Repression Of Gene Expression Research Articles

Transcription factor Ikzf1 associates with Foxp3 to repress gene expression in Treg cells and limit autoimmunity and anti-tumor immunity

The master transcription factor of regulatory T (Treg) cells, forkhead box protein P3 (Foxp3), controls Treg cell function by targeting certain genes for activation or repression, but the specific mechanisms by which it mediates this activation or repression under different conditions remain unclear. We found that Ikzf1 associates with Foxp3 via its exon 5 (IkE5) and that IkE5-deficient Treg cells highly expressed genes that would otherwise be repressed by Foxp3 upon Tcell receptor stimulation, including Ifng. Treg-specific IkE5-deletion caused interferon-γ (IFN-γ) overproduction, which destabilized Foxp3 expression and impaired Treg suppressive function, leading to systemic autoimmune disease and strong anti-tumor immunity. Pomalidomide, which degrades IKZF1 and IKZF3, induced IFN-γ overproduction in human Treg cells. Mechanistically, the Foxp3-Ikzf1-Ikzf3 complex competed with epigenetic co-activators, such as p300, for binding to target gene loci via chromatin remodeling. Therefore, the Ikzf1 association with Foxp3 is essential for the gene-repressive function of Foxp3 and could be exploited to treat autoimmune disease and cancer.

Optogenetic control of phosphate-responsive genes using single component fusion proteins in Saccharomyces cerevisiae.

Blue light illumination can be detected by Light-Oxygen-Voltage (LOV) photosensing proteins and translated into a range of biochemical responses, facilitating the generation of novel optogenetic tools to control cellular function. Here we develop new variants of our previously described VP-EL222 light-dependent transcription factor and apply them to study the phosphate-responsive signaling (PHO) pathway in the budding yeast Saccharomyces cerevisiae, exemplifying the utilities of these new tools. Focusing first on the VP-EL222 protein itself, we quantified the tunability of gene expression as a function of light intensity and duration, and demonstrated that this system can tolerate the addition of substantially larger effector domains without impacting function. We further demonstrated the utility of several EL222-driven transcriptional controllers in both plasmid and genomic settings, using the PHO5 and PHO84 promoters in their native chromosomal contexts as examples. These studies highlight the utility of light-controlled gene activation using EL222 tethered to either artificial transcription domains or yeast activator proteins (Pho4). Similarly, we demonstrate the ability to optogenetically repress gene expression with EL222 fused to the yeast Ume6 protein. We finally investigated the effects of moving EL222 recruitment sites to different locations within the PHO5 and PHO84 promoters, as well as determining how this artificial light-controlled regulation could be integrated with the native controls dependent on inorganic phosphate (Pi) availability. Taken together, our work expands the applicability of these versatile optogenetic tools in the types of functionality they can deliver and biological questions that can be probed.

Potential Use of MicroRNA Technology in Thalassemia Therapy.

Thalassemia encompasses a group of inherited hemoglobin disorders characterized by reduced or absent production of the α- or β-globin chains, leading to anemia and other complications. Current management relies on lifelong blood transfusions and iron chelation, which is burdensome for patients. This review summarizes the emerging therapeutic potential of modulating microRNAs (miRNAs) to treat thalassemia. MiRNAs are small non-coding RNAs that regulate gene expression through sequence-specific binding to messenger RNAs (mRNAs). While they commonly repress gene expression by binding to the 3' untranslated regions (UTRs) of target mRNAs, miRNAs can also interact with 5'UTRs and gene promoters to activate gene expression. Many miRNAs are now recognized as critical regulators of erythropoiesis and are abnormally expressed in β-thalassemia. Therapeutically restoring levels of deficient miRNAs or inhibiting overexpression through miRNA mimics or inhibitors (antagomir), respectively, has shown preclinical efficacy in ameliorating thalassemic phenotypes. The miR-144/451 cluster is especially compelling for targeted upregulation to reactivate fetal hemoglobin synthesis. Advances in delivery systems are addressing previous challenges in stability and targeting of miRNA-based drugs. While still early, gene therapy studies suggest combinatorial approaches with miRNA modulation may provide synergistic benefits. Several key considerations remain including enhancing delivery, minimizing off-target effects, and demonstrating long-term safety and efficacy. While no miRNA therapies have yet progressed to clinical testing for thalassemia specifically, important lessons are being learned through clinical trials for other diseases and conditions, such as cancer, cardiovascular diseases, and viral. If limitations can be overcome through multi-disciplinary collaboration, miRNAs hold great promise to expand and transform treatment options for thalassemia in the future by precisely targeting pathogenic molecular networks. Ongoing innovations, such as advancements in miRNA delivery systems, improved targeting mechanisms, and enhanced understanding of miRNA biology, continue to drive progress in this emerging field towards realizing the clinical potential of miRNA-based medicines for thalassemia patients.

Widespread changes to the translational landscape in a maize microRNA biogenesis mutant.

MicroRNAs are short, non-coding RNAs that repress gene expression in both plants and animals and have diverse functions related to growth, development, and stress responses. The ribonuclease, DICER-LIKE1 (DCL1) is required for two steps in plant miRNA biogenesis: cleavage of the primary miRNAs (pri-miRNAs) to release a hairpin structure, called the precursor miRNA (pre-miRNA) and cleavage of the pre-miRNA to generate the miRNA/miRNA* duplex. The mature miRNA guides the RNA-induced silencing complex to target RNAs with complementary sequences, resulting in translational repression and/or RNA cleavage of target mRNAs. However, the relative contribution of translational repression versus mRNA degradation by miRNAs remains unknown at the genome-level in crops, especially in maize. The maize fuzzy tassel (fzt) mutant contains a hypomorphic mutation in DCL1 resulting in broad developmental defects. While most miRNAs are reduced in fzt, the levels of miRNA-targeted mRNAs are not dramatically increased, suggesting that translational regulation by miRNAs may be common. To gain insight into the repression mechanism of plant miRNAs, we combined ribosome profiling and RNA-sequencing to globally survey miRNA activities in maize. Our data indicate that translational repression contributes significantly to regulation of most miRNA targets and that approximately one-third of miRNA targets are regulated primarily at the translational level. Surprisingly, ribosomes appear altered in fzt mutants suggesting that DCL1 may also have a role in ribosome biogenesis. Thus, DICER-LIKE1 shapes the translational landscape in plants through both miRNA-dependent and miRNA-independent mechanisms.

Elucidating regulation of polyhydroxyalkanoate metabolism in Ralstonia eutropha: Identification of transcriptional regulators from phasin and depolymerase genes

Despite the ever-growing research interest in polyhydroxyalkanoates (PHAs) as green plastic alternatives, our understanding of the regulatory mechanisms governing PHA synthesis, storage, and degradation in the model organism Ralstonia eutropha remains limited. Given its importance for central carbon metabolism, PHA homeostasis is probably controlled by a complex network of transcriptional regulators. Understanding this fine-tuning is key for developing improved PHA production strains thereby boosting the application of PHAs. We conducted promoter pull-down assays with crude protein extracts from R. eutropha Re2058/pCB113, followed by LC-MS/MS, to identify putative transcriptional regulators involved in the expression control of PHA metabolism, specifically targeting phasin phaP1 and depolymerase phaZ3 and phaZ5 genes. The impact on promoter activity was studied in vivo using β-galactosidase assays and the most promising candidates were heterologously produced in Escherichia coli and their interaction with the promoters investigated in vitro by Electrophoretic Mobility Shift Assays. We could show that R. eutropha DNA-binding XRE-family-like protein H16_B1672, specifically binds the phaP1 promoter in vitro with a KD of 175 nM and represses gene expression from this promoter in vivo. Protein H16_B1672 also showed interaction with both depolymerase promoters in vivo and in vitro suggesting a broader role in the regulation of PHA metabolism. Furthermore, in vivo assays revealed that the H-NS-like DNA-binding protein H16_B0227 and the peptidyl-prolyl cis-trans isomerase PpiB, strongly repress gene expression from PphaP1 and PphaZ3, respectively. In summary, this study provides new insights into the regulation of PHA metabolism in R. eutropha, uncovering specific interactions of novel transcriptional regulators.

Epigenetic insights into an epimutant colorless non-ripening: from fruit ripening to stress responses.

The epigenetic machinery has received extensive attention due to its involvement in plant growth, development, and adaptation to environmental changes. Recent studies often highlight the epigenetic regulatory network by discussing various epigenetic mutants across various plant species. However, a systemic understanding of essential epigenetic regulatory mechanisms remains limited due to a lack of representative mutants involved in multiple biological processes. Colorless Non-ripening (Cnr), a spontaneous epimutant isolated from a commercial population, was initially characterized for its role in fruit ripening regulation. Cnr fruits exhibit an immature phenotype with yellow skin, attributed to hypermethylation of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE-CNR (SlSPL-CNR) promoter, resulting in the repression of gene expression. In addition to DNA methylation, this process also involves histone modification and microRNA, integrating multiple epigenetic regulatory factors. Interestingly, knockout mutants of SlSPL-CNR display phenotypical distinctions from Cnr in fruit ripening, indicating complex genetic and epigenetic control over the non-ripening phenotype in Cnr fruits. Accumulating evidence suggests that Cnr epimutation is pleiotropic, participating in various biological processes such as Cd stress, Fe deficiency, vivipary, and cell death. Therefore, the Cnr epimutant serve as an excellent model for unveiling how epigenetic mechanisms are involved in diverse biological processes. This review paper focuses on recent research advances regarding the Cnr epimutant, delving into its complex genetic and epigenetic regulatory mechanisms, with the aim of enhancing our understanding and facilitating the development of high-quality, high-yield crops through epigenetic modification.

An Updated Review on the Significance of DNA and Protein Methyltransferases and De-methylases in Human Diseases: From Molecular Mechanism to Novel Therapeutic Approaches.

Epigenetic mechanisms are crucial in regulating gene expression. These mechanisms include DNA methylation and histone modifications, like methylation, acetylation, and phosphorylation. DNA methylation is associated with gene expression suppression; however, histone methylation can stimulate or repress gene expression depending on the methylation pattern of lysine or arginine residues on histones. These modifications are key factors in mediating the environmental effect on gene expression regulation. Therefore, their aberrant activity is associated with the development of various diseases. The current study aimed to review the significance of DNA and histone methyltransferases and demethylases in developing various conditions, like cardiovascular diseases, myopathies, diabetes, obesity, osteoporosis, cancer, aging, and central nervous system conditions. A better understanding of the epigenetic roles in developing diseases can pave the way for developing novel therapeutic approaches for affected patients.

Unveiling phenylpropanoid regulation: the role of DzMYB activator and repressor in durian (Durio zibethinus) fruit.

DzMYB2 functions as an MYB activator, while DzMYB3 acts as an MYB repressor. They bind to promoters, interact with DzbHLH1, and influence phenolic contents, revealing their roles in phenylpropanoid regulation in durian pulps. Durian fruit has a high nutritional value attributed to its enriched bioactive compounds, including phenolics, carotenoids, and vitamins. While various transcription factors (TFs) regulate phenylpropanoid biosynthesis, MYB (v-myb avian myeloblastosis viral oncogene homolog) TFs have emerged as pivotal players in regulating key genes within this pathway. This study aimed to identify additional candidate MYB TFs from the transcriptome database of the Monthong cultivar at five developmental/postharvest ripening stages. Candidate transcriptional activators were discerned among MYBs upregulated during the ripe stage based on the positive correlation observed between flavonoid biosynthetic genes and flavonoid contents in ripe durian pulps. Conversely, MYBs downregulated during the ripe stage were considered candidate repressors. This study focused on a candidate MYB activator (DzMYB2) and a candidate MYB repressor (DzMYB3) for functional characterization. LC-MS/MS analysis using Nicotiana benthamiana leaves transiently expressing DzMYB2 revealed increased phenolic compound contents compared with those in leaves expressing green fluorescence protein controls, while those transiently expressing DzMYB3 showed decreased phenolic compound contents. Furthermore, it was demonstrated that DzMYB2 controls phenylpropanoid biosynthesis in durian by regulating the promoters of various biosynthetic genes, including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). Meanwhile, DzMYB3 regulates the promoters of PAL, 4-coumaroyl-CoA ligase (4CL), CHS, and CHI, resulting in the activation and repression of gene expression. Moreover, it was discovered that DzMYB2 and DzMYB3 could bind to another TF, DzbHLH1, in the regulation of flavonoid biosynthesis. These findings enhance our understanding of the pivotal role of MYB proteins in regulating the phenylpropanoid pathway in durian pulps.

Mobile-CRISPRi as a powerful tool for modulating Vibrio gene expression.

CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.

A New-Generation Base Editor with an Expanded Editing Window for Microbial Cell Evolution In Vivo Based on CRISPR‒Cas12b Engineering.

Base editors (BEs) are widely used as revolutionary genome manipulation tools for cell evolution. To screen the targeted individuals, it is often necessary to expand the editing window to ensure highly diverse variant library. However, current BEs suffer from a limited editing window of 5-6 bases, corresponding to only 2-3 amino acids. Here, by engineering the CRISPR‒Cas12b, the study develops dCas12b-based CRISPRi system, which can efficiently repress gene expression by blocking the initiation and elongation of gene transcription. Further, based on dCas12b, a new-generation of BEs with an expanded editing window is established, covering the entire protospacer or more. The expanded editing window results from the smaller steric hindrance compared with other Cas proteins. The universality of the new BE is successfully validated in Bacillus subtilis and Escherichia coli. As a proof of concept, a spectinomycin-resistant E. coli strain (BL21) and a 6.49-fold increased protein secretion efficiency in E. coli JM109 are successfully obtained by using the new BE. The study, by tremendously expanding the editing window of BEs, increased the capacity of the variant library exponentially, greatly increasing the screening efficiency for microbial cell evolution.

A nuclease-dead Cas9-derived tool represses target gene expression.

Manipulation of gene expression is central to understanding gene function, engineering cell behavior, and altering biological traits according to production demands. Nuclease-dead Cas9 (dCas9), a variant of active Cas9, offers a versatile platform for the precise control of genome function without DNA cleavage. Notably, however, an effective and universal dCas9-based transcriptional repression system remains unavailable in plants. The non-canonical histone acetyltransferase TENDRIL-LESS (CsTEN) is responsible for chromatin loosening and histone modification in cucumber (Cucumis sativus). In this study, we engineered a gene regulation tool by fusing TEN and its truncated proteins with dCas9. The full-length dCas9-TEN protein substantially repressed gene expression, with the N-terminal domain identified as the core repression domain. We subsequently validated the specificity and efficacy of this system through both transient infection and genetic transformation in cucumber and Arabidopsis (Arabidopsis thaliana). Electrophoretic mobility shift assay (EMSA) revealed the ability of the N-terminal domain of TEN to bind to chromatin, which may promote target binding of the dCas9 complex and enhance the transcriptional repression effect. Our tool enriches the arsenal of genetic regulation tools available for precision breeding in crops.

RNA cis-regulators are important for Streptococcus pneumoniae in vivo success.

Bacteria have evolved complex transcriptional regulatory networks, as well as many diverse regulatory strategies at the RNA level, to enable more efficient use of metabolic resources and a rapid response to changing conditions. However, most RNA-based regulatory mechanisms are not well conserved across different bacterial species despite controlling genes important for virulence or essential biosynthetic processes. Here, we characterize the activity of, and assess the fitness benefit conferred by, twelve cis-acting regulatory RNAs (including several riboswitches and a T-box), in the opportunistic pathogen Streptococcus pneumoniae TIGR4. By evaluating native locus mutants of each regulator that result in constitutively active or repressed expression, we establish that growth defects in planktonic culture are associated with constitutive repression of gene expression, while constitutive activation of gene expression is rarely deleterious. In contrast, in mouse nasal carriage and pneumonia models, strains with either constitutively active and repressed gene expression are significantly less fit than matched control strains. Furthermore, two RNA-regulated pathways, FMN synthesis/transport and pyrimidine synthesis/transport display exceptional sensitivity to mis-regulation or constitutive gene repression in both planktonic culture and in vivo environments. Thus, despite lack of obvious phenotypes associated with constitutive gene expression in vitro, the fitness benefit conferred on bacteria via fine-tuned metabolic regulation through cis-acting regulatory RNAs is substantial in vivo, and therefore easily sufficient to drive the evolution and maintenance of diverse RNA regulatory mechanisms.

Flip the switch: the role of FleQ in modulating the transition between the free-living and sessile mode of growth in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen causing chronic infections that are associated with the sessile/biofilm mode of growth rather than the free-living/planktonic mode of growth. The transcriptional regulator FleQ contributes to both modes of growth by functioning both as an activator and repressor and inversely regulating flagella genes associated with the planktonic mode of growth and genes contributing to the biofilm mode of growth. Here, we review findings that enhance our understanding of the molecular mechanism by which FleQ enables the transition between the two modes of growth. We also explore recent advances in the mechanism of action of FleQ to both activate and repress gene expression from a single promoter. Emphasis will be on the role of sigma factors, cyclic di-GMP, and the transcriptional regulator AmrZ in inversely regulating flagella and biofilm-associated genes and converting FleQ from a repressor to an activator.

Lack of the Histone Deacetylase SIRT1 Leads to Protection against Endoplasmic Reticulum Stress through the Upregulation of Heat Shock Proteins.

Histone deacetylase SIRT1 represses gene expression through the deacetylation of histones and transcription factors and is involved in the protective cell response to stress and aging. However, upon endoplasmic reticulum (ER) stress, SIRT1 impairs the IRE1α branch of the unfolded protein response (UPR) through the inhibition of the transcriptional activity of XBP-1 and SIRT1 deficiency is beneficial under these conditions. We hypothesized that SIRT1 deficiency may unlock the blockade of transcription factors unrelated to the UPR promoting the synthesis of chaperones and improving the stability of immature proteins or triggering the clearance of unfolded proteins. SIRT1+/+ and SIRT1-/- fibroblasts were exposed to the ER stress inducer tunicamycin and cell survival and expression of heat shock proteins were analyzed 24 h after the treatment. We observed that SIRT1 loss significantly reduced cell sensitivity to ER stress and showed that SIRT1-/- but not SIRT1+/+ cells constitutively expressed high levels of phospho-STAT3 and heat shock proteins. Hsp70 silencing in SIRT1-/- cells abolished the resistance to ER stress. Furthermore, accumulation of ubiquitinated proteins was lower in SIRT1-/- than in SIRT1+/+ cells. Our data showed that SIRT1 deficiency enabled chaperones upregulation and boosted the proteasome activity, two processes that are beneficial for coping with ER stress.

Modeling neurodevelopmental disorder-associated human AGO1 mutations in Caenorhabditis elegans Argonaute alg-1

MicroRNAs (miRNA) associate with Argonaute (AGO) proteins and repress gene expression by base pairing to sequences in the 3' untranslated regions of target genes. De novo coding variants in the human AGO genes AGO1 and AGO2 cause neurodevelopmental disorders (NDD) with intellectual disability, referred to as Argonaute syndromes. Most of the altered amino acids are conserved between the miRNA-associated AGO in Homo sapiens and Caenorhabditis elegans, suggesting that the human mutations could disrupt conserved functions in miRNA biogenesis or activity. We genetically modeled four human AGO1 mutations in C. elegans by introducing identical mutations into the C. elegans AGO1 homologous gene, alg-1. These alg-1 NDD mutations cause phenotypes in C. elegans indicative of disrupted miRNA processing, miRISC (miRNA silencing complex) formation, and/or target repression. We show that the alg-1 NDD mutations are antimorphic, causing developmental and molecular phenotypes stronger than those of alg-1 null mutants, likely by sequestrating functional miRISC components into non-functional complexes. The alg-1 NDD mutations cause allele-specific disruptions in mature miRNA profiles, accompanied by perturbation of downstream gene expression, including altered translational efficiency and/or messenger RNA abundance. The perturbed genes include those with human orthologs whose dysfunction is associated with NDD. These cross-clade genetic studies illuminate fundamental AGO functions and provide insights into the conservation of miRNA-mediated post-transcriptional regulatory mechanisms.

EZH2-interacting lncRNAs contribute to gastric tumorigenesis; a review on the mechanisms of action.

Gastric cancer (GC) remains one of the deadliest malignancies worldwide, demanding new targets to improve its diagnosis and treatment. Long non-coding RNAs (lncRNAs) are dysregulated through gastric tumorigenesis and play a significant role in GC progression and development. Recent studies have revealed that lncRNAs can interact with histone-modifying polycomb protein, enhance Zeste Homolog 2 (EZH2), and mediate its site-specific functioning. EZH2, which functions as an oncogene in GC, is the catalytic subunit of the PRC2 complex that induces H3K27 trimethylation and epigenetically represses gene expression. EZH2-interacting lncRNAs can recruit EZH2 to the promoter regions of various tumor suppressor genes and cause their transcriptional deactivation via histone methylation. The interactions between EZH2 and this lncRNA modulate different processes, such as cell cycle, cell proliferation and growth, migration, invasion, metastasis, and drug resistance, in vitro and in vivo GC models. Therefore, EZH2-interacting lncRNAs are exciting targets for developing novel targeted therapies for GC. Subsequently, this review aims to focus on the roles of these interactions in GC progression to understand the therapeutic value of EZH2-interacting lncRNAs further.

A9 LOSS OF INTESTINAL EPITHELIAL NCOR1 INCREASES OXIDATIVE METABOLISM

Abstract Background The nuclear co-repressor NCOR1 acts as a central platform of a complex that represses gene expression associated with inflammatory response. Literature also recently uncovered its possible role in oxidative metabolism in some tissues and organs. Although intestinal epithelial NCOR1 is crucial to restrict IBD symptoms during experimental colitis, its precise role in controlling intestinal epithelial cell biology has not been established. Aims We aimed to investigate the role of intestinal epithelial NCOR1 in oxidative metabolism. Methods Crossing Villin-Cre and Ncor1loxP/loxP mice was performed to delete Ncor1 in the whole intestinal epithelium conditionally. Crypts from control and NCOR1ΔIEC littermates were isolated to generate colonoids 3D cultures. Phenotypic, genotypic, and transcriptomic analyses were performed on those colonoids. Results Colonoids from NCOR1ΔIEC mice were phenotypically smaller but were found to display drastic higher intestinal stem cell propagation features during organoid clonogenic assays. A Gene Set Enrichment Analysis from RNA sequencing predicted an increase in oxidative metabolism in NCOR1ΔIEC colonoids. Several genes associated with oxidative metabolism were confirmed to be modulated in both NCOR1ΔIEC colonoids and the colon of NCOR1ΔIEC mice. Mitochondrial mass was also significantly increased in colonoids when NCOR1 was absent. Loss of the co-repressor did not affect the expression of PGC1-α, a known antagonist of NCOR1. However, transcripts of cytochrome c somatic (Cycs), a target gene of PGC1-α, were found higher in NCOR1ΔIEC colonoids, suggesting that the coactivator activity is increased in the absence of NCOR1. Conclusions Our results highlight a new role for NCOR1 in the oxidative metabolism of intestinal epithelial cells. Its action via PGC1-α activity could potentially be targeted as a therapeutic measure during inflammatory bowel diseases. Funding Agencies CIHR

The BAS chromatin remodeler determines brassinosteroid-induced transcriptional activation and plant growth in Arabidopsis

Brassinosteroid (BR) signaling leads to the nuclear accumulation of the BRASSINAZOLE-RESISTANT 1 (BZR1) transcription factor, which plays dual roles in activating or repressing the expression of thousands of genes. BZR1 represses gene expression by recruiting histone deacetylases, but how it activates transcription of BR-induced genes remains unclear. Here, we show that BR reshapes the genome-wide chromatin accessibility landscape, increasing the accessibility of BR-induced genes and reducing the accessibility of BR-repressed genes in Arabidopsis. BZR1 physically interacts with the BRAHMA-associated SWI/SNF (BAS)-chromatin-remodeling complex on the genome and selectively recruits the BAS complex to BR-activated genes. Depletion of BAS abrogates the capacities of BZR1 to increase chromatin accessibility, activate gene expression, and promote cell elongation without affecting BZR1's ability to reduce chromatin accessibility and expression of BR-repressed genes. Together, these data identify that BZR1 recruits the BAS complex to open chromatin and to mediate BR-induced transcriptional activation of growth-promoting genes.

Gene Regulation and Mitochondrial Activity During White and Brown Adipogenesis Are Modulated by KDM5 Histone Demethylase.

Body fat accumulation differs between males and females and is influenced by both gonadal sex (ovaries vs testes) and chromosomal sex (XX vs XY). We previously showed that an X chromosome gene, Kdm5c, is expressed at higher levels in females compared to males and correlates with adiposity in mice and humans. Kdm5c encodes a KDM5 histone demethylase that regulates gene expression by modulating histone methylation at gene promoters and enhancers. Here, we use chemical inhibition and genetic knockdown to identify a role for KDM5 activity during early stages of white and brown preadipocyte differentiation, with specific effects on white adipocyte clonal expansion, and white and brown adipocyte gene expression and mitochondrial activity. In white adipogenesis, KDM5 activity modulates H3K4 histone methylation at the Dlk1 gene promoter to repress gene expression and promote progression from preadipocytes to mature adipocytes. In brown adipogenesis, KDM5 activity modulates H3K4 methylation and gene expression of Ucp1, which is required for thermogenesis. Unbiased transcriptome analysis revealed that KDM5 activity regulates genes associated with cell cycle regulation and mitochondrial function, and this was confirmed by functional analyses of cell proliferation and cellular bioenergetics. Using genetic knockdown, we demonstrate that KDM5C is the likely KDM5 family member that is responsible for regulation of white and brown preadipocyte programming. Given that KDM5C levels are higher in females compared to males, our findings suggest that sex differences in white and brown preadipocyte gene regulation may contribute to sex differences in adipose tissue function.

Tyrosine 1-phosphorylated RNA polymerase II transcribes PROMPTs to facilitate proximal promoter pausing and induce global transcriptional repression in response to DNA damage.

DNA damage triggers a complex transcriptional response that involves both activation and repression of gene expression. In this study, we investigated global changes in transcription in response to ionizing irradiation (IR), which induces double-strand breaks in DNA. We used mNET-seq to profile nascent transcripts bound to different phosphorylated forms of the RNA polymerase II (RNA Pol II) C-terminal domain (CTD). We found that IR leads to global transcriptional repression of protein-coding genes, accompanied by an increase in antisense transcripts near promoters, called PROMPTs, transcribed by RNA Pol II phosphorylated on tyrosine 1 (Y1P) residue of the CTD. These Y1P-transcribed PROMPTs are enriched for PRC2 binding sites and associated with RNA Pol II proximal promoter pausing. We show the interaction between Y1P RNA Pol II and PRC2, as well as PRC2 binding to PROMPTs. Inhibition of PROMPTs or depletion of PRC2 leads to loss of transcriptional repression. Our results reveal a novel function of Y1P-dependent PROMPTs in mediating PRC2 recruitment to chromatin and RNA Pol II promoter pausing in response to DNA damage.