Demethylase FTO-mediated m6A modification of SIK1 modulates placental cytotrophoblast syncytialization in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) represents a common complication during pregnancy that affects fetoplacental development. We demonstrated the existence of impaired trophoblast syncytialization under hyperglycemic conditions. However, the exact mechanism remains unknown. RNA N6-methyladenosine (m6A) is an emerging regulatory mechanism of mRNA and participates in various biological processes. We described the global m6A modification pattern in T2DM placenta by the combined analysis of methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and RNA sequencing (RNA-Seq). Both the m6A modification and expression of SIK1, which is critical for syncytialization, were significantly decreased in trophoblast exposed to hyperglycemic conditions. In addition, the m6A demethylase fat mass and obesity-associated protein (FTO) affects the expression and mRNA stability of SIK1 by binding to its 3'-untranslated region (UTR) m6A site. This work reveals that the FTO-m6A-SIK1 axis plays critical roles in regulating syncytialization in the placenta.

Xbp1 targets canonical UPRER and non-canonical pathways in separate tissues to promote longevity.

Transcription factors can reprogram gene expression to promote longevity. Here, we investigate the role of Drosophila Xbp1. Xbp1 is activated by splicing of its primary transcript, Xbp1u, to generate Xbp1s, a key activator of the endoplasmic reticulum unfolded protein response (UPRER). We show that Xbp1s induces the conical UPRER in the gut, promoting longevity from the resident stem cells. In contrast, in the fat body, Xbp1s does not appear to trigger UPRER but alters metabolic gene expression and is still able to extend lifespan. In the fat body, Xbp1s and dFOXO impinge on the same target genes, including the PGC-1α orthologue Srl, and dfoxo requires Xbp1 to extend lifespan. Interestingly, unspliceable version of the Xbp1 mRNA, Xbp1u can also extend lifespan, hinting at roles in longevity for the poorly characterized Xbp1u transcription factor. These findings reveal the diverse functions of Xbp1 in longevity in the fruit fly.

Protein degradation of Lsd1 is mediated by Bre1 yet opposed by Lsd1-interacting lncRNAs during fly follicle development.

Tissue development, homeostasis, and repair all require efficient progenitor expansion. Lysine-specific demethylase 1 (Lsd1) maintains plastic epigenetic states to promote progenitor proliferation while overexpressed Lsd1 protein causes oncogenic gene expression in cancer cells. However, the precise regulation of Lsd1 protein expression at the molecular level to drive progenitor differentiation remains unclear. Here, using Drosophila melanogaster oogenesis as our experimental system, we discovered molecular machineries that modify Lsd1 protein stability in vivo. Through genetic and biochemical analyses, an E3 ubiquitin ligase, Bre1, was identified as required for follicle progenitor differentiation, likely by mediating Lsd1 protein degradation. Interestingly, specific Lsd1-interacting long non-coding RNAs (LINRs) were found to antagonize Bre1-mediated Lsd1 protein degradation. The intricate interplay discovered among the Lsd1 complex, LINRs and Bre1 provides insight into how Lsd1 protein stability is fine-tuned to underlie progenitor differentiation in vivo.

YTHDF3 modulates the Cbln1 level by recruiting BTG2 and is implicated in the impaired cognition of prenatal hypoxia offspring.

The "Fetal Origins of Adult Disease (FOAD)" hypothesis holds that adverse factors during pregnancy can increase the risk of chronic diseases in offspring. Here, we investigated the effects of prenatal hypoxia (PH) on brain structure and function in adult offspring and explored the role of the N6-methyladenosine (m6A) pathway. The results suggest that abnormal cognition in PH offspring may be related to the dysregulation of the m6A pathway, specifically increased levels of YTHDF3 in the hippocampus. YTHDF3 interacts with BTG2 and is involved in the decay of Cbln1 mRNA, leading to the down-regulation of Cbln1 expression. Deficiency of Cbln1 may contribute to abnormal synaptic function, which in turn causes cognitive impairment in PH offspring. This study provides a scientific clues for understanding the mechanisms of impaired cognition in PH offspring and provides a theoretical basis for the treatment of cognitive impairment in offspring exposed to PH.

A helicase-independent role of DHX15 promotes MYC stability and acute leukemia cell survival.

DHX15 has been implicated in RNA splicing and ribosome biogenesis, primarily functioning as an RNA helicase. To systematically assess the cellular role of DHX15, we conducted proteomic analysis to investigate the landscape of DHX15 interactome, and identified MYC as a binding partner. DHX15 co-localizes with MYC in cells and directly interacts with MYC in vitro. Importantly, DHX15 contributes to MYC protein stability at the post-translational level and independent of its RNA binding capacity. Mechanistic investigation reveals that DHX15 interferes the interaction between MYC and FBXW7, thereby preventing MYC polyubiquitylation and proteasomal degradation. Consequently, the abrogation of DHX15 drastically inhibits MYC-mediated transcriptional output. While DHX15 depletion blocks T cell development and leukemia cell survival as we recently reported, overexpression of MYC significantly rescues the phenotypic defects. These findings shed light on the essential role of DHX15 in mammalian cells and suggest that maintaining sufficient MYC expression is a significant contributor to DHX15-mediated cellular functions.

Nudt21-mediated alternative polyadenylation of MZT1 3'UTR contributes to pancreatic cancer progression.

Alternative polyadenylation (APA) is an important post-transcriptional regulatory mechanism and is involved in many diseases, but its function and mechanism in regulating pancreatic cancer (PC) pathogenesis remain unclear. In this study, we found that the 3' UTR shortening of MZT1 was the most prominent APA event in PC liver metastases. The short-3'UTR isoform exerted a stronger effect in promoting cell proliferation and migration both in vitro and in vivo. NUDT21, a core cleavage factor involved in APA, promoted the usage of proximal polyadenylation sites (PASs) on MZT1 mRNA by binding to the UGUA element located upstream of the proximal PAS. High percentage of distal polyA site usage index of MZT1 was significantly associated with a better prognosis. These findings demonstrate a crucial mechanism that NUDT21-mediated APA of MZT1 could promote the progression of PC. Our findings provided a better understanding of the connection between PC progression and APA machinery.

MTOR modulation induces selective perturbations in histone methylation which influence the anti-proliferative effects of mTOR inhibitors.

Emerging data suggest a significant cross-talk between metabolic and epigenetic programs. However, the relationship between the mechanistic target of rapamycin (mTOR), which is a pivotal metabolic regulator, and epigenetic modifications remains poorly understood. Our results show that mTORC1 activation caused by the abrogation of its negative regulator tuberous sclerosis complex 2 (TSC2) coincides with increased levels of the histone modification H3K27me3 but not H3K4me3 or H3K9me3. This selective H3K27me3 induction was mediated via 4E-BP-dependent increase in EZH2 protein levels. Surprisingly, mTOR inhibition also selectively induced H3K27me3. This was independent of TSC2, and was paralleled by reduced EZH2 and increased EZH1 protein levels. Notably, the ability of mTOR inhibitors to induce H3K27me3 levels was positively correlated with their anti-proliferative effects. Collectively, our findings demonstrate that both activation and inhibition of mTOR selectively increase H3K27me3 by distinct mechanisms, whereby the induction of H3K27me3 may potentiate the anti-proliferative effects of mTOR inhibitors.

Alternating magnetic fields drive stimulation of gene expression via generation of reactive oxygen species.

Magnetogenetics represents a method for remote control of cellular function. Previous work suggests that generation of reactive oxygen species (ROS) initiates downstream signaling. Herein, a chemical biology approach was used to elucidate further the mechanism of radio frequency-alternating magnetic field (RF-AMF) stimulation of a TRPV1-ferritin magnetogenetics platform that leads to Ca2+ flux. RF-AMF stimulation of HEK293T cells expressing TRPV1-ferritin resulted in ∼30% and ∼140% increase in intra- and extracellular ROS levels, respectively. Mutations to specific cysteine residues in TRPV1 responsible for ROS sensitivity eliminated RF-AMF driven Ca2+-dependent transcription of secreted embryonic alkaline phosphatase (SEAP). Using a non-tethered (to TRPV1) ferritin also eliminated RF-AMF driven SEAP production, and using specific inhibitors, ROS-activated TRPV1 signaling involves protein kinase C, NADPH oxidase, and the endoplasmic reticulum. These results suggest ferritin-dependent ROS activation of TRPV1 plays a key role in the initiation of magnetogenetics, and provides relevance for potential applications in medicine and biotechnology.

TOP1 and R-loops facilitate transcriptional DSBs at hypertranscribed cancer driver genes.

DNA double-stranded breaks (DSBs) pose a significant threat to genomic integrity, and their generation during essential cellular processes like transcription remains poorly understood. In this study, we employ several techniques to map DSBs, R-loops, and topoisomerase 1 cleavage complex (TOP1cc) to comprehensively investigate the interplay between transcription, DSBs, topoisomerase 1 (TOP1), and R-loops. Our findings reveal the presence of DSBs at highly expressed genes enriched with TOP1 and R-loops. Remarkably, transcription-associated DSBs at these loci are significantly reduced upon depletion of R-loops and TOP1, uncovering the pivotal roles of TOP1 and R-loops in transcriptional DSB formation. By elucidating the intricate interplay between TOP1cc trapping, R-loops, and DSBs, our study provides insights into the mechanisms underlying transcription-associated genomic instability. Moreover, we establish a link between transcriptional DSBs and early molecular changes driving cancer development, highlighting the distinct etiology and molecular characteristics of driver mutations compared to passenger mutations.

A library-based approach allows systematic and rapid evaluation of seed region length and reveals design rules for synthetic bacterial small RNAs.

All organisms must respond to environmental changes. In bacteria, small RNAs (sRNAs) are an important aspect of the regulation network underlying the adaptation to such changes. sRNAs base-pair with their target mRNAs, allowing rapid modulation of the proteome. This post-transcriptional regulation is usually facilitated by RNA chaperones, such as Hfq. sRNAs have a potential as synthetic regulators that can be modulated by rational design. In this study, we use a library-based approach and oxacillin susceptibility assays to investigate the importance of the seed region length for synthetic sRNAs based on RybB and SgrS scaffolds in Escherichia coli. In the presence of Hfq we show that 12 nucleotides are sufficient for regulation. Furthermore, we observe a scaffold-specific Hfq-dependency and processing by RNase E. Our results provide information for design considerations of synthetic sRNAs in basic and applied research.

Epitranscriptomic reader YTHDF2 regulates SEK1(MAP2K4)-JNK-cJUN inflammatory signaling in astrocytes during neurotoxic stress.

As the most abundant glial cells in the central nervous system (CNS), astrocytes dynamically respond to neurotoxic stress, however, the key molecular regulators controlling the inflammatory status of these sentinels during neurotoxic stress are many and complex. Herein, we demonstrate that the m6A epitranscriptomic mRNA modification tightly regulates the pro-inflammatory functions of astrocytes. Specifically, the astrocytic neurotoxic stressor, manganese (Mn), downregulated the m6A reader YTHDF2 in human and mouse astrocyte cultures and in the mouse brain. Functionally, YTHDF2 knockdown augmented, while its overexpression dampened, the neurotoxic stress-induced proinflammatory response, suggesting YTHDF2 serves as a key upstream regulator of inflammatory responses in astrocytes. Mechanistically, YTHDF2 RIP-sequencing identified MAP2K4 (MKK4; SEK1) mRNA as a YTHDF2 target influencing inflammatory signaling. Our target validation revealed that Mn-exposed astrocytes mediate proinflammatory responses by activating the phosphorylation of SEK1, JNK, and cJUN signaling. Collectively, YTHDF2 serves as a key upstream 'molecular switch' controlling SEK1(MAP2K4)-JNK-cJUN proinflammatory signaling in astrocytes.

YAP condensates are highly organized hubs.

YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. YAP condensates are stabilized by the transcription factor TEAD1, and recruit BRD4, a coactivator that is enriched at active enhancers. Using single-particle tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.

Engineered FSHD mutations results in D4Z4 heterochromatin disruption and feedforward DUX4 network activation.

Facioscapulohumeral dystrophy (FSHD) is linked to contraction of D4Z4 repeats on chromosome 4q with SMCHD1 mutations acting as a disease modifier. D4Z4 heterochromatin disruption and abnormal upregulation of the transcription factor DUX4, encoded in the D4Z4 repeat, are the hallmarks of FSHD. However, defining the precise effect of D4Z4 contraction has been difficult because D4Z4 repeats are primate-specific and DUX4 expression is very rare in highly heterogeneous patient myocytes. We generated isogenic mutant cell lines harboring D4Z4 and/or SMCHD1 mutations in a healthy human skeletal myoblast line. We found that the mutations affect D4Z4 heterochromatin differently, and that SMCHD1 mutation or disruption of DNA methylation stabilizes otherwise variegated DUX4 target activation in D4Z4 contraction mutant cells, demonstrating the critical role of modifiers. Our study revealed amplification of the DUX4 signal through downstream targets, H3.X/Y and LEUTX. Our results provide important insights into how rare DUX4 expression leads to FSHD pathogenesis.

Chromatin target of protein arginine methyltransferases alleviates cerebral ischemia/reperfusion-induced injury by regulating RNA alternative splicing.

RNA splicing is a post-transcriptional event that regulates many physiological and pathological events. However, whether RNA splicing regulates cerebral I/R-induced brain injury remains largely unknown. In this study, we found that the chromatin target of Prmts (CHTOP) was highly expressed in neurons, and anti-inflammatory cytokine interleukin-10 (IL-10) upregulates its expression after ischemia. In addition, overexpression or knockdown of CHTOP alleviated or exacerbated neuronal death in both experimental stroke mice and cultured neurons. Mechanistically, RNA alternative splicing is altered early after oxygen and glucose deprivation/reoxygenation (OGD/R). CHTOP interacted with nuclear speckle-related proteins to regulate alternative mRNA splicing of neuronal survival-related genes after OGD/R. In addition, I/R injury-induced cytokines IL-10 regulate CHTOP-mediated RNA splicing to alleviate ischemic brain injury. Taken together, this study reveals the alteration of RNA splicing after OGD/R and identifies the IL-10-CHTOP-RNA splicing axis as a modulator of brain injury, which may be promising therapeutic targets for ischemic stroke.

Predicting transcription factor activity using prior biological information.

Dysregulation of normal transcription factor activity is a common driver of disease. Therefore, the detection of aberrant transcription factor activity is important to understand disease pathogenesis. We have developed Priori, a method to predict transcription factor activity from RNA sequencing data. Priori has two key advantages over existing methods. First, Priori utilizes literature-supported regulatory information to identify transcription factor-target gene relationships. It then applies linear models to determine the impact of transcription factor regulation on the expression of its target genes. Second, results from a third-party benchmarking pipeline reveals that Priori detects aberrant activity from 124 single-gene perturbation experiments with higher sensitivity and specificity than 11 other methods. We applied Priori and other top-performing methods to predict transcription factor activity from two large primary patient datasets. Our work demonstrates that Priori uniquely discovered significant determinants of survival in breast cancer and identified mediators of drug response in leukemia.

TET2 regulation of alcoholic fatty liver via Srebp1 mRNA in paraspeckles.

Epigenetic modifications have emerged as key regulators of metabolism-related complex diseases including the alcoholic fatty liver disease (AFLD) prevalent chronic liver disorder with significant economic implications. Building upon previous research that emphasizes ten-eleven translocation (TET) proteins' involvement in adipocyte insulin sensitization and fatty acid oxidation, we explored the role of TET2 protein in AFLD pathogenesis which catalyzes 5-methylcytosine into 5-hydroxymethylcytosine in DNA/RNA. Our findings revealed that TET2 deficiency exacerbates AFLD progression. And TET2 influenced the expression and activity of sterol regulatory element binding protein 1 (SREBP1), a key regulator of hepatic lipid synthesis, by modulating Srebp1 mRNA retention. Employing RIP-qPCR and bisulfite sequencing techniques, we provided evidence of TET2-mediated epigenetic modifications on Srebp1 mRNA, thereby affecting lipid metabolism. Through elucidating the role of methylation in RNA nuclear retention via paraspeckles, our study enhances understanding of AFLD pathogenesis from an epigenetic perspective, paving the way for identifying potential therapeutic targets.

Remodeling of the endothelial cell transcriptional program via paracrine and DNA-binding activities of MPO.

Myeloperoxidase (MPO) is an enzyme that functions in host defense. MPO is released into the vascular lumen by neutrophils during inflammation and may adhere and subsequently penetrate endothelial cells (ECs) coating vascular walls. We show that MPO enters the nucleus of ECs and binds chromatin independently of its enzymatic activity. MPO drives chromatin decondensation at its binding sites and enhances condensation at neighboring regions. It binds loci relevant for endothelial-to-mesenchymal transition (EndMT) and affects the migratory potential of ECs. Finally, MPO interacts with the RNA-binding factor ILF3 thereby affecting its relative abundance between cytoplasm and nucleus. This interaction leads to change in stability of ILF3-bound transcripts. MPO-knockout mice exhibit reduced number of ECs at scar sites following myocardial infarction, indicating reduced neovascularization. In summary, we describe a non-enzymatic role for MPO in coordinating EndMT and controlling the fate of endothelial cells through direct chromatin binding and association with co-factors.

Deep molecular learning of transcriptional control of a synthetic CRE enhancer and its variants.

Massively parallel reporter assay measures transcriptional activities of various cis-regulatory modules (CRMs) in a single experiment. We developed a thermodynamic computational model framework that calculates quantitative levels of gene expression directly from regulatory DNA sequences. Using the framework, we investigated the molecular mechanisms of cis-regulatory mutations of a synthetic enhancer that cause abnormal gene expression. We found that, in a human cell line, competitive binding between family transcription factors (TFs) with slightly different binding preferences significantly increases the accuracy of recapitulating the transcriptional effects of thousands of single- or multi-mutations. We also discovered that even if various harmful mutations occurred in an activator binding site, CRM could stably maintain or even increase gene expression through a certain form of competitive binding between family TFs. These findings enhance understanding the effect of SNPs and indels on CRMs and would help building robust custom-designed CRMs for biologics production and gene therapy.

Epigenetic and transcriptional responses in circulating leukocytes are associated with future decompensation during SARS-CoV-2 infection.

To elucidate host response elements that define impending decompensation during SARS-CoV-2 infection, we enrolled subjects hospitalized with COVID-19 who were matched for disease severity and comorbidities at the time of admission. We performed combined single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) on peripheral blood mononuclear cells (PBMCs) at admission and compared subjects who improved from their moderate disease with those who later clinically decompensated and required invasive mechanical ventilation or died. Chromatin accessibility and transcriptomic immune profiles were markedly altered between the two groups, with strong signals in CD4+ T cells, inflammatory T cells, dendritic cells, and NK cells. Multiomic signature scores at admission were tightly associated with future clinical deterioration (auROC 1.0). Epigenetic and transcriptional changes in PBMCs reveal early, broad immune dysregulation before typical clinical signs of decompensation are apparent and thus may act as biomarkers to predict future severity in COVID-19.

Comprehensive identification of onco-exaptation events in bladder cancer cell lines revealed L1PA2-SYT1 as a prognosis-relevant event.

Transposable elements (TEs) can provide ectopic promoters to drive the expression of oncogenes in cancer, a mechanism known as onco-exaptation. Onco-exaptation events have been extensively identified in various cancers, with bladder cancer showing a high frequency of onco-exaptation events (77%). However, the effect of most of these events in bladder cancer remains unclear. This study identified 44 onco-exaptation events in 44 bladder cancer cell lines in 137 RNA-seq datasets from six publicly available cohorts, with L1PA2 contributing the most events. L1PA2-SYT1, L1PA2-MET, and L1PA2-XCL1 had the highest frequency not only in cell lines but also in TCGA-BLCA samples. L1PA2-SYT1 showed significant tumor specificity and was found to be activated by CpG island demethylation in its promoter. The upregulation of L1PA2-SYT1 enhances the in vitro invasion of bladder cancer and is an independent risk factor for patient's overall survival, suggesting L1PA2-SYT1 being an important event that promotes the development of bladder cancer.