Transcriptional Repressor Research Articles

SREBP1c-Mediated Transcriptional Repression of YME1L1 Contributes to Acute Kidney Injury by Inducing Mitochondrial Dysfunction in Tubular Epithelial Cells.

Acute kidney injury (AKI) is a prevalent clinical syndrome with high morbidity and mortality. Accumulating studies suggest mitochondrial dysfunction as the typical characteristics and key process of AKI, but the underlying mechanism remains elusive. The YME1-like 1 (YME1L1) ATPase, an inner mitochondrial membrane protein, is screened and identified to be downregulated in renal tubular epithelial cells of various mouse models and patients of AKI. Dramatically, restoration of YME1L1 expression significantly alleviates cisplatin-induced AKI and subsequent chronic kidney disease (CKD) through attenuating mitochondrial dysfunction via maintaining optic atrophy 1 (OPA1)-mediated mitochondrial energy metabolism homeostasis. Mechanistically, the upregulated expression of sterol regulatory element binding transcription factor 1c (SREBP1c) is demonstrated to be responsible for cisplatin-mediated transcriptional inhibition of YME1L1 via directly binding to its promoter region. Moreover, cisplatin-induced methyltransferase-like 3 (METTL3)-mediated m6A modification enhances SREBP1c mRNA stability, thereby upregulating its expression. Notably, both depletion of SREBP1c and renal tubule-specific overexpression of YME1L1 markedly ameliorate cisplatin-induced AKI and its transition to CKD. Taken together, these findings suggest that METTL3-mediated SREBP1c upregulation contributes to AKI and its progression to CKD through disrupting mitochondrial energy metabolism via transcriptionally suppressing YME1L1. Targeting the SREBP1c/YME1L1 signaling may serve as a novel therapeutic strategy against AKI.

Epistasis between genetic variations on MdMYB109 and MdHXK1 exerts a large effect on sugar content in apple fruit.

Many quantitative traits are controlled by multiple genetic variations with minor effects, making it challenging to resolve the underlying genetic network and to apply functional markers in breeding. Affected by up to a hundred quantitative trait loci (QTLs), fruit-soluble sugar content is one of the most complex quantitative traits in apple (Malus sp.). Here, QTLs for apple fruit sucrose and fructose content were identified via QTL mapping and bulked-segregant analysis sequencing (BSA-seq) using a population derived from a 'Jonathan' × 'Golden Delicious' cross. Allelic variations and non-allelic interactions were validated in the candidate genes within these defined QTL regions. Three single-nucleotide polymorphisms (SNPs) (SNP -326 C/T, SNP -705 A/G, and SNP -706 G/T) in the MdMYB109 promoter region affected the binding ability of the repressive transcription factor MdWRKY33, leading to increased MdMYB109 expression. MdMYB109 bound directly to the promoter of the sucrose transporter gene MdSUT2.2 and activated its expression, raising fruit sucrose content. A SNP (SNP1060 A/G) in the hexokinase gene MdHXK1 affected the phosphorylation of the transcription factor MdbHLH3, and phosphorylated MdbHLH3 interacted with MdMYB109 to co-activate MdSUT2.2 expression and increase fruit sucrose content. Adding the joint effects of the genotype combinations at the SNP markers based on the SNPs in MdMYB109 and MdHXK1 increased the prediction accuracy of a genomics-assisted prediction (GAP) model for total soluble solid content from 0.3758 to 0.5531. These results uncovered functional variations in MdMYB109 and MdHXK1 regulating apple fruit sucrose content. The updated GAP model with improved predictability can be used efficiently in apple breeding.

Characterization and Transcriptional Regulation of the 2-Ketogluconate Utilization Operon in Pseudomonas plecoglossicida

Pseudomonas plecoglossicida JUIM01 is an industrial 2-keto-d-gluconate (2KGA)-producing strain. However, its regulation mechanism of 2KGA metabolism remains to be clarified. Among other reported Pseudomonas species, the 2-ketogluconate utilization operon (kgu operon) plays key roles in 2KGA catabolism. In this study, the structural genes of the kgu operon and its promoter in P. plecoglossicida JUIM01 were identified using reverse transcription PCR and lacZ reporter gene fusion. The results showed the kgu operon in P. plecoglossicida was composed of four structural genes: kguE, kguK, kguT, and kguD. The ptxS gene located upstream of kguE was excluded from the kgu operon. Then, the knockout and corresponding complementation strains of kguE, kguK, kguT, and kguD were constructed, respectively, to prove the kgu operon was involved in 2KGA catabolism of P. plecoglossicida. The knockout stains, especially JUIM01ΔkguE, showed potential as industrial production strains for 2KGA. Moreover, the transcriptional regulation mechanism of PtxS on the kgu operon was elucidated using multiple methods. In P. plecoglossicida, the LacI-family transcription regulator PtxS could recognize a 14 bp palindrome (5′-TGAAACCGGTTTCA-3′) within the promoter region of the kgu operon and specifically bind to a 26 bp region where the palindrome was located. As the binding sites overlapped with the transcription start site of the kgu operon, the binding of PtxS possibly hindered the binding of RNA polymerase, thus repressing the transcription of the kgu operon and further regulating 2KGA catabolism. 2KGA bound to PtxS as an effector to dissociate it from the kgu operon promoter region, so as to relieve the transcription repression. The results will provide strategies for improving the product accumulation in 2KGA industrial production and theoretical bases for the construction of a Pseudomonas chassis.

The SLR1-OsMADS23-D14 module mediates the crosstalk between strigolactone and gibberellin signaling to control rice tillering.

Strigolactones (SLs) and gibberellins (GAs) have been found to inhibit plant branching or tillering, but molecular mechanisms underlying the interplay between SL and GA signaling to modulate tillering remain elusive. We found that the transcription factor OsMADS23 plays a crucial role in the crosslink between SL and GA signaling in rice tillering. Loss-of-function mutant osmads23 shows normal axillary bud formation but defective bud outgrowth, thus reducing the tiller number in rice, whereas overexpression of OsMADS23 significantly increases tillering by promoting tiller bud outgrowth. OsMADS23 physically interacts with DELLA protein SLENDER RICE1 (SLR1), and the interaction reciprocally stabilizes each other. Genetic evidence showed that SLR1 is required for OsMADS23 to control rice tillering. OsMADS23 acts as an upstream transcriptional repressor to inhibit the expression of SL receptor gene DWARF14 (D14), and addition of SLR1 further enhances OsMADS23-mediated transcriptional repression of D14, indicating that D14 is the downstream target gene of OsMADS23-SLR1 complex. Moreover, application of exogenous SL and GA reduces the protein stability of OsMADS23-SLR1 complex and promotes D14 expression. Our results revealed that SLs and GAs synergistically inhibit rice tillering by destabilizing OsMADS23-SLR1 complex, which provides important insights into the molecular networks of SL-GA synergistic interaction during rice tillering.

The intestine-specific homeobox (ISX) modulates β-carotene-dependent regulation of microsomal triglyceride transfer protein (MTP) in a tissue-specific manner

Vitamin A is an essential nutrient crucial to ensuring proper mammalian embryonic development. β-Carotene is the most prevalent form of vitamin A in food that, when transferred in its intact form from mother to the developing tissues, can serve as an in situ source of retinoic acid, the active form of vitamin A. We have previously provided evidence that the maternal-fetal transfer of β-carotene across the placenta is mediated by lipoproteins and that β-carotene itself regulates placenta lipoprotein biogenesis by means of its derivatives β-apo-10′-carotenoids and retinoic acid. These metabolites exert antagonistic transcriptional activity on placental microsomal triglyceride transfer protein (MTP) and apolipoprotein B (APOB), two key players of lipoprotein biosynthesis. Here, we analyzed the time-dependency of this regulation over the course of 24 h upon a single maternal administration of β-carotene. We also tested the hypothesis that the transcriptional repressor intestine-specific homeobox (ISX) plays a role in the regulation of Mttp in placenta. We observed that ISX is expressed in placenta of mouse dams and is regulated by β-carotene availability. Furthermore, we demonstrated that the absence of Isx disrupts the β-carotene-mediated regulation of placental MTP. We also showed that this mechanism is organ-specific, as it was not observed in enterocytes of the intestine, a major place of Isx expression. Therefore, we identified ISX as a “master” regulator of a placental β-carotene-dependent transcriptional regulatory cascade that fine-tunes the flux of provitamin A carotenoid towards the developing fetus.

The ubiquitination degradation of KLF15 mediated by WSB2 promotes lipogenesis and progression of hepatocellular carcinoma via inhibiting PDLIM2 expression.

Krüppel-like factors15 (KLF15) is a cancer suppressor in many cancers. However, its precise function in the development of hepatocellular carcinoma (HCC) remains unclear. Lipogenesis is necessary for the development of HCC. This research aims to investigate the role of KLF15 in the regulation of hepatic lipid production and HCC progression. The binding relationships among genes were confirmed by ChIP, dual luciferase assays, and Co-IP. Lipogenesis was examined by oil red O staining. Triglyceride and cholesterol levels were measured through commercial kits. The effect of treatment on HCC cell viability, proliferation, migration, and invasion were assessed using CCK-8, clone formation, or transwell assays. A subcutaneous tumorigenic model was utilized to explore the effects of PDLIM2 in HCC in vivo. KLF15 were downregulated in human HCC tissues. KLF15 overexpression reduced lipid droplet production, suppressed the expression of genes associated with lipogenesis, and promoted cell proliferation, migration, and invasion. KLF15 suppressed the NF-κB pathway through transcriptional activation of PDLIM2. PDLIM2 knockdown attenuated the effect of KLF15 overexpression on HCC. WSB2 degraded KLF15 through ubiquitination to promote HCC lipogenesis and development. The ubiquitination degradation of KLF15 was mediated by WSB2, which led to transcriptional repression of PDLIM2 and further activation of the NF-κB pathway, ultimately promoting HCC lipogenesis and development.

Histone methyltransferases MLL2 and SETD1A/B play distinct roles in H3K4me3 deposition during the transition from totipotency to pluripotency.

In early mammalian embryogenesis, a shift from non-canonical histone H3 lysine 4 trimethylation (H3K4me3) linked to transcriptional repression to canonical H3K4me3 indicating active promoters occurs during zygotic genome activation (ZGA). However, the mechanisms and roles of these H3K4me3 states in embryogenesis remain poorly understood. Our research reveals that the histone methyltransferase MLL2 is responsible for installing H3K4me3 (both non-canonical and canonical) in totipotent embryos, while a transition to SETD1A/B-deposited H3K4me3 occurs in pluripotent embryos. Interestingly, MLL2-mediated H3K4me3 operates independently of transcription, fostering a relaxed chromatin state conducive to totipotency rather than directly influencing transcription. Conversely, SETD1A/B-mediated H3K4me3, which depends on transcription, is crucial for facilitating expression of genes essential for pluripotency and pre-implantation development. Our findings highlight the role of the H3K4me3 transition, mediated by an MLL2-to-SETD1A/B relay mechanism, in the regulation of transition from totipotency to pluripotency during early embryogenesis.

The capicua-ataxin-1-like complex regulates Notch-driven marginal zone B cell development and sepsis progression

Follicular B (FOB) and marginal zone B (MZB) cells are pivotal in humoral immune responses against pathogenic infections. MZB cells can exacerbate endotoxic shock via interleukin-6 secretion. Here we show that the transcriptional repressor capicua (CIC) and its binding partner, ataxin-1-like (ATXN1L), play important roles in FOB and MZB cell development. CIC deficiency reduces the size of both FOB and MZB cell populations, whereas ATXN1L deficiency specifically affects MZB cells. B cell receptor signaling is impaired only in Cic-deficient FOB cells, whereas Notch signaling is disrupted in both Cic-deficient and Atxn1l-deficient MZB cells. Mechanistically, ETV4 de-repression leads to inhibition of Notch1 and Notch2 transcription, thereby inhibiting MZB cell development in B cell-specific Cic-deficient (Cicf/f;Cd19-Cre) and Atxn1l-deficient (Atxn1lf/f;Cd19-Cre) mice. In Cicf/f;Cd19-Cre and Atxn1lf/f; Cd19-Cre mice, humoral immune responses and lipopolysaccharide-induced sepsis progression are attenuated but are restored upon Etv4-deletion. These findings highlight the importance of the CIC-ATXN1L complex in MZB cell development and may provide proof of principle for therapeutic targeting in sepsis.

Characterization of DNA methylation reader proteins of Arabidopsis thaliana.

In plants, cytosine DNA methylation (mC) is largely associated with transcriptional repression of transposable elements, but it can also be found in the body of expressed genes, referred to as gene body methylation (gbM). gbM is correlated with ubiquitously expressed genes; however, its function, or absence thereof, is highly debated. The different outputs that mC can have raise questions as to how it is interpreted-or read-differently in these sequence and genomic contexts. To screen for potential mC-binding proteins, we performed an unbiased DNA affinity pull-down assay combined with quantitative mass spectrometry using methylated DNA probes for each DNA sequence context. All mC readers known to date preferentially bind to the methylated probes, along with a range of new mC-binding protein candidates. Functional characterization of these mC readers, focused on the MBD and SUVH families, was undertaken by ChIP-seq mapping of genome-wide binding sites, their protein interactors, and the impact of high-order mutations on transcriptomic and epigenomic profiles. Together, these results highlight specific context preferences for these proteins, and in particular the ability of MBD2 to bind predominantly to gbM. This comprehensive analysis of Arabidopsis mC readers emphasizes the complexity and interconnectivity between DNA methylation and chromatin remodeling processes in plants.

SP1-mediated transcriptional repression of SFRP5 is correlated with cardiac fibroblast activation and atrial myocyte apoptosis in the development of atrial fibrillation

Secreted frizzled related protein 5 (SFRP5) is a recognized cardioprotective protein with diminished expression in atrial fibrillation (AF). This study investigates SFRP5's function in AF-related cardiac fibrosis and cardiomyocyte apoptosis, exploring the underlying dysregulation causes. Utilizing C57BL/6 mice, mouse cardiac fibroblasts (CFs), and HC-1 mouse atrial myocyte cell line, AF models were induced by angiotensin Ⅱ (Ang Ⅱ). SFRP5 levels were consistently decreased in plasma samples from clinical patients, modeled mice, and CF culture supernatants. Treatment with recombinant SFRP5 restored its levels, mitigating Ang Ⅱ-induced AF in mice and ameliorating atrial tissue fibrosis and oxidative stress. In vitro, SFRP5 recombinant protein suppressed CF activation and fibrosis-related markers. The study identified Sp1 transcription factor (SP1) binding to the SFRP5 promoter, causing transcriptional repression. SP1 knockdown reinstated SFRP5 levels in mice and CFs, thus suppressing fibrosis. Additionally, SP1 knockdown attenuated Ang Ⅱ-induced apoptosis in HC-1 cells, but this effect was counteracted by concurrent SFRP5 knockdown. In conclusion, this investigation underscores that SP1 mediates SFRP5 loss during AF by transcriptional repression, contributing to fibrosis and myocyte apoptosis. These findings illuminate potential therapeutic interventions targeting the SFRP5-SP1 axis in AF-related cardiac complications.

Design, synthesis, biological evaluation and molecular docking study of novel quinolone derivatives as potent HDAC1 (Histone Deacetylase) inhibitors and anticancer agents

Histone deacetylases (HDACs), especially HDAC1, are attractive targets for anticancer drug design because of their significant role in transcriptional repression and chromatin remodeling. Benzamide groups are widely reported as the most effective zinc binding groups (ZBGs) in design of HDAC inhibitors. Quinolone derivatives, exhibiting various therapeutic effects, are attractive scaffolds for developing novel HDAC inhibitors with potentially supposed therapeutic profiles. To explore the potential of 4-quinolones as a novel scaffold for a cap of HDACis, 14 novel structures were rationally designed and synthesized. Four cancer cell lines (HCT116, HT29, MCF7, and A549) and one normal cell (CHO) were applied to investigate the cytotoxicity of synthesized compounds. Furthermore, the inhibitory activity of the synthesized compounds was analyzed against pan-HDAC isozymes (HDAC1, HDAC2, HDAC3 and HDAC6). Five compounds (5a, 5b, 5c, 5e and 5h) showed strong cytotoxicity and HDAC inhibition even superior to entinostat as the reference drug. Collectively, these results suggest that compounds with an electron-donating group at the positions 6 or 8 of the quinolone ring (cap) appear to enhance the anti-proliferative potency. After performing induce-fit docking (on HDAC1, 2, 3, 6) for all the synthesized compounds, a significant correlation was identified between the MTT results, the inhibitory activity of pan-HDAC, and the docking scores of HDAC1. Therefore, compounds 5e and 5a (the most potent compounds) were identified as the most favorable leads for further investigation of HDAC1-specific enzyme inhibition. Compounds 5a and 5e showed impressive HDAC1 inhibitory potency with IC50 values of 0.48 μM and 0.36 μM, respectively, equal or more than that of Entinostat. Compound 5e (3 µM) also exhibited significant induction of apoptosis in the HCT116 cell line (the percentage of apoptotic cells was 81.87%), even stronger than the reference drug Entinostat (the percentage of apoptotic cells was 37.5%) at the same concentration which is consistent with their antiproliferative activities as well. 100 ns molecular dynamic (MD) simulation revealed that compound 5e can form stable interactions with HDAC1 through coordination with zinc ion, strong hydrophobic interactions and formation of hydrogen bonds. These results suggested that compound 5e can be promising lead for further development of potent anticancer and HDAC inhibitor drugs.

A polycomb group protein EED epigenetically regulates responses in lipopolysaccharide tolerized macrophages

BackgroundTo avoid exaggerated inflammation, innate immune cells adapt to become hypo-responsive or “tolerance” in response to successive exposure to stimuli, which is a part of innate immune memory. Polycomb repressive complex 2 (PRC2) mediates the transcriptional repression by catalyzing histone H3 lysine 27 trimethylation (H3K27me3) but little is known about its role in lipopolysaccharide (LPS)-induced tolerance in macrophages.ResultWe examined the unexplored roles of EED, a component of the PRC2, in LPS tolerant macrophages. In Eed KO macrophages, significant reduction in H3K27me3 and increased active histone mark, H3K27ac, was observed. Eed KO macrophages exhibited dampened pro-inflammatory cytokine productions (TNF-α and IL-6) while increasing non-tolerizable genes upon LPS tolerance. Pharmacological inhibition of EED also reduced TNF-α and IL-6 during LPS tolerance. Mechanistically, LPS tolerized Eed KO macrophages failed to increase glycolytic activity. RNA-Seq analyses revealed that the hallmarks of hypoxia, TGF-β, and Wnt/β-catenin signaling were enriched in LPS tolerized Eed KO macrophages. Among the upregulated genes, the promoter of Runx3 was found to be associated with EED. Silencing Runx3 in Eed KO macrophages partially rescued the dampened pro-inflammatory response during LPS tolerance. Enrichment of H3K27me3 was decreased in a subset of genes that are upregulated in Eed KO LPS tolerized macrophages, indicating the direct regulatory roles of PRC2 on such genes. Motif enrichment analysis identified the ETS family transcription factor binding sites in the absence of EED in LPS tolerized macrophages.ConclusionOur results provided mechanistic insight into how the PRC2 via EED regulates LPS tolerance in macrophages by epigenetically silencing genes that play a crucial role during LPS tolerance such as those of the TGF-β/Runx3 axis.

Epigenetic dysregulation of H19/IGF2 in hepatic cells exposed to toxic metal mixtures in vitro

Exposure to mixtures of toxic metals is known to cause adverse health effects through epigenetic alterations. Here we aimed to examine the unexplored area of aberrant DNA methylation in the H19/IGF2 domain following combined toxic metal exposure. An in vitro epigenotoxicity assay using the human normal liver epithelial cell line THLE-3 was conducted. When THLE-3 cells were exposed to specific concentrations of either organic arsenic or MeHgCl, an increase in the H19 lncRNA levels and a marked reduction in the IGF2 mRNA levels were observed. In contrast, combined exposures coupled with CdCl2 resulted in the transcriptional repression of H19 and transcriptional activation of IGF2. It should be noted that the correlation between the dysregulated expression of H19/IGF2 and the hypermethylated CpG sites within the H19 differentially methylated region (DMR) was statistically significant. Furthermore, we performed transcriptomic analysis of the hepatocytes exposed to toxic metal combinations indicating enrichment of pro-inflammatory and anti-proliferative pathways compared to the unexposed cells. Our results suggest that hazardous metal mixtures may trigger epigenetic aberrations at the H19/IGF2 locus. We propose that altered CpG methylation in the H19 DMR could be a candidate biomarker for hepatic epigenotoxicity, in part, due to environmental exposure.

Bacterial WYL domain transcriptional repressors sense single-stranded DNA to control gene expression.

Bacteria encode a wide array of immune systems to protect themselves against ubiquitous bacteriophages and foreign DNA elements. While these systems' molecular mechanisms are becoming increasingly well known, their regulation remains poorly understood. Here, we show that an immune system-associated transcriptional repressor of the wHTH-WYL-WCX family, CapW, directly binds single-stranded DNA to sense DNA damage and activate expression of its associated immune system. We show that CapW mediates increased expression of a reporter gene in response to DNA damage in a host cell. CapW directly binds single-stranded DNA by-products of DNA repair through its WYL domain, causing a conformational change that releases the protein from double-stranded DNA. In an Escherichiacoli CBASS system with an integrated capW gene, we find that CapW-mediated transcriptional activation is important for this system's ability to prevent induction of a λ prophage. Overall, our data reveal the molecular mechanisms of WYL-domain transcriptional repressors, and provide an example of how bacteria can balance the protective benefits of carrying anti-phage immune systems against the inherent risk of these systems' aberrant activation.

Toll-Like receptor 3-mediated interferon-β production is suppressed by oncostatin m and a broader epithelial-mesenchymal transition program

BackgroundPatients with Triple Negative Breast Cancer (TNBC) currently lack targeted therapies, and consequently face higher mortality rates when compared to patients with other breast cancer subtypes. The tumor microenvironment (TME) cytokine Oncostatin M (OSM) reprograms TNBC cells to a more stem-like/mesenchymal state, conferring aggressive cancer cell properties such as enhanced migration and invasion, increased tumor-initiating capacity, and intrinsic resistance to the current standards of care. In contrast to OSM, Interferon-β (IFN-β) promotes a more differentiated, epithelial cell phenotype in addition to its role as an activator of anti-tumor immunity. Importantly, OSM suppresses the production of IFN-β, although the mechanism of IFN-β suppression has not yet been elucidated.MethodsIFN-β production and downstream autocrine signaling were assessed via quantitative real-time PCR (qRT-PCR) and Western blotting in TNBC cells following exposure to OSM. RNA-sequencing (RNA-seq) was used to assess an IFN-β metagene signature, and to assess the expression of innate immune sensors, which are upstream activators of IFN-β. Cell migration was assessed using an in vitro chemotaxis assay. Additionally, TNBC cells were exposed to TGF-β1, Snail, and Zeb1, and IFN-β production and downstream autocrine signaling were assessed via RNA-seq, qRT-PCR, and Western blotting.ResultsHere, we identify the repression of Toll-like Receptor 3 (TLR3), an innate immune sensor, as the key molecular event linking OSM signaling and the repression of IFN-β transcription, production, and autocrine IFN signaling. Moreover, we demonstrate that additional epithelial-mesenchymal transition-inducing factors, such as TGF-β1, Snail, and Zeb1, similarly suppress TLR3-mediated IFN-β production and signaling.ConclusionsOur findings provide a novel insight into the regulation of TLR3 and IFN-β production in TNBC cells, which are known indicators of treatment responses to DNA-damaging therapies. Furthermore, strategies to stimulate TLR3 in order to increase IFN-β within the TME may be ineffective in stem-like/mesenchymal cells, as TLR3 is strongly repressed. Rather, we propose that therapies targeting OSM or OSM receptor would reverse the stem-like/mesenchymal program and restore TLR3-mediated IFN-β production within the TME, facilitating improved responses to current therapies.

Haplotype-resolved reconstruction and functional interrogation of cancer karyotypes.

Genomic characterization has revealed widespread structural complexity in cancer karyotypes, however shotgun sequencing cannot resolve genomic rearrangements with chromosome-length continuity. Here, we describe a two-tiered approach to determine the segmental composition of rearranged chromosomes with haplotype resolution. First, we present refLinker , a new method for robust determination of chromosomal haplotypes using cancer Hi-C data. Second, we use haplotype-specific Hi-C contacts to determine the segmental structure of rearranged chromosomes. By contrast with existing methods for diploid haplotype inference, our approach is robust to the confounding effects of large-scale DNA deletions, duplications, and high-level amplification in cancer sequencing. Using this approach, we examine haplotype-specific expression changes on rearranged homologs and provide direct evidence for long-range transcriptional activation and repression associated with rearrangements of the inactive X chromosome (Xi). Together, these results reveal the significant transcriptional consequences of somatic Xi rearrangements, highlighting refLinker 's broad utility for studying the functional consequences of chromosomal rearrangements.

Spatiotemporal bifurcation of HY5-mediated blue-light signaling regulates wood development during secondary growth

Plants have evolved photoreceptors to optimize their development during primary growth, including germination, hypocotyl elongation, cotyledon opening, and root growth, allowing them to adapt to challenging light conditions. The light signaling transduction pathway during seedling establishment has been extensively studied, but little molecular evidence is available for light-regulated secondary growth, and how light regulates cambium-derived tissue production remains largely unexplored. Here, we show that CRYPTOCHROME (CRY)-dependent blue light signaling and the subsequent attenuation of ELONGATED HYPOCOTYL 5 (HY5) movement to hypocotyls are key inducers of xylem fiber differentiation in Arabidopsis thaliana. Using grafted chimeric plants and hypocotyl-specific transcriptome sequencing of light signaling mutants under controlled light conditions, we demonstrate that the perception of blue light by CRYs in shoots drives secondary cell wall (SCW) deposition at xylem fiber cells during the secondary growth of hypocotyls. We propose that HY5 is a blue light–responsive mobile protein that inhibits xylem fiber formation via direct transcriptional repression of NAC SECONDARY WALL THICKENING PROMOTING 3 ( NST3 ). CRYs retain HY5 in the nucleus, impede its long-distance transport from leaf to hypocotyl, and they initiate NST3- driven SCW gene expression, thereby triggering xylem fiber production. Our findings shed light on the long-range CRYs-HY5-NST3 signaling cascade that shapes xylem fiber development, highlighting the activity of HY5 as a transcriptional repressor during secondary growth.

Human CCR4-NOT globally regulates gene expression and is a novel silencer of retrotransposon activation.

CCR4-NOT regulates multiple steps in gene regulation and has been well studied in budding yeast, but much less is known about the human complex. Auxin-induced degradation was used to rapidly deplete the scaffold subunit CNOT1, and CNOT4, to characterize the functions of human CCR4-NOT in gene regulation. Depleting CNOT1 increased RNA levels and caused a widespread decrease in RNA decay. In contrast, CNOT4 depletion only modestly changed steady-state RNA levels and, surprisingly, led to a global acceleration in mRNA decay. Further, depleting either subunit resulted in a global increase in RNA synthesis. In contrast to most of the genome, the transcription of KRAB-Zinc-Finger-protein (KZNFs) genes, especially those on chromosome 19, was repressed. KZNFs are transcriptional repressors of retrotransposable elements (rTEs), and consistent with the decreased KZNFs expression, rTEs, mainly Long Interspersed Nuclear Elements (LINEs), were activated. These data establish CCR4-NOT as a global regulator of gene expression and a novel silencer of rTEs.

CRISPRi Screen Uncovers lncRNA Regulators of Human Monocyte Growth.

Long noncoding RNAs are emerging as critical regulators of biological processes. While there are over 20,000 lncRNAs annotated in the human genome we do not know the function for the majority. Here we performed a high-throughput CRISPRi screen to identify those lncRNAs that are important in viability in human monocytes using the cell line THP1. We identified a total of 38 hits from the screen and validated and characterized two of the top intergenic hits. The first is a lncRNA neighboring the macrophage viability transcription factor IRF8 ( RP11-542M13 . 2 hereafter referred to as long noncoding RNA regulator of monocyte proliferation, LNCRMP ) and the second is a lncRNA called OLMALINC (oligodendrocyte maturation-associated long intervening non-coding RNA) that was previously found to be important in oligodendrocyte maturation. Transcriptional repression of LNCRMP and OLMALINC from monocytes severely limited their proliferation capabilities. RNA-seq analysis of knockdown lines showed that LNCRMP regulated proapoptotic pathways while knockdown of OLMALINC impacted genes associated with the cell cycle. Data supports both LNCRMP and OLMALINC functioning in cis to regulate their neighboring proteins that are also essential for THP1 cell growth. This research highlights the importance of high-throughput screening as a powerful tool for quickly discovering functional long non-coding RNAs (lncRNAs) that play a vital role in regulating monocyte viability.

The function of the ZFP189 transcription factor in the nucleus accumbens facilitates cocaine-specific transcriptional and behavioral adaptations.

Distinguishing the brain mechanisms affected by distinct addictive drugs may inform targeted therapies against specific substance use disorders (SUDs). Here, we explore the function of a drug-associated, transcriptionally repressive transcription factor (TF), ZFP189, whose expression in the nucleus accumbens (NAc) facilitates cocaine-induced molecular and behavioral adaptations. To uncover the necessity of ZFP189-mediated transcriptional control in driving cocaine-induced behaviors, we created synthetic ZFP189 TFs of distinct transcriptional function, including ZFP189VPR, which activates the expression of target genes and exerts opposite transcriptional control to the endogenously repressive ZFP189. By virally delivering synthetic ZFP189 TFs to the NAc of mice, we discover that the transcriptional control exerted by synthetic or endogenous ZFP189 solely alters behavioral adaptations to cocaine but not morphine, saline, or sucrose. Further, these synthetic ZFP189 TFs are only capable of producing gene-expression changes in rodents exposed to cocaine, but not morphine or saline. In these cocaine exposed mice, the gene-expression profile produced by ZFP189VPR is inversely related to the cocaine-induced transcriptional response, as characterized by Upstream Regulator Analysis in Ingenuity Pathway Analysis. Lastly, we demonstrate that NAc ZFP189WT increases vulnerability to cocaine reinforcement through selective sensitization to the reinforcing effects of small cocaine doses. In contrast, ZFP189VPR treated mice do not experience changes in cocaine sensitivity and had lower rates of cocaine self-administration. Collectively, this research describes the brain mechanisms by which a TF specifically coordinates the molecular adaptations that produce increased cocaine addiction-like behaviors. The use of synthetic ZFP189VPR uncovers novel strategies for therapeutic interventions to potentially halt these cocaine-induced transcriptional processes.