Genome-wide Binding Profiles Research Articles

Nuclear receptor E75/NR1D2 promotes tumor malignant transformation by integrating Hippo and Notch pathways

AbstractHormone therapy resistance and the ensuing aggressive tumor progression present a significant clinical challenge. However, the mechanisms underlying the induction of tumor malignancy upon inhibition of steroid hormone signaling remain poorly understood. Here, we demonstrate that Drosophila malignant epithelial tumors show a similar reduction in ecdysone signaling, the main steroid hormone pathway. Our analysis of ecdysone-induced downstream targets reveals that overexpression of the nuclear receptor E75, particularly facilitates the malignant transformation of benign tumors. Genome-wide DNA binding profiles and biochemistry data reveal that E75 not only binds to the transcription factors of both Hippo and Notch pathways, but also exhibits widespread co-binding to their target genes, thus contributing to tumor malignancy. We further validated these findings by demonstrating that depletion of NR1D2, the mammalian homolog of E75, inhibits the activation of Hippo and Notch target genes, impeding glioblastoma progression. Together, our study unveils a novel mechanism by which hormone inhibition promotes tumor malignancy, and describes an evolutionarily conserved role of the oncogene E75/NR1D2 in integration of Hippo and Notch pathway activity during tumor progression.

Genome-wide profiling of soybean WRINKLED1 transcription factor binding sites provides insight into seed storage lipid biosynthesis

Understanding the regulatory mechanisms controlling storage lipid accumulation will inform strategies to enhance seed oil quality and quantity in crop plants. The WRINKLED1 transcription factor (WRI1 TF) is a central regulator of lipid biosynthesis. We characterized the genome-wide binding profile of soybean (Gm)WRI1 and show that the TF directly regulates genes encoding numerous enzymes and proteins in the fatty acid and triacylglycerol biosynthetic pathways. GmWRI1 binds primarily to regions downstream of target gene transcription start sites. We showed that GmWRI1-bound regions are enriched for the canonical WRI1 DNA binding element, the ACTIVATOR of Spomin::LUC1/WRI1 (AW) Box (CNTNGNNNNNNNCG), and another DNA motif, the CNC Box (CNCCNCC). Functional assays showed that both DNA elements mediate transcriptional activation by GmWRI1. We also show that GmWRI1 works in concert with other TFs to establish a regulatory state that promotes fatty acid and triacylglycerol biosynthesis. In particular, comparison of genes targeted directly by GmWRI1 and by GmLEC1, a central regulator of the maturation phase of seed development, reveals that the two TFs act in a positive feedback subcircuit to control fatty acid and triacylglycerol biosynthesis. Together, our results provide unique insights into the genetic circuitry in which GmWRI1 participates to regulate storage lipid accumulation during seed development.

Genome-wide mapping of the binding sites of myocyte enhancer factor 2A in chicken primary myoblasts

Myocyte enhancer factor 2A (MEF2A) is a transcription factor that plays a critical role in cell proliferation, differentiation and apoptosis. In contrast to the wide characterization of its regulation mechanism in mammalian skeletal muscle, its role in chickens is limited. Especially, its wide target genes remain to be identified. Therefore, we utilized Cleavage Under Targets and Tagmentation (CUT&Tag) technology to reveal the genome-wide binding profile of MEF2A in chicken primary myoblasts thus gaining insights into its potential role in muscle development. Our results revealed that MEF2A binding sites were primarily distributed in intergenic and intronic regions. Within the promoter region, although only 8.87% of MEF2A binding sites were found, these binding sites were concentrated around the transcription start site (TSS). Following peak annotation, a total of 1903 genes were identified as potential targets of MEF2A. Gene Ontology (GO) enrichment analysis further revealed that MEF2A target genes may be involved in the regulation of embryonic development in multiple organ systems, including muscle development, gland development, and visual system development. Moreover, a comparison of the MEF2A target genes identified in chicken primary myoblasts with those in mouse C2C12 cells revealed 388 target genes are conserved across species, 1515 target genes are chicken specific. Among these conserved genes, ankyrin repeat and SOCS box containing 5 (ASB5), transmembrane protein 182 (TMEM182), myomesin 2 (MYOM2), leucyl and cystinyl aminopeptidase (LNPEP), actinin alpha 2 (ACTN2), sorbin and SH3 domain containing 1 (SORBS1), ankyrin 3 (ANK3), sarcoglycan delta (SGCD), and ORAI calcium release-activated calcium modulator 1 (ORAI1) exhibited consistent expression patterns with MEF2A during embryonic muscle development. Finally, TMEM182, as an important negative regulator of muscle development, has been validated to be regulated by MEF2A by dual-luciferase and quantitative real-time PCR (qPCR) assays. In summary, our study for the first time provides a wide landscape of MEF2A target genes in chicken primary myoblasts, which supports the active role of MEF2A in chicken muscle development.

A phosphorylation signal activates genome-wide transcriptional control by BfmR, the global regulator of Acinetobacter resistance and virulence.

The nosocomial pathogen Acinetobacter baumannii is a major threat to human health. The sensor kinase-response regulator system, BfmS-BfmR, is essential to multidrug resistance and virulence in the bacterium and represents a potential antimicrobial target. Important questions remain about how the system controls resistance and pathogenesis. Although BfmR knockout alters expression of >1000 genes, its direct regulon is undefined. Moreover, how phosphorylation controls the regulator is unclear. Here, we address these problems by combining mutagenesis, ChIP-seq, and in vitro phosphorylation to study the functions of phospho-BfmR. We show that phosphorylation is required for BfmR-mediated gene regulation, antibiotic resistance, and sepsis development in vivo. Consistent with activating the protein, phosphorylation induces dimerization and target DNA affinity. Integrated analysis of genome-wide binding and transcriptional profiles of BfmR led to additional key findings: (1) Phosphorylation dramatically expands the number of genomic sites BfmR binds; (2) DNA recognition involves a direct repeat motif widespread across promoters; (3) BfmR directly regulates 303 genes as activator (eg, capsule, peptidoglycan, and outer membrane biogenesis) or repressor (pilus biogenesis); (4) BfmR controls several non-coding sRNAs. These studies reveal the centrality of a phosphorylation signal in driving A. baumannii disease and disentangle the extensive pathogenic gene-regulatory network under its control.

Functional Divergence in Orthologous Transcription Factors: Insights from AtCBF2/3/1 and OsDREB1C.

Despite traditional beliefs of orthologous genes maintaining similar functions across species, growing evidence points to their potential for functional divergence. C-repeat binding factors/dehydration-responsive element binding protein 1s (CBFs/DREB1s) are critical in cold acclimation, with their overexpression enhancing stress tolerance but often constraining plant growth. In contrast, a recent study unveiled a distinctive role of rice OsDREB1C in elevating nitrogen use efficiency (NUE), photosynthesis, and grain yield, implying functional divergence within the CBF/DREB1 orthologs across species. Here, we delve into divergent molecular mechanisms of OsDREB1C and AtCBF2/3/1 by exploring their evolutionary trajectories across rice and Arabidopsis genomes, regulatomes, and transcriptomes. Evolutionary scrutiny shows discrete clades for OsDREB1C and AtCBF2/3/1, with the Poaceae-specific DREB1C clade mediated by a transposon event. Genome-wide binding profiles highlight OsDREB1C's preference for GCCGAC compared to AtCBF2/3/1's preference for A/GCCGAC, a distinction determined by R12 in the OsDREB1C AP2/ERF domain. Cross-species multiomic analyses reveal shared gene orthogroups (OGs) and underscore numerous specific OGs uniquely bound and regulated by OsDREB1C, implicated in NUE, photosynthesis, and early flowering, or by AtCBF2/3/1, engaged in hormone and stress responses. This divergence arises from gene gains/losses (∼16.7% to 25.6%) and expression reprogramming (∼62.3% to 66.2%) of OsDREB1C- and AtCBF2/3/1-regulated OGs during the extensive evolution following the rice-Arabidopsis split. Our findings illustrate the regulatory evolution of OsDREB1C and AtCBF2/3/1 at a genomic scale, providing insights on the functional divergence of orthologous transcription factors following gene duplications across species.

Abstract P28: Comprehensive Molecular Phenotyping of ARID1A-deficient Gastric Cancer Reveals Pervasive Epigenomic Reprogramming and Therapeutic Opportunities

Abstract Objective Gastric cancer (GC) is a leading cause of cancer mortality, with ARID1A being the second most frequently mutated driver gene in GC. We sought to decipher ARID1A-specific GC regulatory networks and examine therapeutic vulnerabilities arising from ARID1A loss. Design Genomic profiling of GC patients including a Singapore cohort (>200 patients) was performed to derive mutational signatures of ARID1A inactivation across molecular subtypes. Single-cell transcriptomic profiles of ARID1A-mutated GCs were analyzed to examine tumor microenvironmental changes arising from ARID1A loss. Genome-wide ARID1A binding and chromatin profiles (H3K27ac, H3K4me3, H3K4me1, ATAC-seq) of gastric cell lines were generated to identify gastric-specific epigenetic landscapes regulated by ARID1A. Distinct cancer hallmarks of ARID1A-mutated GCs were converged at the genomic, single-cell, and epigenomic level, and targeted by pharmacological inhibition. Results We observed prevalent ARID1A inactivation across GC molecular subtypes, with distinct mutational signatures and linked to a NFKB-driven pro-inflammatory tumour microenvironment. ARID1A-depletion caused loss of H3K27ac activation signals at ARID1A-occupied distal enhancers, but unexpectedly gain of H3K27ac at ARID1A-occupied promoters in genes such as NFKB1 and NFKB2. Promoter activation in ARID1A-mutated GCs was associated with enhanced gene expression, increased BRD4 binding, and reduced HDAC1 and CTCF occupancy. Combined targeting of promoter activation and tumour inflammation via bromodomain and NFKB inhibitors confirmed therapeutic synergy specific to ARID1A-genomic status. Conclusion Our results suggest a therapeutic strategy for ARID1A-mutated GCs targeting both tumour-intrinsic (BRD4-assocatiated promoter activation) and extrinsic (NFKB immunomodulation) cancer phenotypes. Citation Format: Chang Xu, Kie Kyon Huang, Jia Hao Law, Joy Shijia Chua, Taotao Sheng, Natasha M. Flores, Melissa Pool Pizzi, Atsushi Okabe, Angie Lay Keng Tan, Feng Zhu, Vikrant Kumar, Xiaoyin Lu, Ana Morales Benitez, Benedict Shi Xiang Lian, Haoran Ma, Shamaine Wei Ting Ho, Kalpana Ramnarayanan, Chukwuemeka George Anene-Nzelu, Milad Razavi-Mohseni, Siti Aishah Binte Abdul Ghani, Su Ting Tay, Xuewen Ong, Ming Hui Lee, Yu Amanda Guo, Hassan Ashktorab, Duane Smoot, Shang Li, Anders Jacobsen Skanderup, Michael A. Beer, Roger Sik Yin Foo, Joel Shi Hao Wong, Kaushal Sanghvi, Wei Peng Yong, Raghav Sundar, Atsushi Kaneda, Shyam Prabhakar, Pawel Karol Mazur, Jaffer A. Ajani, Khay Guan Yeoh, Jimmy Bok-Yan So, Patrick Tan, Singapore Gastric Cancer Consortium. Comprehensive Molecular Phenotyping of ARID1A-deficient Gastric Cancer Reveals Pervasive Epigenomic Reprogramming and Therapeutic Opportunities [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P28.

Identification of a chromatin-bound ERRα interactome network in mouse liver

ObjectivesEstrogen-related-receptor α (ERRα) plays a critical role in the transcriptional regulation of cellular bioenergetics and metabolism, and perturbations in its activity have been associated with metabolic diseases. While several coactivators and corepressors of ERRα have been identified to date, a knowledge gap remains in understanding the extent to which ERRα cooperates with coregulators in the control of gene expression. Herein, we mapped the primary chromatin-bound ERRα interactome in mouse liver. MethodsRIME (Rapid Immuno-precipitation Mass spectrometry of Endogenous proteins) analysis using mouse liver samples from two circadian time points was used to catalog ERRα-interacting proteins on chromatin. The genomic crosstalk between ERRα and its identified cofactors in the transcriptional control of precise gene programs was explored through cross-examination of genome-wide binding profiles from chromatin immunoprecipitation-sequencing (ChIP-seq) studies. The dynamic interplay between ERRα and its newly uncovered cofactor Host cell factor C1 (HCFC1) was further investigated by loss-of-function studies in hepatocytes. ResultsCharacterization of the hepatic ERRα chromatin interactome led to the identification of 48 transcriptional interactors of which 42 were previously unknown including HCFC1. Interrogation of available ChIP-seq binding profiles highlighted oxidative phosphorylation (OXPHOS) under the control of a complex regulatory network between ERRα and multiple cofactors. While ERRα and HCFC1 were found to bind to a large set of common genes, only a small fraction showed their colocalization, found predominately near the transcriptional start sites of genes particularly enriched for components of the mitochondrial respiratory chain. Knockdown studies demonstrated inverse regulatory actions of ERRα and HCFC1 on OXPHOS gene expression ultimately dictating the impact of their loss-of-function on mitochondrial respiration. ConclusionsOur work unveils a repertoire of previously unknown transcriptional partners of ERRα comprised of chromatin modifiers and transcription factors thus advancing our knowledge of how ERRα regulates metabolic transcriptional programs.

ChEC-Seq: A Comprehensive Guide for Scalable and Cost-Efficient Genome-Wide Profiling in Saccharomyces cerevisiae.

Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles.

Mask exhibits trxG-like behavior and associates with H3K27ac marked chromatin

The Trithorax group (trxG) proteins counteract the repressive effect of Polycomb group (PcG) complexes and maintain transcriptional memory of active states of key developmental genes. Although chromatin structure and modifications appear to play a fundamental role in this process, it is not clear how trxG prevents PcG-silencing and heritably maintains an active gene expression state. Here, we report a hitherto unknown role of Drosophila Multiple ankyrin repeats single KH domain (Mask), which emerged as one of the candidate trxG genes in our reverse genetic screen. The genome-wide binding profile of Mask correlates with known trxG binding sites across the Drosophila genome. In particular, the association of Mask at chromatin overlaps with CBP and H3K27ac, which are known hallmarks of actively transcribed genes by trxG. Importantly, Mask predominantly associates with actively transcribed genes in Drosophila. Depletion of Mask not only results in the downregulation of trxG targets but also correlates with diminished levels of H3K27ac. The fact that Mask positively regulates H3K27ac levels in flies was also found to be conserved in human cells. Strong suppression of Pc mutant phenotype by mutation in mask provides physiological relevance that Mask contributes to the anti-silencing effect of trxG, maintaining expression of key developmental genes. Since Mask is a downstream effector of multiple cell signaling pathways, we propose that Mask may connect cell signaling with chromatin mediated epigenetic cell memory governed by trxG.

Tho2 is critical for the recruitment of Rrp6 to chromatin in response to perturbed mRNP biogenesis.

The eukaryotic THO complex coordinates the assembly of so-called messenger RNA-ribonucleoprotein particles (mRNPs), a process that involves cotranscriptional coating of nascent mRNAs with proteins. Once formed, mRNPs undergo a quality control step that marks them either for active transport to the cytoplasm, or Rrp6/RNA exosome-mediated degradation in the nucleus. However, the mechanism behind the quality control of nascent mRNPs is still unclear. We investigated the cotranscriptional quality control of mRNPs in budding yeast by expressing the bacterial Rho helicase, which globally perturbs yeast mRNP formation. We examined the genome-wide binding profiles of the THO complex subunits Tho2, Thp2, Hpr1, and Mft1 upon perturbation of the mRNP biogenesis, and found that Tho2 plays two roles. In addition to its function as a subunit of the THO complex, upon perturbation of mRNP biogenesis Tho2 targets Rrp6 to chromatin via its carboxy-terminal domain. Interestingly, other THO subunits are not enriched on chromatin upon perturbation of mRNP biogenesis and are not necessary for localizing Rrp6 at its target loci. Our study highlights the potential role of Tho2 in cotranscriptional mRNP quality control, which is independent of other THO subunits. Considering that both the THO complex and the RNA exosome are evolutionarily highly conserved, our findings are likely relevant for mRNP surveillance in mammals.

BloodChIP Xtra: an expanded database of comparative genome-wide transcription factor binding and gene-expression profiles in healthy human stem/progenitor subsets and leukemic cells.

The BloodChIP Xtra database (http://bloodchipXtra.vafaeelab.com/) facilitates genome-wide exploration and visualization of transcription factor (TF) occupancy and chromatin configuration in rare primary human hematopoietic stem (HSC-MPP) and progenitor (CMP, GMP, MEP) cells and acute myeloid leukemia (AML) cell lines (KG-1, ME-1, Kasumi1, TSU-1621-MT), along with chromatin accessibility and gene expression data from these and primary patient AMLs. BloodChIP Xtra features significantly more datasets than our earlier database BloodChIP (two primary cell types and two cell lines). Improved methodologies for determining TF occupancy and chromatin accessibility have led to increased availability of data for rare primary cell types across the spectrum of healthy and AML hematopoiesis. However, there is a continuing need for these data to be integrated in an easily accessible manner for gene-based queries and use in downstream applications. Here, we provide a user-friendly database based around genome-wide binding profiles of key hematopoietic TFs and histone marks in healthy stem/progenitor cell types. These are compared with binding profiles and chromatin accessibility derived from primary and cell line AML and integrated with expression data from corresponding cell types. All queries can be exported to construct TF-gene and protein-protein networks and evaluate the association of genes with specific cellular processes.

A hybrid RNA FISH immunofluorescence protocol on Drosophila polytene chromosomes

ObjectivesInvestigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors; however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF.ResultsWe developed a hybrid RNA FISH-IF protocol for use on Drosophila melanogaster polytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level in Drosophila melanogaster polytene chromosomes.

An NFIX-mediated regulatory network governs the balance of hematopoietic stem and progenitor cells during hematopoiesis.

The transcription factor (TF) nuclear factor I-X (NFIX) is a positive regulator of hematopoietic stem and progenitor cell (HSPC) transplantation. Nfix-deficient HSPCs exhibit a severe loss of repopulating activity, increased apoptosis, and a loss of colony-forming potential. However, the underlying mechanism remains elusive. Here, we performed cellular indexing of transcriptomes and epitopes by high-throughput sequencing (CITE-seq) on Nfix-deficient HSPCs and observed a loss of long-term hematopoietic stem cells and an accumulation of megakaryocyte and myelo-erythroid progenitors. The genome-wide binding profile of NFIX in primitive murine hematopoietic cells revealed its colocalization with other hematopoietic TFs, such as PU.1. We confirmed the physical interaction between NFIX and PU.1 and demonstrated that the 2 TFs co-occupy super-enhancers and regulate genes implicated in cellular respiration and hematopoietic differentiation. In addition, we provide evidence suggesting that the absence of NFIX negatively affects PU.1 binding at some genomic loci. Our data support a model in which NFIX collaborates with PU.1 at super-enhancers to promote the differentiation and homeostatic balance of hematopoietic progenitors.

The CUT&RUN suspect list of problematic regions of the genome

BackgroundCleavage Under Targets and Release Using Nuclease (CUT&RUN) is an increasingly popular technique to map genome-wide binding profiles of histone modifications, transcription factors, and co-factors. The ENCODE project and others have compiled blacklists for ChIP-seq which have been widely adopted: these lists contain regions of high and unstructured signal, regardless of cell type or protein target, indicating that these are false positives. While CUT&RUN obtains similar results to ChIP-seq, its biochemistry and subsequent data analyses are different. We found that this results in a CUT&RUN-specific set of undesired high-signal regions.ResultsWe compile suspect lists based on CUT&RUN data for the human and mouse genomes, identifying regions consistently called as peaks in negative controls. Using published CUT&RUN data from our and other labs, we show that the CUT&RUN suspect regions can persist even when peak calling is performed with SEACR or MACS2 against a negative control and after ENCODE blacklist removal. Moreover, we experimentally validate the CUT&RUN suspect lists by performing reiterative negative control experiments in which no specific protein is targeted, showing that they capture more than 80% of the peaks identified.ConclusionsWe propose that removing these problematic regions can substantially improve peak calling in CUT&RUN experiments, resulting in more reliable datasets.

SIX1 and EWS/FLI1 co-regulate an anti-metastatic gene network in Ewing Sarcoma

Ewing sarcoma (ES), which is characterized by the presence of oncogenic fusion proteins such as EWS/FLI1, is an aggressive pediatric malignancy with a high rate of early dissemination and poor outcome after distant spread. Here we demonstrate that the SIX1 homeoprotein, which enhances metastasis in most tumor types, suppresses ES metastasis by co-regulating EWS/FLI1 target genes. Like EWS/FLI1, SIX1 promotes cell growth/transformation, yet dramatically inhibits migration and invasion, as well as metastasis in vivo. We show that EWS/FLI1 promotes SIX1 protein expression, and that the two proteins share genome-wide binding profiles and transcriptional regulatory targets, including many metastasis-associated genes such as integrins, which they co-regulate. We further show that SIX1 downregulation of integrins is critical to its ability to inhibit invasion, a key characteristic of metastatic cells. These data demonstrate an unexpected anti-metastatic function for SIX1, through coordinate gene regulation with the key oncoprotein in ES, EWS/FLI1.

Tissue-specific chromatin-binding patterns of Caenorhabditis elegans heterochromatin proteins HPL-1 and HPL-2 reveal differential roles in the regulation of gene expression.

Heterochromatin is characterized by an enrichment of repetitive elements and low gene density and is often maintained in a repressed state across cell division and differentiation. The silencing is mainly regulated by repressive histone marks such as H3K9 and H3K27 methylated forms and the heterochromatin protein 1 (HP1) family. Here, we analyzed in a tissue-specific manner the binding profile of the two HP1 homologs in Caenorhabditis elegans, HPL-1 and HPL-2, at the L4 developmental stage. We identified the genome-wide binding profile of intestinal and hypodermal HPL-2 and intestinal HPL-1 and compared them with heterochromatin marks and other features. HPL-2 associated preferentially to the distal arms of autosomes and correlated positively with the methylated forms of H3K9 and H3K27. HPL-1 was also enriched in regions containing H3K9me3 and H3K27me3 but exhibited a more even distribution between autosome arms and centers. HPL-2 showed a differential tissue-specific enrichment for repetitive elements conversely with HPL-1, which exhibited a poor association. Finally, we found a significant intersection of genomic regions bound by the BLMP-1/PRDM1 transcription factor and intestinal HPL-1, suggesting a corepressive role during cell differentiation. Our study uncovers both shared and singular properties of conserved HP1 proteins, providing information about genomic binding preferences in relation to their role as heterochromatic markers.

Comprehensive molecular phenotyping of ARID1A-deficient gastric cancer reveals pervasive epigenomic reprogramming and therapeutic opportunities

ObjectiveGastric cancer (GC) is a leading cause of cancer mortality, with ARID1A being the second most frequently mutated driver gene in GC. We sought to decipher ARID1A-specific GC regulatory networks...

ETS1-HMGA2 Axis Promotes Human Limbal Epithelial Stem Cell Proliferation.

This study aimed to investigate the role and molecular mechanism of ETS1 in the proliferation and differentiation of human limbal epithelial stem cells (LESCs). RNA-seq and quantitative real-time PCR were used to determine gene expression changes when ETS1 and HMGA2 was knocked down using short-hairpin RNAs or overexpressed by lentivirus. Immunofluorescence and flow cytometry experiments were performed to assess the roles of ETS1 and HMGA2 in LESC proliferation. ETS1-bound cis-regulatory elements and target genes in LESCs were identified using chromatin immunoprecipitation sequencing. The epigenetic features of ETS1-binding sites were assessed by the published histone modification and chromatin accessibility profiles. ETS1 was robustly expressed in LESCs but dramatically reduced on differentiation into corneal epithelial cells (CECs). ETS1 knockdown in LESCs inhibited cellular proliferation and activated CEC markers (KRT3, KRT12, CLU, and ALDH3A1). When ETS1 was overexpressed during CEC differentiation, LESC-associated genes were upregulated while CEC-associated genes were downregulated. The genome-wide binding profile of ETS1 was identified in LESCs. ETS1 occupied H3K4me3-marked promoters and H3K27ac/H3K4me1-marked enhancers. ETS1-binding sites were also enriched for chromatin accessibility signal. HMGA2 showed a consistent expression pattern with ETS1. ETS1 activates HMAG2 by binding to its promoter. Knockdown and overexpression experiments suggested that HMGA2 can promote LESC proliferation and inhibits its differentiation. ETS1 promotes LESC proliferation and inhibits its differentiation via activating HMGA2.

Psi promotes Drosophila wing growth via direct transcriptional activation of cell cycle targets and repression of growth inhibitors.

The first characterised FUSE Binding Protein family member, FUBP1, binds single-stranded DNA to activate MYC transcription. Psi, the sole FUBP protein in Drosophila, binds RNA to regulate P-element and mRNA splicing. Our previous work revealed pro-growth functions for Psi, which depend, in part, on transcriptional activation of Myc. Genome-wide functions for FUBP family proteins in transcriptional control remain obscure. Here, through the first genome-wide binding and expression profiles obtained for a FUBP family protein, we demonstrate that, in addition to being required to activate Myc to promote cell growth, Psi also directly binds and activates stg to couple growth and cell division. Thus, Psi knockdown results in reduced cell division in the wing imaginal disc. In addition to activating these pro-proliferative targets, Psi directly represses transcription of the growth inhibitor tolkin (tok, a metallopeptidase implicated in TGFβ signalling). We further demonstrate tok overexpression inhibits proliferation, while tok loss of function increases mitosis alone and suppresses impaired cell division caused by Psi knockdown. Thus, Psi orchestrates growth through concurrent transcriptional activation of the pro-proliferative genes Myc and stg, in combination with repression of the growth inhibitor tok.

Innovations and stepwise evolution of CBFs/DREB1s and their regulatory networks in angiosperms.

The C-repeat binding factors/dehydration-responsive element binding protein 1s (CBFs/DREB1s) have been identified as major regulators of cold acclimation in many angiosperm plants. However, their origin and evolutionary process associated to cold responsiveness are still lacking. By integrating multi-omics data of genomes, transcriptomes, and CBFs/DREB1s genome-wide binding profiles, we unveil the origin and evolution of CBFs/DREB1s and their regulatory network. Gene collinearity and phylogeny analyses show that CBF/DREB1 is an innovation evolved from tandem duplication-derived DREB III gene. A subsequent event of ε-whole genome duplication led to two CBF/DREB1 archetypes (Clades I and II) in ancient angiosperms. In contrast to cold-insensitivity of Clade I and their parent DREB III genes, Clade II evolved a further innovation in cold-sensitive response and was stepwise expanded in eudicots and monocots by independent duplications. In geological time, the duplication events were mainly enriched around the Cretaceous-Paleogene (K-Pg) boundary and/or in the Late Cenozoic Ice Age, when the global average temperature significantly decreased. Consequently, the duplicated CBF/DREB1 genes contributed to the rewiring of CBFs/DREB1s-regulatory network for cold tolerance. Altogether, our results highlight an origin and convergent evolution of CBFs/DREB1s and their regulatory network probably for angiosperms adaptation to global cooling.