Gene Transcription Programs Research Articles

Evidence for a hydrogen sulfide-sensing E3 ligase in yeast.

In yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor that activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCFMet30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the trans-sulfuration pathway to regulate the MET gene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4 in vivo. Additionally, we identify important cysteine residues in Met30's WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCFMet30 dynamically senses the flow of sulfur metabolites through the trans-sulfuration pathway to regulate the synthesis of these special amino acids.

Genome binding properties of Zic transcription factors underlie their changing functions during neuronal maturation

BackgroundThe Zic family of transcription factors (TFs) promote both proliferation and maturation of cerebellar granule neurons (CGNs), raising the question of how a single, constitutively expressed TF family can support distinct developmental processes. Here we use an integrative experimental and bioinformatic approach to discover the regulatory relationship between Zic TF binding and changing programs of gene transcription during postnatal CGN differentiation.ResultsWe first established a bioinformatic pipeline to integrate Zic ChIP-seq data from the developing mouse cerebellum with other genomic datasets from the same tissue. In newborn CGNs, Zic TF binding predominates at active enhancers that are co-bound by developmentally regulated TFs including Atoh1, whereas in mature CGNs, Zic TF binding consolidates toward promoters where it co-localizes with activity-regulated TFs. We then performed CUT&RUN-seq in differentiating CGNs to define both the time course of developmental shifts in Zic TF binding and their relationship to gene expression. Mapping Zic TF binding sites to genes using chromatin looping, we identified the set of Zic target genes that have altered expression in RNA-seq from Zic1 or Zic2 knockdown CGNs.ConclusionsOur data show that Zic TFs are required for both induction and repression of distinct, developmentally regulated target genes through a mechanism that is largely independent of changes in Zic TF binding. We suggest that the differential collaboration of Zic TFs with other TF families underlies the shift in their biological functions across CGN development.

Bacteria can anticipate the seasons: photoperiodism in cyanobacteria.

Photoperiodic Time Measurement is the ability of plants and animals to measure differences in day/night-length (photoperiod) and use that information to anticipate critical seasonal transformations such as annual temperature cycles. This timekeeping phenomenon triggers adaptive responses in higher organisms such as gonadal growth/regression, flowering, and hibernation. Unexpectedly, we discovered this capability in cyanobacteria, unicellular prokaryotes with generation times of only 5-6 h. Cyanobacteria in short winter-like days develop enhanced resistance to cold that involves desaturation of membrane lipids and differential programs of gene transcription, including stress response pathways. As in eukaryotes, this photoperiodic timekeeping requires an intact circadian clockwork and develops over multiple cycles. Therefore, photoperiodic timekeeping evolved in much simpler organisms than previously appreciated, and involved genetic responses to stresses that recur seasonally.

Abstract Tu082: Novel Structural and Biochemical Effects of Nexilin Associated Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy is primarily a genetic disease that results in abnormal cardiac muscle phenotypes characterized by ventricular hypertrophy, fibrosis, and contractile dysfunction. Both sarcomere structural and signaling genes are associated with HCM. Among various genes, one of the implicated genes for HCM is Nexilin (NEXN). Nexilin is an F-actin binding protein widely expressed in muscle cells and localized at sarcomere and T-tubules. It is known to have a surprising role in insulin signaling. The role of Nexilin mutations is poorly studied. We hypothesized that Nexilin missense variants in genetically engineered human pluripotent stem cells (hPSCs) perturb essential cardiac metabolic signaling pathways leading to HCM. In our Indian HCM cohort, we identified seven Nexilin mutations through exome sequencing and categorized them as pathogenic per ACMG/AMP classification. A representative novel mutant in Nexilin p.Q131T was introduced in hPSC with the CRISPR-Cas9 knock-in method and was differentiated into cardiomyocytes, recapitulating patient-specific heterozygous mutation. Our data shows that Nexilin p.Q131T causes an increase in cell size and activation of fetal gene transcriptional programs in hPSC-derived cardiomyocytes. Global transcriptomics was performed to understand transcriptional changes and investigate further. We observe the significant activation of insulin-related signaling pathways, including AKT/MTOR and MAPK, confirmed by immunoblotting. Akt-mTOR and MAPK cascades are well-known to be regulated by IRS-1 signaling. Our studies show hyperactivation of these cascades, suggesting IRS-1 involvement in the pathogenesis. Nexilin and IRS-1 interaction were probed and found to be perturbed, hence causing higher activation of IRS-1 and its downstream targets. In addition, Nexilin wild type and mutant protein structure-functional analysis were performed with purified proteins. Using cryo-EM structural analysis, we identified unique F-actin binding sites for Nexilin for the first time, and the mutant p.Q131T showed decreased binding affinity to F-actin. We report Nexilin's high-resolution cryo-EM structure and a novel metabolic signaling mechanism where mutations in Nexilin cause dysregulation of IRS-1 signaling in the gene-edited cardiomyocytes, leading to HCM pathogenesis.

Elucidating immune-related gene transcriptional programs via factorization of large-scale RNA-profiles

Recent developments in immunotherapy, including immune checkpoint blockade (ICB) and adoptive cell therapy (ACT), have encountered challenges such as immune-related adverse events and resistance, especially in solid tumors. To advance the field, a deeper understanding of the molecular mechanisms behind treatment responses and resistance is essential. However, the lack of functionally characterized immune-related gene sets has limited data-driven immunological research. To address this gap, we adopted non-negative matrix factorization on 83 human bulk RNA sequencing (RNA-seq) datasets and constructed 28 immune-specific gene sets. After rigorous immunologist-led manual annotations and orthogonal validations across immunological contexts and functional omics data, we demonstrated that these gene sets can be applied to refine pan-cancer immune subtypes, improve ICB response prediction and functionally annotate spatial transcriptomic data. These functional gene sets, informing diverse immune states, will advance our understanding of immunology and cancer research.

Abstract 873: Elucidating immune-related gene transcriptional programs via factorization of large-scale RNA profiles

Abstract Background: Despite the recent success in Immune checkpoint blockade (ICB) therapy, limited patients benefit due to immune-related adverse events or treatment resistance, especially in solid tumors. To overcome these hurdles, understanding the molecular mechanisms behind treatment response is essential. However, the current lack of immune gene transcriptional programs (GTPs) hampers data-driven immunological discovery. Constructing immunity-specific knowledgebases with rigorously curated gene sets (irGSs) and rich immunological language can unlock new insights, enabling interpretation of high-throughput immune microenvironment profiling studies and fostering personalized, effective cancer treatments. Methods: We collected 83 BulkRNAseq datasets from the ImmuneSigDB. These datasets contain 1826 samples challenged with infections, cytokines or immunological perturbations of different kinds and magnitude, possessing yet-to-be discovered immune functions that lie beneath the transcriptomic profiles. Using non-negative matrix factorization (NMF), we identified gene sets with coordinated expression. We used CITE-seq and selected scRNAseq to annotate the immunological functions of each gene set and validated the clinical utilities of these gene sets from different aspects using data from Cancer Genome Atlas Program (TCGA) pan-cancer, ICB cohorts and a 10X Genomics Visium FFPE Human Breast Cancer spatial slide. Results: We presented 19 lymphoid-data derived and 9 myeloid-data derived gene sets (irGSs), encompassing a diverse array of immune functions. Through extensive validation using BulkRNAseq, scRNAseq and spatial transcriptomics data, we revealed six irGSs-defined pan-cancer microenvironment subtypes in TCGA with significantly distinct survival patterns regardless of cancer types. Moreover, irGSs are associated with T cell exhaustion phenotypes, which in turn well predicted (classification accuracy of 72%) Nivolumab response using 104 treatment-naïve melanoma patients. Lastly, irGSs simultaneously isolated out tumor regions and highlighted immune infiltration with high specificity in breast cancer spatial transcriptomics H&E image. Conclusions: These pioneering gene sets, originally derived from non-cancerous experiments, hold tremendous promise for cancer research across diverse contexts. Similarities between cancerous and sepsis immune microenvironments underscore their wide applicability. By studying gene set activities, immunologists can gain profound insights into cancer survival drivers, unravel the intricacies of ICB treatment mechanisms, and potentially conquer therapeutic resistance. These translational utilities herald a transformative era in cancer immunotherapy and open new frontiers in the fight against cancer. Citation Format: Shan He, Vakul Mohanty, Matthew Gubin, Hind Rafei, Rafet Basar, Merve Dede, Xianli Jiang, Yukun Tan, Maura Gillison, Katayoun Rezvani, Weiyi Peng, Ken Chen. Elucidating immune-related gene transcriptional programs via factorization of large-scale RNA profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 873.

Abstract 5831: The TLE1 transcriptional and epigenetic machinery contributes to EGFR targeted therapy resistance in human lung adenocarcinoma

Abstract In human lung adenocarcinoma that carry Epidermal Growth Factor Receptor (EGFR) sensitizing mutations, targeting the EGFR pathway with the use of EGFR tyrosine kinase inhibitors (EGFR-TKIs) has emerged as a promising therapeutic strategy providing initial clinical benefit to many patients. Unfortunately, resistance to EGFR-TKIs eventually develops leading to disease progression. In addition to genetic mutations, transcriptional and epigenetic mediated changes in gene expression remain an important mechanism contributing to EGFR-TKI resistance. Here, we report a novel function of the transcriptional corepressor Transducin-Like Enhancer of Split-1 (TLE1) in mediating EGFR-TKI resistance in EGFR mutant LUAD cells through its survival promoting gene transcriptional program. In EGFR mutant, EGFR TKI sensitive LUAD cells, sole activation of the TLE1 nuclear function attenuates EGFR TKI sensitivity with concomittant reduction of EGFR-TKI-mediated apoptosis. Conversely, downregulation of endogenous TLE1 expression via siRNAs/shRNAs and deletion of TLE1 gene via CRISPR-Cas9 in EGFR-mutant, EGFR-TKI resistant cells partially restore EGFR-TKI sensitivity via induction of EGFR-TKI apoptosis. Mechanistically, the anti-apoptosis promoting gene transcriptional program of TLE1 is dependent on its functional interaction with the transcriptional repressor HES-1, a downstream Notch target which has been associated with relapse in LUAD patients following EGFR-TKI treatment. Collectively, these studies indicate the TLE1 corepressor as a determinant of resistance to EGFR targeted therapy and raise the possibility that inhibiting TLE1 nuclear function may serve as an adjuct therapy to circumvent EGFR-TKI resistance. Citation Format: Hector Ramos Biliran, Xin Yao, Nasir Roberts, Prince Iheukwumere, Renwei Chen, Ma. Carmela Dela Cruz. The TLE1 transcriptional and epigenetic machinery contributes to EGFR targeted therapy resistance in human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5831.

Abstract 3013: Alzheimer’s-linked gene BIN1: Unraveling its unexpected role as a tumor suppressor in prostate cancer and influence on androgen receptor signaling

Abstract Androgen-receptor signaling inhibitors (ARSI) have been shown to significantly alter the natural history of castration-resistant PCa (CRPC). Unfortunately, resistance to ARSIs is inevitable. Unraveling the underlying mechanisms to identify potential therapeutic interventions to treat these lethal tumors has become a critical challenge in the post-ARSI era. Recent genomic sequencing has revealed a subclass of PCa harboring deletion of bridging integrator-1 (BIN1) and is associated with increased AR activity. Notably, BIN1 deletion frequently co-occurs with SPOP mutation and deletion of SPOPL, collectively suggesting BIN1 may function as tumor suppressor. To elucidate the role of BIN1 in prostate cells, we generated a prostate-specific BIN1 knockout murine model (PB-CreBIN1FL/FL). This model revealed increased prostate mass at 6 months of age, accompanied by increased cell proliferation. We next crossed this strain to SPOP mutant (SPOPF133V) model to generate a prostate specific BIN1 knockout, SPOP mutant model (PB-CreSPOPF133VBIN1FL/FL) and found increased proliferation and greater prostate mass compared to animals harboring SPOP mutation or BIN1 deletion alone. Next, we generated a doxycycline inducible BIN1 overexpressing PCa model (22Rv1BINOE) to evaluate AR signaling in vitro. We found that overexpression of BIN1 modestly reduced prostate cancer cell proliferation but profoundly suppressed androgen receptor protein level. To evaluate the transcriptional activity of AR and ARv7, we also utilized a luciferase reporter system harboring ARE consensus sequences found in the KLK3 gene (AR-FL target) promoter or in the EDN2 gene (ARv7 target) promoter, respectively. These reporter assays illustrated suppression of both AR-FL and AR-v7 driven gene transcriptional program upon BIN1 overexpression. Co-expression of both BIN1 and SPOPwt showed a synergistic knockdown of AR activity, while SPOPmut did not show this effect. These observations were further confirmed using global RNAseq. The RNAseq analysis also showed downregulation of several AR pathways as well as gene sets associated with cancer progression and metastasis. Interestingly, the RNAseq data showed downregulation of genes that are normally upregulated after SPOP mutation, further suggesting a synergistic role between SPOP and BIN1. Citation Format: Collin McColl, Darlene Skapura, Elisa Ruiz Echartea, Jenny Deng, Aleksandra Rusin, Jinna Shin, Alexey Tyryshkin, Christel Davis, Erik Ehil, Salma Kaochar. Alzheimer’s-linked gene BIN1: Unraveling its unexpected role as a tumor suppressor in prostate cancer and influence on androgen receptor signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3013.

Modulation of canonical Wnt signaling regulates peribiliary mesenchymal identity during homeostasis and injury.

The matrix and associated mesenchyme of the extrahepatic bile ducts are distinct, which could drive diseases with a predilection for these ducts, such as primary sclerosing cholangitis. We aimed to understand the molecular drivers of peribiliary mesenchymal cell (PMC) identity in the extrahepatic bile ducts and dissect how this changed in the context of injury using an entirely in vivo approach with transcriptomic analysis. Single-cell sequencing with a receptor-ligand analysis showed that PMCs had the most interactions with surrounding cells. Wnt4, Wnt5a, and Wnt7b were identified as the major ligands secreted from PMCs and cholangiocytes that interacted in both paracrine and autocrine fashion. Bile duct ligation caused an increase in all 3 Wingless/Integrated ligands and Axin2 with an associated increase in the transcription factors T-box transcription factor (Tbx)2 and Tbx3. Conversely, Indian hedgehog secretion decreased without an associated decrease in hedgehog signaling effectors. Loss of smoothened within PMCs did not impact hedgehog signaling effectors or cellular identity, whereas smoothened gain of function led to myofibroblast transdifferentiation with upregulation of Tbx2 and Tbx3 without injury. Loss of β-catenin caused a decrease in expression of all 3 Gli transcription factors and associated mesenchymal gene expression, which was phenocopied with compound Gli2 and Gli3 loss in uninjured PMCs. With injury, loss of β-catenin resulted in decreased myofibroblast transdifferentiation with reduced Tbx2 and Tbx3 expression. Our results show how modulation of canonical Wingless/Integrated signaling in PMCs is important for regulating basal mesenchymal gene expression and initiating a myogenic gene transcriptional program during injury. They also highlight reciprocating interactions between the hedgehog and Wingless/Integrated signaling pathways within PMCs.

Abstract 18286: Histone Acetyltransferase P300/CBP in Pulmonary Arterial Hypertension and Associated Right Ventricular Failure

INTRODUCTION Pulmonary arterial hypertension (PAH) is defined by increased pulmonary artery (PA) pressure and vascular remodeling, partly due to excessive proliferation and survival of PA smooth muscle cells (PASMCs). Initially, RV hypertrophy allows adaptation to increased resistance, but as the disease progresses, maladaptive remodeling takes place, leading to RV failure and death. Current therapies aim to promote vasodilatation, but none of them directly targets pathological lungs or RV remodeling. The histone acetyltransferases P300/CBP have been identified as central players driving gene expression in various cellular processes such as proliferation/apoptosis and hypertrophy/fibrosis, all of which are critical features of pathological lung and RV remodeling in PAH. Given their role in controlling gene transcription programs, we hypothesized that P300/CBP contributes to maladaptive remodeling. METHODS & RESULTS We show by western blot (WB) and immunofluorescence (IF) increased P300 expression in isolated PASMCs and distal PAs from PAH patients compared to controls (p<0.01) as well as in monocrotaline (MCT) and sugen-hypoxia rat models (p<0.05). Similar results are observed in remodeled RV from PAH patients, MCT- and pulmonary artery banding-subjected rats (WB). In vitro, P300/CBP inhibition (CCS-1477 or siRNA) reduces H3K27 acetylation (WB, p<0.05) in PAH-PASMCs and RV fibroblasts (RVFbs). This effect is accompanied by a decrease in PAH-PASMC proliferation and resistance to apoptosis [WB (PCNA, PLK1, Survivin), IF (Ki67, AnexinV), p<0.01]. Similarly, P300/CBP inhibition reduces proliferation (WB, PCNA, Survivin) and activation/extracellular matrix production (WB, pSMAD2/3, aSMA, Fn, Col1, MMP2) in RVFbs. In addition, P300/CBP inhibition prevents phenylephrine-induced hypertrophy in H9C2 cells and adult rat cardiomyocytes (IF, p<0.05). In vivo, administration of CCS-1477 reduces pulmonary vascular remodeling (Elastica Van Gieson, p<0.05), improves pulmonary hemodynamics (p<0.01) and attenuates RV fibrosis (Masson’s Trichrome, p<0.05) in MCT rats with established PAH. Conclusion: We provide evidence that targeting P300/CBP may represent a promising avenue to tackle both lung and RV maladaptive remodeling in PAH.

A Glimpse into Chromatin Organization and Nuclear Lamina Contribution in Neuronal Differentiation.

During embryonic development, stem cells undergo the differentiation process so that they can specialize for different functions within the organism. Complex programs of gene transcription are crucial for this process to happen. Epigenetic modifications and the architecture of chromatin in the nucleus, through the formation of specific regions of active as well as inactive chromatin, allow the coordinated regulation of the genes for each cell fate. In this mini-review, we discuss the current knowledge regarding the regulation of three-dimensional chromatin structure during neuronal differentiation. We also focus on the role the nuclear lamina plays in neurogenesis to ensure the tethering of the chromatin to the nuclear envelope.

Bidirectional regulation of postmitotic H3K27me3 distributions underlie cerebellar granule neuron maturation dynamics.

The functional maturation of neurons is a prolonged process that extends past the mitotic exit and is mediated by the chromatin-dependent orchestration of gene transcription programs. We find that expression of this maturation gene program in mouse cerebellar granule neurons (CGNs) requires dynamic changes in the genomic distribution of histone H3 lysine 27 trimethylation (H3K27me3), demonstrating a function for this chromatin modification beyond its role in cell fate specification. The developmental loss of H3K27me3 at promoters of genes activated as CGNs mature is facilitated by the lysine demethylase and ASD-risk gene, Kdm6b. Interestingly, inhibition of the H3K27 methyltransferase EZH2 in newborn CGNs not only blocks the repression of progenitor genes but also impairs the induction of mature CGN genes, showing the importance of bidirectional H3K27me3 regulation across the genome. These data demonstrate that H3K27me3 turnover in developing postmitotic neurons regulates the temporal coordination of gene expression programs that underlie functional neuronal maturation.

Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin.

Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) invitro and onto rDNA invivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at the melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment. In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100-500bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.

TMIC-74. THERAPEUTICALLY REPROGRAMMING MICROGLIA TO ANTI-TUMORAL ACTIVATION STATES IN DIFFUSE MIDLINE GLIOMA, H3K27 MUTANT

Abstract Diffuse midline glioma (DMG), H3K27M mutant, is one of the most challenging paediatric cancers to date with poor treatment options and dismal survival outcomes. Therefore, new treatment options are desperately needed. Previously we found that activation of the microglial tumour-supportive phenotype by DIPG is associated with a transient H3K27me3 downregulation suggesting a role for this pathway in inducing this activation state. However, it should be noted, that microglia do not carry the H3-K27M mutation when analysed in DIPG patient tissue, otherwise characteristic of the cancer cells. Ezh2 is a histone methyltransferase and the catalytic subunit of the polycomb repressor complex 2 (PRC2), responsible for tri-methylation of histone H3 at lysine 27. We found that repression of Ezh2 in microglia induced an anti-tumour phenotype resulting in decreased cancer cell invasion capability, increased microglial phagocytosis, and tumour-related cell death. Antisense RNA strategies were used to target Ezh2 gene expression in BV2 microglia and we have now carried out RNA sequencing on scramble BV2 or siEzh2 microglia co-cultured with DIPG tumour cells to further understand how EZH2, PRC2 and H3K27me3 regulates DIPG-induced microglia activation. Further work has found that activation of the microglial tumour supportive phenotype, in this context, is determined by a complex epigenetic mechanism involving both H3K4me3 and H3K27me3 and their associated methyltransferases. These histone marks are often associated with bivalent regulation of distinct gene transcription programs, and we are currently investigating if this is the case for DIPG-induced activation states using chromatin immunoprecipitation techniques. Overall, our results indicate that microglia, can be reprogrammed epigenetically by EZH2 inhibition to exert anti-tumoral responses and that microglia represent a new therapeutic avenue in DIPG. This future work should provide novel mechanisms governing pro-tumoral microglial activation states and new therapeutic targets that can be combined with EZH2 inhibition to enhance anti-tumoral microglia activation states.

The pleiotropic roles of EZH2 in T-cell immunity and immunotherapy.

EZH2 is a histone methyltransferase. It catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3) to control gene transcription critical for cell proliferation, differentiation, expansion, and function. For instance, EZH2 plays a central role in regulating T-cell immune responses. EZH2 restrains terminal differentiation of effector CD8 T cells, promotes formation of precursor and mature memory CD8 T cells, regulates appropriate lineage-specification and identity maintenance of helper CD4 T cells, and maintains survival of differentiated antigen-specific T cells. Most importantly, EZH2 is shown to be important for reinvigoration of exhausted chimeric antigen receptor (CAR) T cells. Dysregulated EZH2 function has been linked to many forms of cancer, including lymphomas and solid tumors. In B-cell lymphoid malignancies, EZH2 is overexpressed to drive tumorigenesis. These specific effects of EZH2, in the context of its roles in catalyzing H3K27me3 and orchestrating gene transcription programs in both normal and malignant cells, establishes EZH2 as a unique target for drug development. Here, we will discuss Ezh2 regulation of T-cell immunity, EZH2-mediated lymphomagenesis, and therapeutic benefits of EZH2 inhibitors to the treatment of lymphoma.

Myocardin regulates exon usage in smooth muscle cells through induction of splicing regulatory factors

Differentiation of smooth muscle cells (SMCs) depends on serum response factor (SRF) and its co-activator myocardin (MYOCD). The role of MYOCD for the SMC program of gene transcription is well established. In contrast, the role of MYOCD in control of SMC-specific alternative exon usage, including exon splicing, has not been explored. In the current work we identified four splicing factors (MBNL1, RBPMS, RBPMS2, and RBFOX2) that correlate with MYOCD across human SMC tissues. Forced expression of MYOCD family members in human coronary artery SMCs in vitro upregulated expression of these splicing factors. For global profiling of transcript diversity, we performed RNA-sequencing after MYOCD transduction. We analyzed alternative transcripts with three different methods. Exon-based analysis identified 1637 features with differential exon usage. For example, usage of 3´ exons in MYLK that encode telokin increased relative to 5´ exons, as did the 17 kDa telokin to 130 kDa MYLK protein ratio. Dedicated event-based analysis identified 239 MYOCD-driven splicing events. Events involving MBNL1, MCAM, and ACTN1 were among the most prominent, and this was confirmed using variant-specific PCR analyses. In support of a role for RBPMS and RBFOX2 in MYOCD-driven splicing we found enrichment of their binding motifs around differentially spliced exons. Moreover, knockdown of either RBPMS or RBFOX2 antagonized splicing events stimulated by MYOCD, including those involving ACTN1, VCL, and MBNL1. Supporting an in vivo role of MYOCD-SRF-driven splicing, we demonstrate altered Rbpms expression and splicing in inducible and SMC-specific Srf knockout mice. We conclude that MYOCD-SRF, in part via RBPMS and RBFOX2, induce a program of differential exon usage and alternative splicing as part of the broader program of SMC differentiation.

Selenium: a potent regulator of ferroptosis and biomass production

Emerging evidence supports the notion that selenium (Se) plays a beneficial role in plant development for modern crop production and is considered an essential micronutrient and the predominant source of plants. However, the essential role of selenium in plant metabolism remains unclear. When used in moderate concentrations, selenium promotes plant physiological processes such as enhancing plant growth, increasing antioxidant capacity, reducing reactive oxygen species and lipid peroxidation and offering stress resistance by preventing ferroptosis cell death. Ferroptosis, a recently discovered mechanism of regulated cell death (RCD) with unique features such as iron-dependant accumulation of lipid peroxides, is distinctly different from other known forms of cell death. Glutathione peroxidase (GPX) activity plays a significant role in scavenging the toxic by-products of lipid peroxidation in plants. A low level of GPX activity in plants causes high oxidative stress, which leads to ferroptosis. An integrated view of ferroptosis and selenium in plants and the selenium-mediated nanofertilizers (SeNPs) have been discussed in more recent studies. For instance, selenium supplementation enhanced GPX4 expression and increased TFH cell (Follicular helper T) numbers and the gene transcriptional program, which prevent lipid peroxidase and protect cells from ferroptosis. However, though ferroptosis in plants is similar to that in animals, only few studies have focused on plant-specific ferroptosis; the research on ferroptosis in plants is still in its infancy. Understanding the implication of selenium with relevance to ferroptosis is indispensable for plant bioresource technology. In this review, we hypothesize that blocking ferroptosis cell death improves plant immunity and protects plants from abiotic and biotic stresses. We also examine how SeNPs can be the basis for emerging unconventional and advanced technologies for algae/bamboo biomass production. For instance, algae treated with SeNPs accumulate high lipid profile in algal cells that could thence be used for biodiesel production. We also suggest that further studies in the field of SeNPs are essential for the successful application of this technology for the large-scale production of plant biomass.

VHL Ser65 mutations enhance HIF2\u03b1 signaling and promote epithelial-mesenchymal transition of renal cancer cells

BackgroundVon Hippel-Lindau (VHL) disease is an autosomal dominant genetic neoplastic disorder caused by germline mutation or deletion of the VHL gene, characterized by the tendency to develop multisystem benign or malignant tumors. The mechanism of VHL mutants in pathogenicity is poorly understand.ResultsHere we identified heterozygous missense mutations c.193T > C and c.194C > G in VHL in several patients from two Chinese families. These mutations are predicted to cause Serine (c.193T > C) to Proline and Tryptophan (c.194C > G) substitution at residue 65 of VHL protein (p.Ser65Pro and Ser65Trp). Ser65 residue, located within the β-domain and nearby the interaction sites with hypoxia-inducing factor α (HIFα), is highly conserved among different species. We observed gain of functions in VHL mutations, thereby stabilizing HIF2α protein and reprograming HIF2α genome-wide target gene transcriptional programs. Further analysis of independent cohorts of patients with renal carcinoma revealed specific HIF2α gene expression signatures in the context of VHL Ser65Pro or Ser65Trp mutation, showing high correlations with hypoxia and epithelial-mesenchymal transition signaling activities and strong associations with poor prognosis.ConclusionsTogether, our findings highlight the crucial role of pVHL-HIF dysregulation in VHL disease and strengthen the clinical relevance and significance of the missense mutations of Ser65 residue in pVHL in the familial VHL disease.

The zinc-finger transcription factor LSL-1 is a major regulator of the germline transcriptional program in Caenorhabditis elegans.

Specific gene transcriptional programs are required to ensure the proper proliferation and differentiation processes underlying the production of specialized cells during development. Gene activity is mainly regulated by the concerted action of transcription factors and chromatin proteins. In the nematode Caenorhabditis elegans, mechanisms that silence improper transcriptional programs in germline and somatic cells have been well studied, however, how are tissue-specific sets of genes turned on is less known. LSL-1 is herein defined as a novel crucial transcriptional regulator of germline genes in C. elegans. LSL-1 is first detected in the P4 blastomere and remains present at all stages of germline development, from primordial germ cell proliferation to the end of meiotic prophase. lsl-1 loss-of-function mutants exhibit many defects including meiotic prophase progression delay, a high level of germline apoptosis, and production of almost no functional gametes. Transcriptomic analysis and ChIP-seq data show that LSL-1 binds to promoters and acts as a transcriptional activator of germline genes involved in various processes, including homologous chromosome pairing, recombination, and genome stability. Furthermore, we show that LSL-1 functions by antagonizing the action of the heterochromatin proteins HPL-2/HP1 and LET-418/Mi2 known to be involved in the repression of germline genes in somatic cells. Based on our results, we propose LSL-1 to be a major regulator of the germline transcriptional program during development.

The H2A N-terminal tail is required to alleviate copper-induced stress in Saccharomyces cerevisiae

Histone tail residues drive many biological processes by regulating genome-wide transcription. Functions of histone H3 and H4 tail residues in stress-responsive gene transcriptional programs have been extensively studied. The H2A tail residues have been shown to regulate DNA damage repair and oxidative stress response, but the involvement of N-terminal tail of H2A (H2ANtT) in proteostasis regulation is unknown. The unfolded protein response pathway (UPR) is an essential mechanism adopted by cells to prevent protein toxicity in response to ER stress. The disturbance in ER can occur by various factors such as heat stress, redox imbalance, exposure to xenobiotics and metals. Copper is utilized as a cofactor by cellular enzymes, but excessive copper affects ER homeostasis. We found that cells lacking 1-20 residues of H2ANtT are intolerant to copper stress, owing to the accumulation of misfolded proteins in the mutant cells. H2A 1-20 truncation also reduces the physiological UPR, and copper exposure further aggravates this effect. Furthermore, the expression of a spliced version of HAC1 mRNA in H2A∆(1-20) cells, encoding the downstream transcription factor of UPR signalling, rescues their growth under copper stress. Altogether these results provide evidence that H2ANtT reduces copper-induced ER stress by regulating UPR signalling.