Repress Target Gene Expression Research Articles

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.

Cooperation of MLL1 and Jun in controlling H3K4me3 on enhancers in colorectal cancer

BackgroundEnhancer dysregulation is one of the important features for cancer cells. Enhancers enriched with H3K4me3 have been implicated to play important roles in cancer. However, their detailed features and regulatory mechanisms have not been well characterized.ResultsHere, we profile the landscape of H3K4me3-enriched enhancers (m3Es) in 43 pairs of colorectal cancer (CRC) samples. M3Es are widely distributed in CRC and averagely possess around 10% of total active enhancers. We identify 1322 gain variant m3Es and 367 lost variant m3Es in CRC. The target genes of the gain m3Es are enriched in immune response pathways. We experimentally prove that repression of CBX8 and RPS6KA5 m3Es inhibits target gene expression in CRC. Furthermore, we find histone methyltransferase MLL1 is responsible for depositing H3K4me3 on the identified Vm3Es. We demonstrate that the transcription factor AP1/JUN interacts with MLL1 and regulates m3E activity. Application of a small chemical inhibitor for MLL1 activity, OICR-9429, represses target gene expression of the identified Vm3Es, enhances anti-tumor immunity and inhibits CRC growth in an animal model.ConclusionsTaken together, our study illustrates the genome-wide landscape and the regulatory mechanisms of m3Es in CRC, and reveals potential novel strategies for cancer treatment.

HDAC1 regulates the chromatin landscape to control transcriptional dependencies in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a quiescent B-cell malignancy that depends on transcriptional dysregulation for survival. The histone deacetylases are transcriptional regulators whose role within the regulatory chromatin and consequence on the CLL transcriptome is poorly characterized. Here, we profiled and integrated the genome-wide occupancy of HDAC1, BRD4, H3K27Ac, and H3K9Ac signals with chromatin accessibility, Pol2 occupancy, and target expression signatures in CLL cells. We identified that when HDAC1 was recruited within super-enhancers (SEs) marked by acetylated H3K27 and BRD4, it functioned as a transcriptional activator that drove the de novo expression of select genes to facilitate survival and progression in CLL. Targeting HDACs reduced BRD4 and Pol2 engagement to downregulate the transcript and proteins levels of specific oncogenic driver genes in CLL such as BLK, a key mediator of the B-cell receptor pathway, core transcription factors such as PAX5 and IKZF3, and the antiapoptotic gene, BCL2. Concurrently, HDAC1, when recruited in the absence of SEs, repressed target gene expression. HDAC inhibition reversed silencing of a defined set of protein-coding and noncoding RNA genes. We focused on a specific set of microRNA genes and showed that their upregulation was inversely correlated with the expression of CLL-specific survival, transcription factor, and signaling genes. Our findings identify that the transcriptional activator and repressor functions of HDACs cooperate within the same tumor to establish the transcriptional dependencies essential for survival in CLL.

Lack of expression of miR-29a/b1 impairs bladder function in male mice.

Lower urinary tract symptoms (LUTS) refer to various urological diseases, and incomplete bladder emptying is common among affected patients. Etiology of LUTS is largely unknown, and investigations of LUTS suggest that bladder fibrosis contributes to pathogenesis of LUTS. MicroRNAs (miRNAs) are short (∼22 nucleotides), non-coding RNAs that repress target gene expression by a combination of mRNA degradation and translation inhibition. The miR-29 family is best known for its anti-fibrotic role in various organs. miR-29 was decreased in bladders of patients with outlet obstruction and a rat model of bladder outlet obstruction, suggesting that miR-29 may contribute to impaired bladder function subsequent to tissue fibrosis. We characterized bladder function in male mice lacking expression of MIR29A and MIR29B1 (miR-29a/b1). Lack of miR-29a/b1 resulted in severe urinary retention, increased voiding duration with reduced flow rate, and these mice failed to void or voided irregularly during anesthetized cytometry. Collagens and elastin were increased in bladders of mice lacking miR-29a/b1. These findings reveal an important role of mir-29 in bladder homeostasis and suggest therapeutic potential of miR-29 to improve symptoms in patients with LUTS.

Critical role of lncEPAT in coupling dysregulated EGFR pathway and histone H2A deubiquitination during glioblastoma tumorigenesis.

Histone 2A (H2A) monoubiquitination is a fundamental epigenetics mechanism of gene expression, which plays a critical role in regulating cell fate. However, it is unknown if H2A ubiquitination is involved in EGFR-driven tumorigenesis. In the current study, we have characterized a previously unidentified oncogenic lncRNA (lncEPAT) that mediates the integration of the dysregulated EGFR pathway with H2A deubiquitination in tumorigenesis. LncEPAT was induced by the EGFR pathway, and high-level lncEPAT expression positively correlated with the glioma grade and predicted poor survival of glioma patients. Mass spectrometry analyses revealed that lncEPAT specifically interacted with deubiquitinase USP16. LncEPAT inhibited USP16’s recruitment to chromatin, thereby blocking USP16-mediated H2A deubiquitination and repressing target gene expression, including CDKN1A and CLUSTERIN. Depletion of lncEPAT promoted USP16-induced cell cycle arrest and cellular senescence, and then repressed GBM cell tumorigenesis. Thus, the EGFR-lncEPAT-ubH2A coupling represents a previously unidentified mechanism for epigenetic gene regulation and senescence resistance during GBM tumorigenesis.

Comparative analysis of exosomal miRNAs derived from lipopolysaccharide and polyinosinic-polycytidylic acid-stimulated chicken macrophage cell line

Exosomes play important roles in cellular communication by delivering exosomal proteins and nucleic acid molecules to cells. In particular, exosomal miRNAs can modulate various biological processes in recipient cells by repressing target gene expression. In this study, to identify the composition of exosomal miRNAs and their regulatory mechanisms against bacterial and viral infections, profiles of exosomal miRNAs from lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly(I:C))-stimulated chicken macrophage cell line (HD11) were analyzed by small RNA sequencing. Exosomes were purified after stimulation with LPS (1 μg/mL) and poly(I:C) (50 μg/mL) for 24 h. Then, exosomal RNA were analyzed for small RNA sequencing using the HiSeq 2500 System. Thirty six differentially expressed miRNAs (DE miRNAs) were obtained by comparing LPS-stimulated exosomes (LPS-EXO) and unstimulated exosomes (CTRL-EXO), 42 DE miRNAs in poly(I:C)-stimulated exosomes (POLY-EXO) and CTRL-EXO, and 45 DE miRNAs in LPS-EXO and POLY-EXO. Target genes of DE miRNAs were predicted using miRDB and TargetScan. KEGG pathway analysis showed that most of the target genes were related to mitogen-activated protein kinase and Wnt signaling pathways. Moreover, results of qRT-PCR for miRNAs (gga-miR-142-3p, gga-miR-19a-3p, gga-miR-21-3p, gga-miR-301a-3p, gga-miR-338-3p, and gga-miR-3523) were consistent with the sequencing results. This study will provide knowledge about immuno-regulatory mechanisms of exosomal miRNAs derived from macrophages against pathological insults such as bacterial and viral infections.

FBP1 /miR-24-1/enhancer axis activation blocks renal cell carcinoma progression via Warburg effect.

Warburg effect is a pivotal hallmark of cancers and appears prevalently in renal cell carcinoma (RCC). FBP1 plays a negative role in Warburg effect as a rate-limiting enzyme in gluconeogenesis, yet its mechanism in RCC remains to be further characterized. Herein, we revealed that FBP1 was downregulated in RCC tissue samples and was related to the poor survival rate of RCC. Strikingly, miR-24-1 whose DNA locus is overlapped with enhancer region chr9:95084940-95087024 was closely linked with the depletion of FBP1 in RCC. Of note, miRNAs like miR-24-1 whose DNA loci are enriched with H3K27ac and H3K4me1 modifications are belonging to nuclear activating miRNAs (NamiRNAs), which surprisingly upregulate target genes in RCC through enhancer beyond the conventional role of repressing target gene expression. Moreover, miR-24-1 reactivated the expression of FBP1 to suppress Warburg effect in RCC cells, and subsequently inhibited proliferation and metastasis of RCC cells. In mechanism, the activating role of miR-24-1 was dependent on enhancer integrity by dual luciferase reporter assay and CRISPR/Cas9 system. Ultimately, animal assay in vivo validated the suppressive function of FBP1 on 786-O and ACHN cells. Collectively, the current study highlighted that activation of FBP1 by enhancer-overlapped miR-24-1 is capable of contributing to Warburg effect repression through which RCC progression is robustly blocked, providing an alternative mechanism for RCC development and as well implying a potential clue for RCC treatment strategy.

Abstract 1494: 5-methyltetrahydrofolate (5-MTHF) as a superior ligand for delivery of small RNAs for cancer therapy

Abstract MicroRNAs (miRNAs) are small noncoding RNAs that regulate a wide array of genes. Because miRNA downregulate translation proteins associated with cancer progression, miRNA-based therapeutics have great potential as anticancer drugs. Nonetheless, the therapeutic potential of miRNA replacement is limited due to lack of safe and efficient delivery vehicles, and inability to target miRNAs to the intended cells/tissues. Thus, identifying molecules that are able to deliver therapeutic miRNA is of critical need. This could be achieved by targeting receptors that are overexpressed on target tumor cells. For example, the folate receptor (FR) is overexpressed on various tumor types including breast, lung, ovarian and brain tumors. Our laboratory has previously developed a unique strategy that directly links a miRNA mimic to the high affinity FR ligand, folate (FolamiRs). In the present work, to further improve the potency of FolamiRs, we introduce the reduced folate metabolite, 5-methyltetrahydrofolate (5-MTHF) that maintains nanomolar affinity for the FR at pH 7, but disengages from the FR at a significantly higher pH than that of folate, which is important when including an endosomal escape agent that typically disrupts endosomal acidification. We synthesize and conjugate 5-MTHF ligand to the potent tumor suppressor miRNA-34a (5-MTHF-miR-34a). We show that 5-MTHF-miR-34a uptake is specific to the cells overexpressing FR and represses target gene expression. Our data supports the use of 5-MTHF in combination with the endosomal escape agent, nigericin, to achieve robust cytosolic localization of therapeutically relevant miRNAs such as miR-34a. This work provides the foundation for future studies using 5-MTHF- conjugated therapeutic agents for targeting additional FR expressing cells, including myeloid derived suppressor cells (MDSCs) and macrophages. Collectively these studies will pave the way for developing 5-MTHF-based small RNA conjugates for treating FR-expressing cancers. The details of these intriguing results will be discussed in the poster. Citation Format: Sudarsan R. Kasireddy, Ahmed M. Abdelaal, Philip S. Low, Andrea L. Kasinski. 5-methyltetrahydrofolate (5-MTHF) as a superior ligand for delivery of small RNAs for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1494.

The Mediator Complex: A Central Coordinator of Plant Adaptive Responses to Environmental Stresses.

As sessile organisms, plants are constantly exposed to a variety of environmental stresses and have evolved adaptive mechanisms, including transcriptional reprogramming, in order to survive or acclimate under adverse conditions. Over the past several decades, a large number of gene-specific transcription factors have been identified in the transcriptional regulation of plant adaptive responses. The Mediator complex plays a key role in transducing signals from gene-specific transcription factors to the transcription machinery to activate or repress target gene expression. Since its first purification about 15 years ago, plant Mediator complex has been extensively analyzed for its composition and biological functions. Mutants of many plant Mediator subunits are not lethal but are compromised in growth, development and response to biotic and abiotic stress, underscoring a particularly important role in plant adaptive responses. Plant Mediator subunits also interact with partners other than transcription factors and components of the transcription machinery, indicating the complexity of the regulation of gene expression by plant Mediator complex. Here, we present a comprehensive discussion of recent analyses of the structure and function of plant Mediator complex, with a particular focus on its roles in plant adaptive responses to a wide spectrum of environmental stresses and associated biological processes.

A VIN3-like Protein OsVIL1 Is Involved in Grain Yield and Biomass in Rice.

In chromatin remodeling, the post-translational modification of histone proteins is mediated by multimeric protein complexes. VERNALIZATION INSENSITIVE3 (VIN3) forms a complex with Polycomb Repressive Complex 2 (PRC2), which mediates the trimethylation of H3K27 to repress target gene expression. In rice, four genes (OsVIL1-OsVIL4) encoding the VIN3-like proteins are expressed ubiquitously in various tissues. Null mutants of osvil2 display pleiotropic phenotypes such as altered flowering time, floral organ defects, and reduced tiller size. In contrast, osvil1 mutants did not show significant phenotypes except in fertilization compared with the wild type. However, transgenic plants overexpressing OsVIL1 showed phenotypes of increased biomass and grain yield. Cross-sections of the basal region of elongating stems revealed that the increased biomass was mediated by inducing cell proliferation in the meristem. Chromatin immunoprecipitation assay indicated that OsVIL1 repressed expression of cytokinin oxidase/dehydrogenase gene (OsCKX2) by binding to the promoter and genic regions of OsCKX2. We also observed that OsVIL1 modified the levels of H3K27me3 in the OsCKX2 chromatin. Because OsCKX2 encodes an enzyme that degrades active cytokinin, we conclude that OsVIL1 functions in the regulation of endogenous active cytokinin levels, thereby increasing plant height and productivity.

MicroRNA-mediated regulation of microRNA machinery controls cell fate decisions.

microRNAs associate with Argonaute proteins, forming the microRNA-induced silencing complex (miRISC), to repress target gene expression post-transcriptionally. Although microRNAs are critical regulators in mammalian cell differentiation, our understanding of how microRNA machinery, such as the miRISC, are regulated during development is still limited. We previously showed that repressing the production of one Argonaute protein, Ago2, by Trim71 is important for mouse embryonic stem cells (mESCs) self-renewal (Liu et al., 2021). Here, we show that among the four Argonaute proteins in mammals, Ago2 is the major developmentally regulated Argonaute protein in mESCs. Moreover, in pluripotency, besides the Trim71-mediated regulation of Ago2 (Liu et al., 2021), Mir182/Mir183 also repress Ago2. Specific inhibition of this microRNA-mediated repression results in stemness defects and accelerated differentiation through the let-7 microRNA pathway. These results reveal a microRNA-mediated regulatory circuit on microRNA machinery that is critical to maintaining pluripotency.

Investigation of Circulating Plasma Micro RNAs As Biomarkers for Iron Status in Blood Donors

Introduction: Ferritin is the major iron storage protein in the body and serum ferritin is the most widely used screening test both for iron deficiency and iron overload conditions. However, as ferritin is a positive acute phase response protein its usefulness is limited in the presence of either acute or chronic inflammation or liver disease. A more specific biomarker of iron status could therefore be of considerable value. MicroRNAs (miRNAs) are small non-coding RNA fragments that repress target gene expression post-transcriptionally within cells. MiRNAs can also be detected outside cells in various body fluids and these circulating forms may be useful as biomarkers for various pathological conditions. Studies indicate that certain miRNAs may play an important role in cellular processes regulating iron homeostasis including, miRNA-320 in post-transcriptional control of Transferrin Receptor 1 (TfR1) gene expression, and miR-200b in regulation of ferritin expression. The aim of this study was to determine whether circulating miRNAs in plasma could serve as biomarkers for iron status in healthy blood donors.Methods: Study participants were selected from new/active blood donors based on their latest historic ferritin results in the blood bank laboratory information system. Participants were divided into two groups, a) high ferritin (>150 μg/l, HF) and b) low ferritin (<15 μg/l, LF) arms. The study was divided into two phases; i) a pilot study (20 participants) and ii) a confirmation study (50 participants). After obtaining informed consent EDTA blood samples were collected and frozen plasma aliquots prepared by a standard method to minimize pre-analytical variation. Ferritin was measured in all participants with an automated immunoassay (Ortho Vitros®). Hepcidin and sTFR plasma levels were measured by ELISA (DRG Instruments GmbH) in the pilot study group to confirm iron status. MiRNA isolation and analysis was performed by a miRNA profiling service (Exiqon A/S, Vedbæk, Denmark). In the pilot-study 179 miRNAs were analyzed using commercially available qPCR assays (Serum/Plasma Focus Panel, Exiqon A/S). In the confirmation study a custom-made Pick-&-Mix miRNA qPCR panel was ordered including 13 candidate miRNAs chosen from the pilot-study data. Standard biostatistical methods were utilized for data analysis.Results: Serum ferritin measured on average 13.8 ± 3.8 μg/l in the pilot study low ferritin (LF) arm, compared to 231 ± 58.8 μg/l in the high ferritin (HF) group (p<0.001). Hepcidin plasma levels were markedly lower in the low ferritin (LF) arm compared to the high ferritin group (HF) (2.3 ± 0.6 vs. 17.8 ± 3.1 ng/ml, p<0.001) whereas sTfR1 levels were higher in the LF group (1.9 ± 0.4 vs. 1.1 ± 0.2 µg/ml, p<0,001) confirming the difference in iron status between the two groups. Quality control of raw miRNA qPCR data indicated good technical performance of the profiling experiments. In the pilot-study 106 of 179 miRNAs in the panel gave signals in all 20 samples and on average 153 miRNAs were detected in each sample. On unsupervised analysis by hierarchical clustering and principal component analysis (PCA) the samples did not cluster according to iron status (figure 1). When comparing HF vs. LF groups 17 miRNAs were found to be differentially expressed when using a significance level of 0.05, but only one assay (hsa-miR-34a-5p) passed Benjamini-Hochberg correction for multiple testing. However, this particular miRNA frequently gave low/absent signals in both groups and was not considered further. To confirm the findings of the pilot study thirteen miRNAs were chosen for a confirmation-study based on technical performance and expression in the pilot study. Unsupervised analysis of 50 samples did not reveal any difference in expression for the 13 selected miRNAs between the LF and HF groups. Furthermore, when comparing the two groups no miRNA was found to be differentially expressed when using a p-value cutoff of 0.05.Conclusion: These data do not suggest that those circulating plasma miRNAs analyzed by qPCR panels in this study should be considered promising biomarkers for iron status in healthy individuals. However, in the future, sensitive and comprehensive detection methods such as next-generation sequencing might reveal novel circulating miRNAs that correlate with iron stores. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The EAR Motif in the Arabidopsis MADS Transcription Factor AGAMOUS-Like 15 Is Not Necessary to Promote Somatic Embryogenesis.

AGAMOUS-like 15 (AGL15) is a member of the MADS domain family of transcription factors (TFs) that can directly induce and repress target gene expression, and for which promotion of somatic embryogenesis (SE) is positively correlated with accumulation. An ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif of form LxLxL within the carboxyl-terminal domain of AGL15 was shown to be involved in repression of gene expression. Here, we examine whether AGL15′s ability to repress gene expression is needed to promote SE. While a form of AGL15 where the LxLxL is changed to AxAxA can still promote SE, another form with a strong transcriptional activator at the carboxy-terminal end, does not promote SE and, in fact, is detrimental to SE development. Select target genes were examined for response to the different forms of AGL15.

RIP-Seq of EZH2 Identifies TCONS-00036665 as a Regulator of Myogenesis in Pigs.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 and contains a SET domain that catalyzes histone H3 trimethylation on lysine 27 (H3K27me3) to generate an epigenetic silencing mark. EZH2 interacts with transcription factors or RNA transcripts to perform its function. In this study, we applied RNA immunoprecipitation sequencing and long intergenic non-coding RNA (lincRNA) sequencing methods to identify EZH2-binding lincRNAs. A total of 356 novel EZH2-binding lincRNAs were identified by bioinformatics analysis and an EZH2-binding lincRNA TCONS-00036665 was characterized. TCONS-00036665 promoted pig skeletal satellite cell proliferation but inhibited cell differentiation, and this function was conserved between pigs and mice. Further mechanistic studies indicated that TCONS-00036665 can bind to EZH2 and recruits EZH2 to the promoters of the target genes p21, MyoG, and Myh4, which leads to the enrichment of H3K27me3 and the repression of target gene expression and pig myogenesis. In conclusion, the lincRNA TCONS-00036665 regulates pig myogenesis through its interaction with EZH2.

No Easy Way Out for EZH2: Its Pleiotropic, Noncanonical Effects on Gene Regulation and Cellular Function.

Enhancer of zeste homolog 2 (EZH2) plays critical roles in a range of biological processes including organ development and homeostasis, epigenomic and transcriptomic regulation, gene repression and imprinting, and DNA damage repair. A widely known function of EZH2 is to serve as an enzymatic subunit of Polycomb repressive complex 2 (PRC2) and catalyze trimethylation of histone H3 lysine 27 (H3K27me3) for repressing target gene expression. However, an increasing body of evidence demonstrates that EZH2 has many “non-conventional” functions that go beyond H3K27 methylation as a Polycomb factor. First, EZH2 can methylate a number of nonhistone proteins, thereby regulating cellular processes in an H3K27me3-independent fashion. Furthermore, EZH2 relies on both methyltransferase-dependent and methyltransferase-independent mechanisms for modulating gene-expression programs and/or epigenomic patterns of cells. Importantly, independent of PRC2, EZH2 also forms physical interactions with a number of DNA-binding factors and transcriptional coactivators to context-dependently influence gene expression. The purpose of this review is to detail the complex, noncanonical roles of EZH2, which are generally less appreciated in gene and (epi)genome regulation. Because EZH2 deregulation is prevalent in human diseases such as cancer, there is increased dependency on its noncanonical function, which shall have important implications in developing more effective therapeutics.

The Contribution of Transcriptional Coregulators in the Maintenance of β-cell Function and Identity

Islet β-cell dysfunction that leads to impaired insulin secretion is a principal source of pathology of diabetes. In type 2 diabetes, this breakdown in β-cell health is associated with compromised islet-enriched transcription factor (TF) activity that disrupts gene expression programs essential for cell function and identity. TF activity is modulated by recruited coregulators that govern activation and/or repression of target gene expression, thereby providing a supporting layer of control. To date, more than 350 coregulators have been discovered that coordinate nucleosome rearrangements, modify histones, and physically bridge general transcriptional machinery to recruited TFs; however, relatively few have been attributed to β-cell function. Here, we will describe recent findings on those coregulators with direct roles in maintaining islet β-cell health and identity and discuss how disruption of coregulator activity is associated with diabetes pathogenesis.

Comprehensive genomic characterization of NAC transcription factor family and their response to salt and drought stress in peanut

BackgroundPeanut is one of the most important oil crop species worldwide. NAC transcription factor (TF) genes play important roles in the salt and drought stress responses of plants by activating or repressing target gene expression. However, little is known about NAC genes in peanut.ResultsWe performed a genome-wide characterization of NAC genes from the diploid wild peanut species Arachis duranensis and Arachis ipaensis, which included analyses of chromosomal locations, gene structures, conserved motifs, expression patterns, and cis-acting elements within their promoter regions. In total, 81 and 79 NAC genes were identified from A. duranensis and A. ipaensis genomes. Phylogenetic analysis of peanut NACs along with their Arabidopsis and rice counterparts categorized these proteins into 18 distinct subgroups. Fifty-one orthologous gene pairs were identified, and 46 orthologues were found to be highly syntenic on the chromosomes of both A. duranensis and A. ipaensis. Comparative RNA sequencing (RNA-seq)-based analysis revealed that the expression of 43 NAC genes was up- or downregulated under salt stress and under drought stress. Among these genes, the expression of 17 genes in cultivated peanut (Arachis hypogaea) was up- or downregulated under both stresses. Moreover, quantitative reverse transcription PCR (RT-qPCR)-based analysis revealed that the expression of most of the randomly selected NAC genes tended to be consistent with the comparative RNA-seq results.ConclusionOur results facilitated the functional characterization of peanut NAC genes, and the genes involved in salt and drought stress responses identified in this study could be potential genes for peanut improvement.

Memory of Divisional History Directs the Continuous Process of Primitive Hematopoietic Lineage Commitment.

SummaryHematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.

The Evening Complex Establishes Repressive Chromatin Domains Via H2A.Z Deposition.

The Evening Complex (EC) is a core component of the Arabidopsis (Arabidopsis thaliana) circadian clock, which represses target gene expression at the end of the day and integrates temperature information to coordinate environmental and endogenous signals. Here we show that the EC induces repressive chromatin structure to regulate the evening transcriptome. The EC component ELF3 directly interacts with a protein from the SWI2/SNF2-RELATED (SWR1) complex to control deposition of H2A.Z-nucleosomes at the EC target genes. SWR1 components display circadian oscillation in gene expression with a peak at dusk. In turn, SWR1 is required for the circadian clockwork, as defects in SWR1 activity alter morning-expressed genes. The EC-SWR1 complex binds to the loci of the core clock genes PSEUDO-RESPONSE REGULATOR7 (PRR7) and PRR9 and catalyzes deposition of nucleosomes containing the histone variant H2A.Z coincident with the repression of these genes at dusk. This provides a mechanism by which the circadian clock temporally establishes repressive chromatin domains to shape oscillatory gene expression around dusk.

First High-Resolution Crystal Structures of the Glucocorticoid Receptor Ligand-Binding Domain-Peroxisome Proliferator-Activated γ Coactivator 1-α Complex with Endogenous and Synthetic Glucocorticoids.

Both synthetic and endogenous glucocorticoids are important pharmaceutic drugs known to bind to the ligand-binding domain (LBD) of glucocorticoid receptor (GR), a member of the nuclear receptor (NR) superfamily. Ligand binding induces conformational changes within GR, resulting in subsequent DNA binding and differential coregulator recruitment, ultimately activating or repressing target gene expression. One of the most crucial coregulators is peroxisome proliferator-activated γ coactivator 1-α (PGC1α), which acts to regulate energy metabolism by directly interacting with GR to modulate gene expression. However, the mechanisms through which PGC1α senses GR conformation to drive transcription are not completely known. Here, an ancestral variant of the GR (AncGR2) LBD was used as a tool to produce stable protein for biochemical and structural studies. PGC1α is found to interact more tightly and form a more stable complex with AncGR2 LBD than nuclear receptor coactivator 2. We report the first high-resolution X-ray crystal structures of AncGR2 LBD in complex with PGC1α and dexamethasone (DEX) or hydrocortisone (HCY). Structural analyses reveal how distinct steroid drugs bind to GR with different affinities by unique hydrogen bonds and hydrophobic interactions. Important charge clamps are formed between the activation function 2 and PGC1α to mediate their specific interactions. These interactions lead to a high level of protection from hydrogen-deuterium exchange at the coregulator interaction site and strong intramolecular allosteric communication to ligand binding site. This is the first structure detailing the GR-PGC1α interaction providing a foundation for future design of specific therapeutic agents targeting these critical metabolic regulators. SIGNIFICANCE STATEMENT: High-resolution structures of AncGR2 LBD bound to DEX and HCY in complex with PGC1α reveal the molecular mechanism of PGC1α binding to AncGR2 LBD as well as the distinct affinities between DEX and HCY binding. Identifying the structural mechanisms that drive drug affinity is of pharmacologic interest to the glucocorticoid receptor field as an avenue to guide future drug design targeting GR-PGC1α signaling, which plays a crucial role in controlling hepatic glucose output.