Start Site Region Research Articles

Age-related and species-specific methylation changes in the protein-coding marmoset sperm epigenome.

The sperm epigenome is thought to affect the developmental programming of the resulting embryo, influencing health and disease in later life. Age-related methylation changes in the sperm of old fathers may mediate the increased risks for reproductive and offspring medical problems. The impact of paternal age on sperm methylation has been extensively studied in humans and, to a lesser extent, in rodents and cattle. Here, we performed a comparative analysis of paternal age effects on protein-coding genes in the human and marmoset sperm methylomes. The marmoset has gained growing importance as a non-human primate model of aging and age-related diseases. Using reduced representation bisulfite sequencing, we identified age-related differentially methylated transcription start site (ageTSS) regions in 204 marmoset and 27 human genes. The direction of methylation changes was the opposite, increasing with age in marmosets and decreasing in humans. None of the identified ageTSS was differentially methylated in both species. Although the average methylation levels of all TSS regions were highly correlated between marmosets and humans, with the majority of TSS being hypomethylated in sperm, more than 300 protein-coding genes were endowed with species-specifically (hypo)methylated TSS. Several genes of the glycosphingolipid (GSL) biosynthesis pathway, which plays a role in embryonic stem cell differentiation and regulation of development, were hypomethylated (<5%) in human and fully methylated (>95%) in marmoset sperm. The expression levels and patterns of defined sets of GSL genes differed considerably between human and marmoset pre-implantation embryo stages and blastocyst tissues, respectively.

Histone H3 posttranslational modified enzymes defined neutrophil plasticity and their vulnerability to IL-10 in the course of the inflammation

BackgroundNeutrophils are a heterogeneous population capable of antimicrobial functions associated with pre-activation/activation and tissue regeneration. The specific polarisation of immune cells is mediated by the modification of ‘chromatin landscapes’, which enables differentiated access and activity of regulatory elements that guarantee their plasticity during inflammation No specific pattern within histone posttranslational modifications (PTMs) controlling this plasticity has been identified.MethodsUsing the in vitro model of inflammation, reflecting different states of neutrophils from resting, pre-activated cells to activated and reducing tissue regeneration, we have analysed 11 different histone posttranslational modifications (PTMs), PTM enzymes associated with remodelling neutrophil chromatin, and H3K4me3 ChIP-Seq Gene Ontology analysis focusing on the processes related to histone PTMs. These findings were verified by extrapolation to adequate clinical status, using neutrophils derived from the patients with sepsis (systemic septic inflammation with LPS-stimulated neutrophils), neuromyelitis optical spectrum disorders (aseptic inflammation with pre-activated neutrophils) and periodontitis (local self-limiting septic inflammation with IL-10-positive neutrophils).ResultsPhysiological activation of neutrophils comprises a pre-activation characterised by histone H3K27ac and H3K4me1, which position enhancers; direct LPS exposure is induced explicitly by H3K4me3 which marked Transcription Start Site (TSS) regions and low-level of H3K9me3, H3K79me2 and H3K27me3 which, in turn, marked repressed genes. Contrary to antimicrobial action, IL-10 positively induced levels of H3S10p and negatively H3K9me3, which characterised processes related to the activation of genes within heterochromatin mediated by CHD1 and H3K9me3 specific demethylase JMJD2A. IL-10 protects changes within histone PTMs induced by TNF or LPS that affected H3K4me3-specific methyltransferase SETD1A and MLL1. Neutrophils previously exposed to inflammatory factors become unvulnerable to IL-10 because previous LPS stimulation interrupts TSS regions marked by H3K4me3 of CHD1 and JMJD2A genes. Therefore, LPS-activated neutrophils are disabled to induce CHD1/JMJD2A enzymes by IL-10, making this process irreversible. Because transcription of JMJD2A and CHD1 also depends on TSS positioning by H3K4me3, neutrophils before LPS stimulation become insensitive to IL-10.ConclusionNeutrophils, once pre-activated by TNF or directly stimulated by LPS, become insensitive to the anti-inflammatory effects of IL-10, and vice versa; IL-10 protects neutrophils against these proinflammatory stimuli. This phenomenon is responsible for disturbing the natural process of resolving inflammation and tissue regeneration.Graphical

The Role of KDM2A and H3K36me2 Demethylation in Modulating MAPK Signaling During Neurodevelopment.

Intellectual disability (ID) is a condition characterized by cognitive impairment and difficulties in adaptive functioning. In our research, we identified two de novo mutations (c.955C>T and c.732C>A) at the KDM2A locus in individuals with varying degrees of ID. In addition, by using the Gene4Denovo database, we discovered five additional cases of de novo mutations in KDM2A. The mutations we identified significantly decreased the expression of the KDM2A protein. To investigate the role of KDM2A in neural development, we used both 2D neural stem cell models and 3D cerebral organoids. Our findings demonstrated that the reduced expression of KDM2A impairs the proliferation of neural progenitor cells (NPCs), increases apoptosis, induces premature neuronal differentiation, and affects synapse maturation. Through ChIP-Seq analysis, we found that KDM2A exhibited binding to the transcription start site regions of genes involved in neurogenesis. In addition, the knockdown of KDM2A hindered H3K36me2 binding to the downstream regulatory elements of genes. By integrating ChIP-Seq and RNA-Seq data, we made a significant discovery of the core genes' remarkable enrichment in the MAPK signaling pathway. Importantly, this enrichment was specifically linked to the p38 MAPK pathway. Furthermore, disease enrichment analysis linked the differentially-expressed genes identified from RNA-Seq of NPCs and cerebral organoids to neurodevelopmental disorders such as ID, autism spectrum disorder, and schizophrenia. Overall, our findings suggest that KDM2A plays a crucial role in regulating the H3K36me2 modification of downstream genes, thereby modulating the MAPK signaling pathway and potentially impacting early brain development.

OVCH1 Antisense RNA 1 is differentially expressed between non-frail and frail old adults.

While some old adults stay healthy and non-frail up to late in life, others experience multimorbidity and frailty often accompanied by a pro-inflammatory state. The underlying molecular mechanisms for those differences are still obscure. Here, we used gene expression analysis to understand the molecular underpinning between non-frail and frail individuals in old age. Twenty-four adults (50% non-frail and 50% frail) from InCHIANTI study were included. Total RNA extracted from whole blood was analyzed by Cap Analysis of Gene Expression (CAGE). CAGE identified transcription start site (TSS) and active enhancer regions. We identified a set of differentially expressed (DE) TSS and enhancer between non-frail and frail and male and female participants. Several DE TSSs were annotated as lncRNA (XIST and TTTY14) and antisense RNAs (ZFX-AS1 and OVCH1 Antisense RNA 1). The promoter region chr6:366,786,54-366,787,97;+ was DE and overlapping the longevity CDKN1A gene. GWAS-LD enrichment analysis identifies overlapping LD-blocks with the DE regions with reported traits in GWAS catalog (isovolumetric relaxation time and urinary tract infection frequency). Furthermore, we used weighted gene co-expression network analysis (WGCNA) to identify changes of gene expression associated with clinical traits and identify key gene modules. We performed functional enrichment analysis of the gene modules with significant trait/module correlation. One gene module is showing a very distinct pattern in hub genes. Glycogen Phosphorylase L (PYGL) was the top ranked hub gene between non-frail and frail. We predicted transcription factor binding sites (TFBS) and motif activity. TF involved in age-related pathways (e.g., FOXO3 and MYC) shows different expression patterns between non-frail and frail participants. Expanding the study of OVCH1 Antisense RNA 1 and PYGL may help understand the mechanisms leading to loss of homeostasis that ultimately causes frailty.

Abstract 1486: The regulatory mechanism of LncRNA CCAT1 via three-dimensional genome structure in colorectal cancer cells

Abstract Kruppel-like factor 5 (KLF5) belongs to zinc finger transcription factors and its expression is up-regulated in specific cancer types, such as gastrointestinal cancer and squamous carcinoma. It is reported that KLF5 is associated with the maintenance of stemness and essential for the integrity of intestinal epithelium and oncogenesis. Colon Cancer Associated Transcript 1 (CCAT1) is a long noncoding RNA that was initially found to be up-regulated in colorectal cancer (CRC), and studies have reported that high CCAT1 expression is associated with poor prognosis in CRC patients. In this study, we performed in vitro enChIP (engineered DNA-binding molecule-mediated chromatin immunoprecipitation)-sequencing to identify the KLF5 enhancer region which interacts with KLF5 promoter region. We successfully identified the KLF5 enhancer region in colorectal cancer cells and further found that KLF5 promoter region interacted with the transcription start site (TSS) region of CCAT1 gene, which is located at the different chromosome. Our results indicated that KLF5 protein participated in the core regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which contributes to construction of the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. We also found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples and that KLF5 regulated CCAT1 expression. In conclusion, our data propose the mechanistic insight that KLF5 protein constructs the core regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC. Citation Format: Yuhki Yokoyama, Hirofumi Yamamoto. The regulatory mechanism of LncRNA CCAT1 via three-dimensional genome structure in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1486.

Unscheduled epigenetic modifications cause genome instability and sterility through aberrant R-loops following starvation.

During starvation, organisms modify both gene expression and metabolism to adjust to the energy stress. We previously reported that Caenorhabditis elegans lacing AMP-activated protein kinase (AMPK) exhibit transgenerational reproductive defects associated with abnormally elevated trimethylated histone H3 at lysine 4 (H3K4me3) levels in the germ line following recovery from acute starvation. Here, we show that these H3K4me3 marks are significantly increased at promoters, driving aberrant transcription elongation resulting in the accumulation of R-loops in starved AMPK mutants. DNA-RNA immunoprecipitation followed byhigh-throughput sequencing (DRIP-seq) analysis demonstrated that a significant proportion of the genome was affected by R-loop formation. This was most pronounced in the promoter-transcription start site regions of genes, in which the chromatin was modified by H3K4me3. Like H3K4me3, the R-loops were also found to be heritable, likely contributing to the transgenerational reproductive defects typical of these mutants following starvation. Strikingly, AMPK mutant germ lines show considerably more RAD-51 (the RecA recombinase) foci at sites of R-loop formation, potentially sequestering them from their roles at meiotic breaks or at sites of induced DNA damage. Our study reveals a previously unforeseen role of AMPK in maintaining genome stability following starvation. The downstream effects of R-loops on DNA damage sensitivity and germline stem cell integrity may account for inappropriate epigenetic modification that occurs in numerous human disorders, including various cancers.

Transient Methionine Deprivation Triggers Histone Modification and Potentiates Differentiation of Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) require high levels of methionine (Met). Met deprivation results in a rapid decrease in intracellular S-adenosyl-methionine (SAM), poising human iPSCs for differentiation and leading to the apoptosis of undifferentiated cells. Met deprivation triggers rapid metabolic changes, including SAM, followed by reversible epigenetic modifications. Here, we show that short-term Met deprivation impairs the pluripotency network through epigenetic modification in a 3D suspension culture. The trimethylation of lysine 4 on histone H3 (H3K4me3) was drastically affected compared with other histone modifications. Short-term Met deprivation specifically affects the transcription start site (TSS) region of genes, such as those involved in the transforming growth factor β pathway and cholesterol biosynthetic process, besides key pluripotent genes such as NANOG and POU5F1. The expression levels of these genes decreased, correlating with the loss of H3K4me3 marks. Upon differentiation, Met deprivation triggers the upregulation of various lineage-specific genes, including key definitive endoderm genes, such as GATA6. Upon differentiation, loss of H3K27me3 occurs in many endodermal genes, switching from a bivalent to a monovalent (H3K4me3) state. In conclusion, Met metabolism maintains the pluripotent network with histone marks, and their loss potentiates differentiation.

Promoter sequence and architecture determine expression variability and confer robustness to genetic variants.

Genetic and environmental exposures cause variability in gene expression. Although most genes are affected in a population, their effect sizes vary greatly, indicating the existence of regulatory mechanisms that could amplify or attenuate expression variability. Here, we investigate the relationship between the sequence and transcription start site architectures of promoters and their expression variability across human individuals. We find that expression variability can be largely explained by a promoter's DNA sequence and its binding sites for specific transcription factors. We show that promoter expression variability reflects the biological process of a gene, demonstrating a selective trade-off between stability for metabolic genes and plasticity for responsive genes and those involved in signaling. Promoters with a rigid transcription start site architecture are more prone to have variable expression and to be associated with genetic variants with large effect sizes, while a flexible usage of transcription start sites within a promoter attenuates expression variability and limits genotypic effects. Our work provides insights into the variable nature of responsive genes and reveals a novel mechanism for supplying transcriptional and mutational robustness to essential genes through multiple transcription start site regions within a promoter.

R-loops at microRNA encoding loci promote co-transcriptional processing of pri-miRNAs in plants

In most organisms, the maturation of nascent RNAs is coupled to transcription. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNA genes (MIRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs coupled to transcription. We found that the entire biogenesis occurs co-transcriptionally for pri-miRNAs processed from the loop of the hairpin but requires a second nucleoplasmic step for those processed from the base. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops, formed near the transcription start site region of MIRNAs, promote co-transcriptional pri-miRNA processing. Furthermore, our results suggest the neofunctionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

EPCO-08. ATRX DEFICIENCY INDUCES DYSFUNCTIONAL HETEROCHROMATIN ARCHITECTURE IN GLIOMAS AND ESTABLISHES DISEASE-DEFINING TRANSCRIPTIONAL NETWORKS

Abstract Loss of ATRX (Alpha Thalassemia/Mental Retardation Syndrome X, a member of SWI/SNF family chromatin regulator is altered in diffuse gliomas and defines molecular subtypes with aggressive behavior. Mechanistically, ATRX regulates incorporation of histone H3.3 into chromatin sites across the genome, maintains alternative lengthening of telomeres and establishes genomic distribution of polycomb responsive genes. We have recently reported Atrx deficiency induces glioma oncogenic features via widespread alterations in chromatin accessibility using mouse Neural Progenitor Cells (mNPCs- Tp53 -/-,Atrx -/-). Surprisingly, Atrx was found to be associated with transcription start site and enhancer regions, suggesting their strong association with epigenome architecture. In this background, we have recently performed ChIP-seq for histone marks that define active transcription, enhancers, repressors and gene bodies and Cohesion molecules on Atrx intact and deficient mNPCs. Our integrated analysis reports depletion of H3K9me3 loci’s with enrichment of H3K27me3 marks that coincidently enriched with Atrx binding sites and Lamina-Associated Domains (LADs). GSEA confirmed that the genes corresponding to “newly formed LADs” in mNPC-to-astrocyte differentiation are significantly enriched for genes down-regulated in Atrx deficient mNPCs and belongs to Gene Ontology categories such as cell cycle, chromosome organization and DNA damage. Alternatively, genes corresponding to decreased LAD association are enriched for up-regulated genes in Atrx deficient mNPCs and for regulation of differentiation, adhesion and cell death. Additionally, whole-genome bisulphite sequencing further demonstrated loss of methylation marks at H3K9me3 sites in Atrx deficient mNPCs, suggesting perturbations of heterochromatin regions leading to activation of canonical signals that define glioma phenotype and disease-state. To summarize, our data establishes tangible links between Atrx deficiency and dysregulated chromatin and heterochromatin architecture in gliomas and suggests the role of Atrx in establishing global chromatin features and transcriptional networks. Further, our data unravel novel therapeutic molecules/pathways for engineering potential.

A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells

BackgroundKLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene. In this study, we aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC).MethodsIn vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)-seq method was used to identify the regions that bind to the KLF5 promoter.ResultsWe revealed that the KLF5 promoter region interacted with the KLF5 enhancer region as well as the transcription start site (TSS) region of the Colon Cancer Associated Transcript 1 (CCAT1) gene. Notably, the heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. In vitro analysis indicated that KLF5 regulated CCAT1 expression and we found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples.ConclusionsOur data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC.

Metabolically controlled histone H4K5 acylation/acetylation ratio drives BRD4 genomic distribution.

In addition to acetylation, histones are modified by a series of competing longer-chain acylations. Most of these acylation marks are enriched and co-exist with acetylation on active gene regulatory elements. Their seemingly redundant functions hinder our understanding of histone acylations' specific roles. Here, by using an acute lymphoblastic leukemia (ALL) cell model and blasts from individuals with B-precusor ALL (B-ALL), we demonstrate a role of mitochondrial activity in controlling the histone acylation/acetylation ratio, especially at histone H4 lysine 5 (H4K5). An increase in the ratio of non-acetyl acylations (crotonylation or butyrylation) over acetylation on H4K5 weakens bromodomain containing protein 4 (BRD4) bromodomain-dependent chromatin interaction and enhances BRD4 nuclear mobility and availability for binding transcription start site regions of active genes. Our data suggest that the metabolism-driven control of the histone acetylation/longer-chain acylation(s) ratio could be a common mechanism regulating the bromodomain factors' functional genomic distribution.

CREB-mediated transcriptional activation of NRMT1 drives muscle differentiation

ABSTRACT The N-terminal methyltransferase NRMT1 is an important regulator of protein/DNA interactions and plays a role in many cellular processes, including mitosis, cell cycle progression, chromatin organization, DNA damage repair, and transcriptional regulation. Accordingly, loss of NRMT1 results in both developmental pathologies and oncogenic phenotypes. Though NRMT1 plays such important and diverse roles in the cell, little is known about its own regulation. To better understand the mechanisms governing NRMT1 expression, we first identified its predominant transcriptional start site and minimal promoter region with predicted transcription factor motifs. We then used a combination of luciferase and binding assays to confirm CREB1 as the major regulator of NRMT1 transcription. We tested which conditions known to activate CREB1 also activated NRMT1 transcription, and found CREB1-mediated NRMT1 expression was increased during recovery from serum starvation and muscle cell differentiation. To determine how NRMT1 expression affects myoblast differentiation, we used CRISPR/Cas9 technology to knock out NRMT1 expression in immortalized C2C12 mouse myoblasts. C2C12 cells depleted of NRMT1 lacked Pax7 expression and were unable to proceed down the muscle differentiation pathway. Instead, they took on characteristics of C2C12 cells that have transdifferentiated into osteoblasts, including increased alkaline phosphatase and type I collagen expression and decreased proliferation. These data implicate NRMT1 as an important downstream target of CREB1 during muscle cell differentiation.

The COMPASS-like complex modulates fungal development and pathogenesis by regulating H3K4me3-mediated targeted gene expression in Magnaporthe oryzae.

Histone‐3‐lysine‐4 (H3K4) methylation is catalysed by the multiprotein complex known as the Set1/COMPASS or MLL/COMPASS‐like complex, an element that is highly evolutionarily conserved from yeast to humans. However, the components and mechanisms by which the COMPASS‐like complex targets the H3K4 methylation of plant‐pathogenic genes in fungi remain elusive. Here we present a comprehensive analysis combining biochemical, molecular, and genome‐wide approaches to characterize the roles of the COMPASS‐like family in the rice blast fungus Magnaporthe oryzae, a model plant pathogen. We purified and identified six conserved subunits of COMPASS from M. oryzae: MoBre2 (Cps60/ASH2L), MoSpp1 (Cps40/Cfp1), MoSwd2 (Cps35), MoSdc1 (Cps25/DPY30), MoSet1 (MLL/ALL), and MoRbBP5 (Cps50), using an affinity tag on MoBre2. We determined the sequence repeat in dual‐specificity kinase splA and ryanodine receptors domain of MoBre2 can interact directly with the DPY30 domain of MoSdc1 in vitro. Furthermore, we found that deletion of the genes encoding COMPASS subunits of MoBre2, MoSPP1, and MoSwd2 caused similar defects regarding invasive hyphal development and pathogenicity. Genome‐wide profiling of H3K4me3 revealed that it has remarkable co‐occupancy at the transcription start site regions of target genes. Significantly, these target genes are often involved in spore germination and pathogenesis. Decreased gene expression caused by the deletion of MoBre2, MoSwd2, or MoSpp1 was highly correlated with a decrease in H3K4me3. These results suggest that MoBre2, MoSpp1, and MoSwd2 function as a whole COMPASS complex, contributing to fungal development and pathogenesis by regulating H3K4me3‐targeted genes in M. oryzae.

Nsd2 Represses Endogenous Retrovirus MERVL in Embryonic Stem Cells.

The facilitates chromatin transcription (FACT) complex is a histone H2A/H2B chaperone, which represses endogenous retroviruses (ERVs) and transcription of ERV-chimeric transcripts. It binds to both transcription start site and gene body region. Here, we investigated the downstream targets of FACT complex to identify the potential regulators of MERVL, which is a key 2-cell marker gene. H3K36me2 profile was positively correlated with that of FACT component Ssrp1. Among H3K36me2 deposition enzymes, Nsd2 was downregulated after the loss of Ssrp1. Furthermore, we demonstrated that Nsd2 repressed the expression of ERVs without affecting the expression of pluripotency genes. The expression of MERVL and 2-cell genes was partially rescued by Nsd2 overexpression. The enrichment of H3K36me2 decreased on MERVL-chimeric gene in ESCs without Ssrp1. Our study discovers that Nsd2 is a repressor of MERVL, and FACT partially represses MERVL expression by regulating the expression of Nsd2 and its downstream H3K36me2.

Elongin A associates with actively transcribed genes and modulates enhancer RNA levels with limited impact on transcription elongation rate in vivo

Elongin A (EloA) is an essential transcription factor that stimulates the rate of RNA polymerase II (Pol II) transcription elongation in vitro. However, its role as a transcription factor in vivo has remained underexplored. Here we show that in mouse embryonic stem cells, EloA localizes to both thousands of Pol II transcribed genes with preference for transcription start site and promoter regions and a large number of active enhancers across the genome. EloA deletion results in accumulation of transcripts from a subset of enhancers and their adjacent genes. Notably, EloA does not substantially enhance the elongation rate of Pol II in vivo. We also show that EloA localizes to the nucleoli and associates with RNA polymerase I transcribed ribosomal RNA gene, Rn45s. EloA is a highly disordered protein, which we demonstrate forms phase-separated condensates in vitro, and truncation mutations in the intrinsically disordered regions (IDR) of EloA interfere with its targeting and localization to the nucleoli. We conclude that EloA broadly associates with transcribed regions, tunes RNA Pol II transcription levels via impacts on enhancer RNA synthesis, and interacts with the rRNA producing/processing machinery in the nucleolus. Our work opens new avenues for further investigation of the role of this functionally multifaceted transcription factor in enhancer and ribosomal RNA biology.

Demethylation of HACE1 gene promoter by propofol promotes autophagy of human A549 cells.

Propofol (2,6-diisopropylphenol) is one of the most commonly used intravenous anesthetics and possesses a number of non-anesthetic effects, including antitumor function. The aim of the present study was to elucidate the antitumor molecular mechanism of propofol on A549 and H1299 cells. A549 and H1299 cells were treated in the presence or absence of different concentrations (0, 60 or 120 µmol) of propofol for different durations (0, 24, 48 or 72 h), and proliferation was detected by MTT and colony formation assays; the protein levels of optineurin (OPTN) ubiquitination, HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1), methyl-CpG binding domain protein 3 (MBD3) and Microtubule-associated protein 1A/1B-light chain 3 were detected by immunoblotting or quantitative (q)PCR; the methylation state of the HACE1 gene promoter was detected by bisulfite DNA sequencing; and binding of MBD3 on HACE1 gene promoter was detected by chromatin immunoprecipitation-qPCR. Propofol inhibited proliferation of A549 and H1299 cells and promoted HACE1-OPTN axis-mediated selective autophagy activity by increasing the protein expression levels of HACE1 via demethylating its promoter region. Furthermore, propofol promoted expression levels of MBD3 and binding to the −1,000 to −1 bp (transcription start site) region of HACE1 gene promoter. MBD3-knockdown experiments indicated that propofol inhibited proliferation of A549 cells in a MBD3-dependent manner. Thus, the findings of the present study provided a potential antitumor molecular mechanism mediated by propofol.

Chromatin-associated SUMOylation controls the transcriptional switch between plant development and heat stress responses

The post-translational protein modification known as SUMOylation has conserved roles in the heat stress responses of various species. The functional connection between the global regulation of gene expression and chromatin-associated SUMOylation in plant cells is unknown. Here, we uncovered a genome-wide relationship between chromatin-associated SUMOylation and transcriptional switches in Arabidopsis thaliana grown at room temperature, exposed to heat stress, and exposed to heat stress followed by recovery. The small ubiquitin-like modifier (SUMO)-associated chromatin sites, characterized by whole-genome ChIP-seq, were generally associated with active chromatin markers. In response to heat stress, chromatin-associated SUMO signals increased at promoter-transcriptional start site regions and decreased in gene bodies. RNA-seq analysis supported the role of chromatin-associated SUMOylation in transcriptional activation during rapid responses to high temperature. Changes in SUMO signals on chromatin were associated with the upregulation of heat-responsive genes and the downregulation of growth-related genes. Disruption of the SUMO ligase gene SIZ1 abolished SUMO signals on chromatin and attenuated rapid transcriptional responses to heat stress. The SUMO signal peaks were enriched in DNA elements recognized by distinct groups of transcription factors under different temperature conditions. These observations provide evidence that chromatin-associated SUMOylation regulates the transcriptional switch between development and heat stress response in plant cells.

Premature Termination Codon-Bearing mRNA Mediates Genetic Compensation Response.

The genetic compensation response (GCR), triggered by deleterious mutations but not by gene knockdown, has been proposed to explain many phenotypic discrepancies between gene-knockout and gene-knockdown models. GCRs have been observed in many model organisms from mice to Arabidopsis. Although the GCR is beneficial for organism survival, it impedes the exploration of gene function as many knockout mutants do not display discernible phenotypes due to the GCR. Uncovering how the mechanism of GCR operates is not only a fundamental goal in biology but also may provide a key solution in the unmasking of phenotypes in mutants displaying GCRs. Using zebrafish as the model, two recent studies have provided a molecular basis to explain this genetic paradox by demonstrating that the nonsense-mediated mRNA decay pathway is essential for nonsense mRNA to upregulate the expression of its homologous genes through an enhancement of histone H3 Lys4 trimethylation (H3K4me3) at the transcription start site regions of the compensatory genes. Here, we summarize the progress on the molecular mechanism of the GCR and make suggestions on how to overcome GCRs in the generation of genetic mutants.

Nonsense mutations and genetic compensation response

The genetic compensation response (GCR) was firstly described in zebrafish to explain the phenotypic discrepancies between gene-knockout and gene-knockdown, whereby a deleterious mutation, but not gene-knockdown, can lead to the transcriptional upregulation of related genes, which can assume the function of the mutated gene. This phenomenon was also found in other model systems including mice and Arabidopsis. However, the underlying molecular mechanism of the GCR remains elusive until two papers were published in Nature on April 3, 2019: one from our lab and the other from Stainier's lab. Using different genetic mutants of various genes in zebrafish or culture cells of mice, both of us reveal that the upregulation of compensatory genes is only triggered by mutations that generate a premature termination codon (PTC); the compensatory genes share nucleotide sequence homology to the mutated genes; nonsense mRNA mediated decay pathway (NMD) is essential for the induction of GCR, and the increased transcription of the compensatory genes is accompanied by an enhancement of H3K4 trimethylation (H3K4me3) at their transcription start site (TSS) regions. In this review, we summarize the mechanisms of the GCR proposed in the two studies.