate transcriptional identity has not been addressed. To fill this gap, we developed driver-predictor, which relies on scoring per gene transcriptional similarity between RNA-Seq datasets to reveal directional bias of differentiation. By comparing against large cell type transcriptome libraries or a desired target expression profile, the tool enables the user to visualize both the changes in transcriptional identity as well as the genes accounting for the cell type changes. This software will be a powerful tool for researchers to explore in vitro experiments that involve cell type conversions., Availability and Implementation: Source code is open source under the MIT license and is freely available on https://github.com/LoaloaF/DPre., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The measurement of gene expression has long provided significant insight into biological functions. The development of high-throughput short-read sequencing technology has revealed transcriptional complexity at an unprecedented scale, and informed almost all areas of biology. However, as researchers have sought to gather more insights from the data, these new technologies have also increased the computational analysis burden. In this review, we describe typical computational pipelines for RNA-Seq analysis and discuss their strengths and weaknesses for the assembly, quantification and analysis of coding and non-coding RNAs. We also discuss the assembly of transposable elements into transcripts, and the difficulty these repetitive elements pose. In summary, RNA-Seq is a powerful technology that is likely to remain a key asset in the biologist's toolkit.

CTCF plays key roles in gene regulation, chromatin insulation, imprinting, X chromosome inactivation and organizing the higher-order chromatin architecture of mammalian genomes. Previous studies have mainly focused on the roles of the canonical CTCF isoform. Here, we explore the functions of an alternatively spliced human CTCF isoform in which exons 3 and 4 are skipped, producing a shorter isoform (CTCF-s). Functionally, we find that CTCF-s competes with the genome binding of canonical CTCF and binds a similar DNA sequence. CTCF-s binding disrupts CTCF/cohesin binding, alters CTCF-mediated chromatin looping and promotes the activation of IFI6 that leads to apoptosis. This effect is caused by an abnormal long-range interaction at the IFI6 enhancer and promoter. Taken together, this study reveals a non-canonical function for CTCF-s that antagonizes the genomic binding of canonical CTCF and cohesin, and that modulates chromatin looping and causes apoptosis by stimulating IFI6 expression.

The majority of mammalian genomes are devoted to transposable elements (TEs). Whilst TEs are increasingly recognized for their important biological functions, they are a potential danger to genomic stability and are carefully regulated by the epigenetic system. However, the full complexity of this regulatory system is not understood. Here, using mouse embryonic stem cells, we show that TEs are suppressed by heterochromatic marks like H3K9me3, and are also labelled by all major types of chromatin modification in complex patterns, including bivalent activatory and repressive marks. We identified 29 epigenetic modifiers that significantly deregulated at least one type of TE. The loss of Setdb1, Ncor2, Rnf2, Kat5, Prmt5, Uhrf1, and Rrp8 caused widespread changes in TE expression and chromatin accessibility. These effects were context-specific, with different chromatin modifiers regulating the expression and chromatin accessibility of specific subsets of TEs. Our work reveals the complex patterns of epigenetic regulation of TEs.

Experimental studies have found the involvement of certain conserved noncoding sequences (CNSs) in the regulation of the proximal protein-coding genes in mammals. However, reported cases of long range enhancer activities and inter-chromosomal regulation suggest that proximity of CNSs to protein-coding genes might not be important for regulation. To test the importance of the CNS genomic location, we extracted the CNSs conserved between chicken and four mammalian species (human, mouse, dog, and cattle). These CNSs were confirmed to be under purifying selection. The intergenic CNSs are often found in clusters in gene deserts, where protein-coding genes are in paucity. The distribution pattern, ChIP-Seq, and RNA-Seq data suggested that the CNSs are more likely to be regulatory elements and not corresponding to long intergenic noncoding RNAs. Physical distances between CNS and their nearest protein coding genes were well conserved between human and mouse genomes, and CNS-flanking genes were often found in evolutionarily conserved genomic neighborhoods. ChIP-Seq signal and gene expression patterns also suggested that CNSs regulate nearby genes. Interestingly, genes with more CNSs have more evolutionarily conserved expression than those with fewer CNSs. These computationally obtained results suggest that the genomic locations of CNSs are important for their regulatory functions. In fact, various kinds of evolutionary constraints may be acting to maintain the genomic locations of CNSs and protein-coding genes in mammals to ensure proper regulation., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Conserved noncoding sequences (CNSs) of vertebrates are considered to be closely linked with protein-coding gene regulatory functions. We examined the abundance and genomic distribution of CNSs in four mammalian orders: primates, rodents, carnivores, and cetartiodactyls. We defined the two thresholds for CNS using conservation level of coding genes; using all the three coding positions and using only first and second codon positions. The abundance of CNSs varied among lineages, with primates and rodents having highest and lowest number of CNSs, respectively, whereas carnivores and cetartiodactyls had intermediate values. These CNSs cover 1.3-5.5% of the mammalian genomes and have signatures of selective constraints that are stronger in more ancestral than the recent ones. Evolution of new CNSs as well as retention of ancestral CNSs contribute to the differences in abundance. The genomic distribution of CNSs is dynamic with higher proportions of rodent and primate CNSs located in the introns compared with carnivores and cetartiodactyls. In fact, 19% of orthologous single-copy CNSs between human and dog are located in different genomic regions. If CNSs can be considered as candidates of gene expression regulatory sequences, heterogeneity of CNSs among the four mammalian orders may have played an important role in creating the order-specific phenotypes. Fewer CNSs in rodents suggest that rodent diversity is related to lower regulatory conservation. With CNSs shown to cluster around genes involved in nervous systems and the higher number of primate CNSs, our result suggests that CNSs may be involved in the higher complexity of the primate nervous system.