live-cell-frster-resonance-energy-transfer-imaging-metabolically

Live-Cell F&#246;rster Resonance Energy Transfer Imaging of Metabolically Regulated Akt Activation Dynamics in HepG2 Cells

Metabolically regulated Akt activation is a critical node in the insulin signaling cascade and provides valuable insights into the relationship between ...

Southern University of Science and Technology

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.

Unified Analysis of Multiple ChIP-Seq Datasets.

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.

β-Catenin safeguards the ground state of mousepluripotency by strengthening the robustness of the transcriptional apparatus.

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.

Identifying transposable element expression dynamics and heterogeneity during development at the single-cell level with a processing pipeline scTE.

A major event in embryonic development is the rearrangement of epigenetic information as the somatic genome is reprogrammed for a new round of organismal development. Epigenetic data are held in chemical modifications on DNA and histones, and there are dramatic and dynamic changes in these marks during embryogenesis. However, the mechanisms behind this intricate process and how it is regulating and responding to embryonic development remain unclear. As embryos develop from totipotency to pluripotency, they pass through several distinct stages that can be captured permanently or transiently . Pluripotent naïve cells resemble the early epiblast, primed cells resemble the late epiblast, and blastomere-like cells have been isolated, although fully totipotent cells remain elusive. Experiments using these  model systems have led to insights into chromatin changes in embryonic development, which has informed exploration of pre-implantation embryos. Intriguingly, human and mouse cells rely on different signaling and epigenetic pathways, and it remains a mystery why this variation exists. In this review, we will summarize the chromatin rearrangements in early embryonic development, drawing from genomic data from  cell lines, and human and mouse embryos.

Chromatin and Epigenetic Rearrangements in Embryonic Stem Cell Fate Transitions.

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.

Transposable element sequence fragments incorporated into coding and noncoding transcripts modulate the transcriptome of human pluripotent stem cells.

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.

Transposable Elements in Pluripotent Stem Cells and Human Disease.

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.

Metabolic and epigenetic dysfunctions underlie the arrest of in vitro fertilized human embryos in a senescent-like state.

RNA N-methyladenosine (mA) is the most abundant internal mRNA modification and forms part of an epitranscriptomic system that modulates RNA function. mA is reversibly catalyzed by specific enzymes, and those modifications can be recognized by RNA-binding proteins that in turn regulate biological processes. Although there are many reports demonstrating mA participation in critical biological functions, this exploration has mainly been conducted through the global KO or knockdown of the writers, erasers, or readers of mA. Consequently, there is a lack of information about the role of mA on single transcripts in biological processes, posing a challenge in understanding the biological functions of mA. Here, we demonstrate a CRISPR/dCas13a-based RNA mA editors, which can target RNAs using a single or multiple CRISPR RNA array to methylate or demethylate mA in human 293T cells and mouse embryonic stem cells. We systematically assay its capabilities to enable the targeted rewriting of mA dynamics, including modulation of circular RNA translation and transcript half-life. Finally, we use the system to specifically modulate mA levels on the noncoding XIST (X-inactive specific transcript) to modulate X chromosome silencing and activation. The editors described here can be used to explore the roles of mA in biological processes.

A programmable system to methylate and demethylate N-methyladenosine (mA) on specific RNA transcripts in mammalian cells.

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.

Restricting epigenetic activity promotes the reprogramming of transformed cells to pluripotency in a line-specific manner.

c-Jun is a proto-oncogene functioning as a transcription factor to activate gene expression under many physiological and pathological conditions, particularly in somatic cells. However, its role in early embryonic development remains unknown.

c-Jun as a one-way valve at the naive to primed interface.

Carrimycin is a potential immune-regulating agent for sepsis in patients with tumors. In this study, we investigated its effects on inflammation and immune function in tumor patients with sepsis. In total, 120 participants were randomized to receive either carrimycin treatment (400 mg/day) (n = 62) or placebo (n = 58) for 7 days. The primary outcomes were immune-related indicators. Subsequently, patients were stratified into two subgroups (CD4 < 38.25% and CD8 < 25.195%). Ninety-nine participants were analyzed: 47 and 52 in the carrimycin and placebo groups, respectively. HLA-DR levels were rapidly increased in the carrimycin group; however, the placebo group initially experienced a decline in HLA-DR level at 1 day after administration. In the subgroup with CD4 < 38.25%, the carrimycin group exhibited significantly higher HLA-DR levels than the placebo group (2.270, P = 0.023) 1 day after administration and the degree of increase in HLA-DR in the carrimycin group was higher than that in the placebo group (2.057, P = 0.040). In the CD8 < 25.195% subgroup, the carrimycin group demonstrated significantly higher levels of CD8 T cells than the placebo group at 3 (2.300P = 0.027) and 5 (2.106, P = 0.035) days after administration. Carrimycin intervention led to significant reductions in the SOFA, APACHE II, PCT, and CRP levels. No adverse events were observed. In tumor patients with sepsis, particularly in those experiencing immunological suppression, carrimycin effectively regulates immune responses by increasing HLA-DR and CD8 T cell levels and plays an anti-infective role, reducing disease severity. (Chictr.org.cn, ID Number: ChiCTR2000032339).

Effects of carrimycin on biomarkers of inflammation and immune function in tumor patients with sepsis: A multicenter double-blind randomized controlled trial.

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.

A primate-specific endogenous retroviral envelope protein sequesters SFRP2 to regulate human cardiomyocyte development.

Epigenetic control of cell fates is a critical determinant to maintain cell type stability and permit differentiation during embryonic development. However, the epigenetic control mechanisms are not well understood. Here, it is shown that the histone acetyltransferase reader protein BRD8 impairs the conversion of primed mouse EpiSCs (epiblast stem cells) to naive mouse ESCs (embryonic stem cells). BRD8 works by maintaining histone acetylation on promoters and transcribed gene bodies. BRD8 is responsible for maintaining open chromatin at somatic genes, and histone acetylation at naive-specific genes. When Brd8 expression is reduced, chromatin accessibility is unchanged at primed-specific genes, but histone acetylation is reduced. Conversely, naive-specific genes has reduced repressive chromatin marks and acquired accessible chromatin more rapidly during the cell type conversion. It is shown that this process requires active histone deacetylation to promote the conversion of primed to naive. This data supports a model for BRD8 reading histone acetylation to accurately localize the genome-wide binding of the histone acetyltransferase KAT5. Overall, this study shows how the reading of the histone acetylation state by BRD8 maintains cell type stability and both enables and impairs stem cell differentiation.

BRD8 Guards the Pluripotent State by Sensing and Maintaining Histone Acetylation.

The nuclear matrix, a proteinaceous gel composed of proteins and RNA, is an important nuclear structure that supports chromatin architecture, but its role in human pluripotent stem cells (hPSCs) has not been described. Here we show that by disrupting heterogeneous nuclear ribonucleoprotein U (HNRNPU) or the nuclear matrix protein, Matrin-3, primed hPSCs adopted features of the naive pluripotent state, including morphology and upregulation of naive-specific marker genes. We demonstrate that HNRNPU depletion leads to increased chromatin accessibility, reduced DNA contacts and increased nuclear size. Mechanistically, HNRNPU acts as a transcriptional co-factor that anchors promoters of primed-specific genes to the nuclear matrix with POLII to promote their expression and their RNA stability. Overall, HNRNPU promotes cell-type stability and when reduced promotes conversion to earlier embryonic states.

Article	Year
Unified Analysis of Multiple ChIP-Seq Datasets. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 32822050	2021
β-Catenin safeguards the ground state of mousepluripotency by strengthening the robustness of the transcriptional apparatus. Science advances\| PubMed ID: 32832621	2020
Identifying transposable element expression dynamics and heterogeneity during development at the single-cell level with a processing pipeline scTE. Nature communications\| PubMed ID: 33674594	2021
Chromatin and Epigenetic Rearrangements in Embryonic Stem Cell Fate Transitions. Frontiers in cell and developmental biology\| PubMed ID: 33681220	2021
Transposable element sequence fragments incorporated into coding and noncoding transcripts modulate the transcriptome of human pluripotent stem cells. Nucleic acids research\| PubMed ID: 34390351	2021
Transposable Elements in Pluripotent Stem Cells and Human Disease. Frontiers in genetics\| PubMed ID: 35719395	2022
Metabolic and epigenetic dysfunctions underlie the arrest of in vitro fertilized human embryos in a senescent-like state. PLoS biology\| PubMed ID: 35771762	2022
A programmable system to methylate and demethylate N-methyladenosine (mA) on specific RNA transcripts in mammalian cells. The Journal of biological chemistry\| PubMed ID: 36162509	2022
Restricting epigenetic activity promotes the reprogramming of transformed cells to pluripotency in a line-specific manner. Cell death discovery\| PubMed ID: 37452056	2023
c-Jun as a one-way valve at the naive to primed interface. Cell & bioscience\| PubMed ID: 37838693	2023
Effects of carrimycin on biomarkers of inflammation and immune function in tumor patients with sepsis: A multicenter double-blind randomized controlled trial. Pharmacological research\| PubMed ID: 37984505	2023
A primate-specific endogenous retroviral envelope protein sequesters SFRP2 to regulate human cardiomyocyte development. Cell stem cell\| PubMed ID: 39146934	2024
BRD8 Guards the Pluripotent State by Sensing and Maintaining Histone Acetylation. Advanced science (Weinheim, Baden-Wurttemberg, Germany)\| PubMed ID: 39656858	2025
The nuclear matrix stabilizes primed-specific genes in human pluripotent stem cells. Nature cell biology\| PubMed ID: 39789220	2025

Gang Ma

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