Primary infection with human cytomegalovirus (HCMV) results in a lifelong infection due to its ability to establish latent infection, with one characterized viral reservoir being hematopoietic cells. ...Although reactivation from latency causes serious disease in immunocompromised individuals, our molecular understanding of latency is limited. Here, we delineate viral gene expression during natural HCMV persistent infection by analyzing the massive transcriptome RNA sequencing (RNA-seq) atlas generated by the Genotype-Tissue Expression (GTEx) project. This systematic analysis reveals that HCMV persistence
is prevalent in diverse tissues. Notably, we find only viral transcripts that resemble gene expression during various stages of lytic infection with no evidence of any highly restricted latency-associated viral gene expression program. To further define the transcriptional landscape during HCMV latent infection, we also used single-cell RNA-seq and a tractable experimental latency model. In contrast to some current views on latency, we also find no evidence for any highly restricted latency-associated viral gene expression program. Instead, we reveal that latency-associated gene expression largely mirrors a late lytic viral program, albeit at much lower levels of expression. Overall, our work has the potential to revolutionize our understanding of HCMV persistence and suggests that latency is governed mainly by quantitative changes, with a limited number of qualitative changes, in viral gene expression.
Human cytomegalovirus is a prevalent pathogen, infecting most of the population worldwide and establishing lifelong latency in its hosts. Although reactivation from latency causes significant morbidity and mortality in immunocompromised hosts, our molecular understanding of the latent state remains limited. Here, we examine the viral gene expression during natural and experimental latent HCMV infection on a transcriptome-wide level. In contrast to the classical views on herpesvirus latency, we find no evidence for a restricted latency-associated viral gene expression program. Instead, we reveal that latency gene expression largely resembles a late lytic viral profile, albeit at much lower levels of expression. Taken together, our data transform the current view of HCMV persistence and suggest that latency is mainly governed by quantitative rather than qualitative changes in viral gene expression.
Recent advances in transcriptome sequencing have enabled the discovery of thousands of long non-coding RNAs (lncRNAs) across many species. Though several lncRNAs have been shown to play important ...roles in diverse biological processes, the functions and mechanisms of most lncRNAs remain unknown. Two significant obstacles lie between transcriptome sequencing and functional characterization of lncRNAs: identifying truly non-coding genes from de novo reconstructed transcriptomes, and prioritizing the hundreds of resulting putative lncRNAs for downstream experimental interrogation.
We present slncky, a lncRNA discovery tool that produces a high-quality set of lncRNAs from RNA-sequencing data and further uses evolutionary constraint to prioritize lncRNAs that are likely to be functionally important. Our automated filtering pipeline is comparable to manual curation efforts and more sensitive than previously published computational approaches. Furthermore, we developed a sensitive alignment pipeline for aligning lncRNA loci and propose new evolutionary metrics relevant for analyzing sequence and transcript evolution. Our analysis reveals that evolutionary selection acts in several distinct patterns, and uncovers two notable classes of intergenic lncRNAs: one showing strong purifying selection on RNA sequence and another where constraint is restricted to the regulation but not the sequence of the transcript.
Our results highlight that lncRNAs are not a homogenous class of molecules but rather a mixture of multiple functional classes with distinct biological mechanism and/or roles. Our novel comparative methods for lncRNAs reveals 233 constrained lncRNAs out of tens of thousands of currently annotated transcripts, which we make available through the slncky Evolution Browser.
Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter ...referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.
Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by ectopic expression of different transcription factors, classically Oct4 (also known as Pou5f1), Sox2, Klf4 and Myc ...(abbreviated as OSKM). This process is accompanied by genome-wide epigenetic changes, but how these chromatin modifications are biochemically determined requires further investigation. Here we show in mice and humans that the histone H3 methylated Lys 27 (H3K27) demethylase Utx (also known as Kdm6a) regulates the efficient induction, rather than maintenance, of pluripotency. Murine embryonic stem cells lacking Utx can execute lineage commitment and contribute to adult chimaeric animals; however, somatic cells lacking Utx fail to robustly reprogram back to the ground state of pluripotency. Utx directly partners with OSK reprogramming factors and uses its histone demethylase catalytic activity to facilitate iPSC formation. Genomic analysis indicates that Utx depletion results in aberrant dynamics of H3K27me3 repressive chromatin demethylation in somatic cells undergoing reprogramming. The latter directly hampers the derepression of potent pluripotency promoting gene modules (including Sall1, Sall4 and Utf1), which can cooperatively substitute for exogenous OSK supplementation in iPSC formation. Remarkably, Utx safeguards the timely execution of H3K27me3 demethylation observed in embryonic day 10.5-11 primordial germ cells (PGCs), and Utx-deficient PGCs show cell-autonomous aberrant epigenetic reprogramming dynamics during their embryonic maturation in vivo. Subsequently, this disrupts PGC development by embryonic day 12.5, and leads to diminished germline transmission in mouse chimaeras generated from Utx-knockout pluripotent cells. Thus, we identify Utx as a novel mediator with distinct functions during the re-establishment of pluripotency and germ cell development. Furthermore, our findings highlight the principle that molecular regulators mediating loss of repressive chromatin during in vivo germ cell reprogramming can be co-opted during in vitro reprogramming towards ground state pluripotency.
Lynch syndrome (LS) is a hereditary cancer syndrome caused by autosomal dominant mutations, with high probability of early onset for several cancers, mainly colorectal cancer (CRC). The gut ...microbiome was shown to be influenced by host genetics and to be altered during cancer development. Therefore, we aimed to determine alterations in gut microbiome compositions of LS patients with and without cancer. We performed fecal microbiome analyses on samples of LS and non-LS members from the Druze ethnoreligious community in Israel, based on both their LS mutation and their cancer history. Our analysis revealed specific bacterial operational taxonomic units (OTUs) overrepresented in LS individuals as well as bacterial OTUs differentiating between the LS individuals with a history of cancer. The identified OTUs align with previous studies either correlating them to pro-inflammatory functions, which can predispose to cancer, or to the cancer itself, and as such, these bacteria can be considered as future therapeutic targets.
The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we ...characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.
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•Unbiased high-resolution profiling of mouse deterministic reprogramming to naive iPSCs•Early DNA demethylation of pluripotency enhancers definitively marks future iPSCs•Myc activity is indispensable for conducive iPS formation from somatic cells•Optimized changes in tRNA codon usage amplify the output of Myc governed program
The epigenetic dynamics of iPSC reprogramming in correctly reprogrammed cells at high-resolution remain largely undefined. Hanna and colleagues now provide comprehensive characterization during the entire course of murine fibroblast reprogramming, by using Gatad2a-Mbd3/NuRD-depleted and radically efficient reprogramming systems. These data provide insights into key questions underlying successful naive iPSC reprogramming.
Ectopic expression of lineage master regulators induces transdifferentiation. Whether cell fate transitions can be induced during various developmental stages has not been systemically examined. Here ...we discover that amongst different developmental stages, mouse embryonic stem cells (mESCs) are resistant to cell fate conversion induced by the melanocyte lineage master regulator MITF. By generating a transgenic system we exhibit that in mESCs, the pluripotency master regulator Oct4, counteracts pro-differentiation induced by Mitf by physical interference with MITF transcriptional activity. We further demonstrate that mESCs must be released from Oct4-maintained pluripotency prior to ectopically induced differentiation. Moreover, Oct4 induction in various differentiated cells represses their lineage identity in vivo. Alongside, chromatin architecture combined with ChIP-seq analysis suggest that Oct4 competes with various lineage master regulators for binding promoters and enhancers. Our analysis reveals pluripotency and transdifferentiation regulatory principles and could open new opportunities in the field of regenerative medicine.
ChatGPT, an advanced language model, is increasingly used in diverse fields, including medicine. This study explores using ChatGPT to optimize postcolonoscopy management by providing guideline-based ...recommendations and addressing low compliance rates and timing issues.
In this proof-of-concept study, 20 clinical scenarios were prepared as structured reports and free-text notes, and ChatGPT's responses were evaluated by 2 senior gastroenterologists. Compliance with guidelines and accuracy were assessed, and inter-rater agreement was calculated using Fleiss' kappa coefficient.
ChatGPT exhibited 90% compliance with guidelines and 85% accuracy, with a very good inter-rater agreement (Fleiss' kappa coefficient of .84, P < .01). ChatGPT handled multiple variations and descriptions and crafted concise patient letters.
Results suggest that ChatGPT could aid healthcare providers in making informed decisions and improve compliance with postcolonoscopy surveillance guidelines. Future research should investigate integrating ChatGPT into electronic health record systems and evaluating its effectiveness in different healthcare settings and populations.
Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N6-methyladenosine ...(m6A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m6A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m6A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.
In vitro cultured stem cells with distinct developmental capacities can contribute to embryonic or extraembryonic tissues after microinjection into pre-implantation mammalian embryos. However, ...whether cultured stem cells can independently give rise to entire gastrulating embryo-like structures with embryonic and extraembryonic compartments remains unknown. Here, we adapt a recently established platform for prolonged ex utero growth of natural embryos to generate mouse post-gastrulation synthetic whole embryo models (sEmbryos), with both embryonic and extraembryonic compartments, starting solely from naive ESCs. This was achieved by co-aggregating non-transduced ESCs, with naive ESCs transiently expressing Cdx2 or Gata4 to promote their priming toward trophectoderm and primitive endoderm lineages, respectively. sEmbryos adequately accomplish gastrulation, advance through key developmental milestones, and develop organ progenitors within complex extraembryonic compartments similar to E8.5 stage mouse embryos. Our findings highlight the plastic potential of naive pluripotent cells to self-organize and functionally reconstitute and model the entire mammalian embryo beyond gastrulation.
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•Advanced synthetic embryos (sEmbryos) self-assembled from ESCs in an ex utero setup•Naive ESCs give rise to all embryonic and extraembryonic compartments in sEmbryos•Post-gastrulation stem cell derived sEmbryos develop organ-specific progenitors•Extraembryonic compartments adequately develop in post-gastrulation whole sEmbryos
Post-gastrulation stages of development with organ progenitors and complex extra-embryonic compartments are achieved ex utero entirely from assembled naive mouse embryonic stem cells.
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