Polycomb protein binding and looping in the ON transcriptional state.

Polycomb group (PcG) proteins mediate epigenetic silencing of important developmental genes by modifying histones and compacting chromatin through two major protein complexes, PRC1 and PRC2. These complexes are recruited to DNA by CpG islands (CGIs) in mammals and Polycomb response elements (PREs) in Drosophila. When PcG target genes are turned OFF, PcG proteins bind to PREs or CGIs, and PREs serve as anchors that loop together and stabilize gene silencing. Here, we address which PcG proteins bind to PREs and whether PREs mediate looping when their targets are in the ON transcriptional state. While the binding of most PcG proteins decreases at PREs in the ON state, one PRC1 component, Ph, remains bound. Further, PREs can loop to each other and with presumptive enhancers in the ON state and, like CGIs, may act as tethering elements between promoters and enhancers. Overall, our data suggest that PREs are important looping elements for developmental loci in both the ON and OFF states.

Hypoxia-inducible factor 1 recruits FACT and RNF20/40 to mediate histone ubiquitination and transcriptional activation of target genes.

Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying enzymes leading to changes in histone acetylation, citrullination, and methylation at target genes. Here, we demonstrate that hypoxia-inducible gene expression in estrogen receptor (ER)-positive MCF7 and ER-negative SUM159 human breast cancer cells requires the histone H2A/H2B chaperone facilitates chromatin transcription (FACT) and the H2B ubiquitin ligase RING finger protein 20/40 (RNF20/40). Knockdown of FACT or RNF20/40 expression leads to decreased transcription initiation and elongation at HIF-1 target genes. Mechanistically, FACT and RNF20/40 are recruited to hypoxia response elements (HREs) by HIF-1 and stabilize binding of HIF-1 (and each other) at HREs. Hypoxia induces the monoubiquitination of histone H2B at lysine 120 at HIF-1 target genes in an HIF-1-dependent manner. Together, these findings delineate a cooperative molecular mechanism by which FACT and RNF20/40 stabilize multiprotein complex formation at HREs and mediate histone ubiquitination to facilitate HIF-1 transcriptional activity.

Placenta-Specific Transcripts Containing Androgen Response Elements Are Altered In Silico by Male Growth Outcomes.

A birthweight centile (BWC) below the 25th is associated with an elevated risk of adverse perinatal outcomes, particularly among males. This male vulnerability may stem from alterations in placenta-specific androgen signalling, a signalling axis that involves the androgen receptor (AR)-mediated regulation of target genes containing androgen response elements (AREs). In this study, we examined global and ARE-specific transcriptomic signatures in term male placentae (≥37 weeks of gestation) across BWC subcategories (<10th, 10th-30th, >30th) using RNA-seq and gene set enrichment analysis. ARE-containing transcripts in placentae with BWCs below the 10th percentile were upregulated compared to those in the 10th-30th and >30th percentiles, which coincided with the enrichment of gene sets related to hypoxia and the suppression of gene sets associated with mitochondrial function. In the absence of ARE-containing transcripts in silico, <10th and 10th-30th BWC subcategory placentae upregulated gene sets involved in vasculature development, immune function, and cell adhesion when compared to those in the >30th BWC subcategory. Collectively, our in silico findings suggest that changes in the expression of ARE-containing transcripts in male placentae may contribute to impaired placental vasculature and therefore result in reduced fetal growth outcomes.

Association study of a single nucleotide polymorphism in the hypoxia response element of the macrophage migration inhibitory factor gene promoter with suicide completers in the Japanese population.

BACKGROUND: More than 800 000 people die by suicide annually. The heritability of suicide is 30%-50%. We focused on the hypoxia response element (HRE), which promotes the expression of macrophage migration inhibitory factor (MIF) via the hypoxia-inducible factor (HIF) pathway, important in neurogenesis and neuroprotection. We examined a genetic polymorphism of rs17004038, a single-nucleotide polymorphism (SNP), in suicide completers and controls. METHODS: The study population included 1336 suicide completers and 814 unrelated healthy controls. All participants were Japanese. We obtained peripheral blood, extracted DNA, and genotyped the patients for SNP rs17004038 (C > A). RESULTS: No significant differences were observed between the two groups in either the allele or genotype analyses. Subgroup analyses by sex, age (<40 or ≥40), and suicide method (violent or nonviolent suicide) were performed with similar results. CONCLUSION: No association was observed between SNP rs17004038 and suicide completion. Although it is challenging to collect a large number of samples from suicide completers, further MIF-related genetic studies, including those of rs17004038, are necessary with larger sample sizes.

X-chromosome and kidney function: evidence from a multi-trait genetic analysis of 908,697 individuals reveals sex-specific and sex-differential findings in genes regulated by androgen response elements.

X-chromosomal genetic variants are understudied but can yield valuable insights into sexually dimorphic human traits and diseases. We performed a sex-stratified cross-ancestry X-chromosome-wide association meta-analysis of seven kidney-related traits (n = 908,697), identifying 23 loci genome-wide significantly associated with two of the traits: 7 for uric acid and 16 for estimated glomerular filtration rate (eGFR), including four novel eGFR loci containing the functionally plausible prioritized genes ACSL4, CLDN2, TSPAN6 and the female-specific DRP2. Further, we identified five novel sex-interactions, comprising male-specific effects at FAM9B and AR/EDA2R, and three sex-differential findings with larger genetic effect sizes in males at DCAF12L1 and MST4 and larger effect sizes in females at HPRT1. All prioritized genes in loci showing significant sex-interactions were located next to androgen response elements (ARE). Five ARE genes showed sex-differential expressions. This study contributes new insights into sex-dimorphisms of kidney traits along with new prioritized gene targets for further molecular research.

DDX5 enhances HIF-1 activity by promoting the interaction of HIF-1α with HIF-1ß and recruiting the resulting heterodimer to its target gene loci.

BACKGROUND INFORMATION: Cancer cells acquire malignant characteristics and therapy resistance by employing the hypoxia-inducible factor 1 (HIF-1)-dependent adaptive response to hypoxic microenvironment in solid tumors. Since the underlying molecular mechanisms remain unclear, difficulties are associated with establishing effective therapeutic strategies. RESULTS: We herein identified DEAD-box helicase 5 (DDX5) as a novel activator of HIF-1 and found that it enhanced the heterodimer formation of HIF-1α and HIF-1ß and facilitated the recruitment of the resulting HIF-1 to its recognition sequence, hypoxia-response element (HRE), leading to the expression of a subset of cancer-related genes under hypoxia. CONCLUSIONS: This study reveals that the regulation of HIF-1 recruitment to HRE is an important regulatory step in the control of HIF-1 activity. SIGNIFICANCE: The present study provides novel insights for the development of strategies to inhibit the HIF-1-dependent expression of cancer-related genes.

Slide-tags enables single-nucleus barcoding for multimodal spatial genomics.

Recent technological innovations have enabled the high-throughput quantification of gene expression and epigenetic regulation within individual cells, transforming our understanding of how complex tissues are constructed1-6. However, missing from these measurements is the ability to routinely and easily spatially localize these profiled cells. We developed a strategy, Slide-tags, in which single nuclei within an intact tissue section are tagged with spatial barcode oligonucleotides derived from DNA-barcoded beads with known positions. These tagged nuclei can then be used as an input into a wide variety of single-nucleus profiling assays. Application of Slide-tags to the mouse hippocampus positioned nuclei at less than 10 µm spatial resolution and delivered whole-transcriptome data that are indistinguishable in quality from ordinary single-nucleus RNA-sequencing data. To demonstrate that Slide-tags can be applied to a wide variety of human tissues, we performed the assay on brain, tonsil and melanoma. We revealed cell-type-specific spatially varying gene expression across cortical layers and spatially contextualized receptor-ligand interactions driving B cell maturation in lymphoid tissue. A major benefit of Slide-tags is that it is easily adaptable to almost any single-cell measurement technology. As a proof of principle, we performed multiomic measurements of open chromatin, RNA and T cell receptor (TCR) sequences in the same cells from metastatic melanoma, identifying transcription factor motifs driving cancer cell state transitions in spatially distinct microenvironments. Slide-tags offers a universal platform for importing the compendium of established single-cell measurements into the spatial genomics repertoire.

The relationship between sexual dimorphism and androgen response element proliferation in primate genomes.

In males of many vertebrate species, sexual selection has led to the evolution of sexually dimorphic traits, which are often developmentally controlled by androgen signaling involving androgen response elements (AREs). Evolutionary changes in the number and genomic locations of AREs can modify patterns of receptor regulation and potentially alter gene expression. Here, we use recently sequenced primate genomes to evaluate the hypothesis that the strength of sexual selection is related to the genome-wide number of AREs in a diversifying lineage. In humans, we find a higher incidence of AREs near male-biased genes and androgen-responsive genes when compared with randomly selected genes from the genome. In a set of primates, we find that gains or losses of AREs proximal to genes are correlated with changes in male expression levels and the degree of sex-biased expression of those genes. In a larger set of primates, we find that an increase in one indicator of sexual selection, canine size sexual dimorphism, is correlated with genome-wide ARE counts. Our results suggest that the responsiveness of the genome to androgens in humans and their close relatives has been shaped by sexual selection that arises from competition among males for mating access to females.

XRE transcription factors conserved in Caulobacter and φCbK modulate adhesin development and phage production.

The xenobiotic response element (XRE) family of transcription factors (TFs), which are commonly encoded by bacteria and bacteriophage, regulate diverse features of bacterial cell physiology and impact phage infection dynamics. Through a pangenome analysis of Caulobacter species isolated from soil and aquatic ecosystems, we uncovered an apparent radiation of a paralogous XRE TF gene cluster, several of which have established functions in the regulation of holdfast adhesin development and biofilm formation in C. crescentus. We further discovered related XRE TFs throughout the class Alphaproteobacteria and its phages, including the φCbK Caulophage, suggesting that members of this cluster impact host-phage interactions. Here we show that a closely related group of XRE transcription factors encoded by both C. crescentus and φCbK can physically interact and function to control the transcription of a common gene set, influencing processes including holdfast development and the production of φCbK virions. The φCbK-encoded XRE paralog, tgrL, is highly expressed at the earliest stages of infection and can directly inhibit transcription of host genes including hfiA, a potent holdfast inhibitor, and gafYZ, an activator of prophage-like gene transfer agents (GTAs). XRE proteins encoded from the C. crescentus chromosome also directly repress gafYZ transcription, revealing a functionally redundant set of host regulators that may protect against spurious production of GTA particles and inadvertent cell lysis. Deleting the C. crescentus XRE transcription factors reduced φCbK burst size, while overexpressing these host genes or φCbK tgrL rescued this burst defect. We conclude that this XRE TF gene cluster, shared by C. crescentus and φCbK, plays an important role in adhesion regulation under phage-free conditions, and influences host-phage dynamics during infection.

A molecular mechanism for the "digital" response of p53 to stress.

The tumor suppressor protein p53 accumulates in response to cellular stress and consequently orchestrates the expression of multiple genes in a p53-level and time-dependent manner to overcome stress consequences, for which a molecular mechanism is currently unknown. Previously, we reported that DNA torsional flexibility distinguishes among p53 response elements (REs) and that transactivation at basal p53 levels is correlated with p53 REs flexibility. Here, we calculated the flexibility of ~200 p53 REs. By connecting functional outcomes of p53-target genes' activation to the calculated flexibility of their REs, we show that genes known to belong to pathways that are activated rapidly upon stress contain REs that are significantly more flexible relative to REs of genes known to be involved in pathways that are activated later in the response to stress. The global structural properties of several p53 REs belonging to different pathways were experimentally validated. Additionally, reporter-gene expression driven by flexible p53 REs occurred at lower p53 levels and with faster rates than expression from rigid REs. Furthermore, analysis of published endogenous mRNA levels of p53-target genes as a function of REs' flexibility showed that early versus late genes differ significantly in their flexibility properties of their REs and that highly flexible p53 REs enable high-activation level exclusively to early-response genes. Overall, we demonstrate that DNA flexibility of p53 REs contributes significantly to functional selectivity in the p53 system by facilitating the initial steps of p53-dependent target-genes expression, thereby contributing to survival versus death decisions in the p53 system.

NanoFIRE: A NanoLuciferase and Fluorescent Integrated Reporter Element for Robust and Sensitive Investigation of HIF and Other Signalling Pathways.

The Hypoxia Inducible Factor (HIF) transcription factors are imperative for cell adaption to low oxygen conditions and development; however, they also contribute to ischaemic disease and cancer. To identify novel genetic regulators which target the HIF pathway or small molecules for therapeutic use, cell-based reporter systems are commonly used. Here, we present a new, highly sensitive and versatile reporter system, NanoFIRE: a NanoLuciferase and Fluorescent Integrated Reporter Element. Under the control of a Hypoxic Response Element (HRE-NanoFIRE), this system is a robust sensor of HIF activity within cells and potently responds to both hypoxia and chemical inducers of the HIF pathway in a highly reproducible and sensitive manner, consistently achieving 20 to 150-fold induction across different cell types and a Z' score > 0.5. We demonstrate that the NanoFIRE system is adaptable via substitution of the response element controlling NanoLuciferase and show that it can report on the activity of the transcriptional regulator Factor Inhibiting HIF, and an unrelated transcription factor, the Progesterone Receptor. Furthermore, the lentivirus-mediated stable integration of NanoFIRE highlights the versatility of this system across a wide range of cell types, including primary cells. Together, these findings demonstrate that NanoFIRE is a robust reporter system for the investigation of HIF and other transcription factor-mediated signalling pathways in cells, with applications in high throughput screening for the identification of novel small molecule and genetic regulators.

A high throughput screen for pharmacological inhibitors of the carbohydrate response element.

A central regulator of metabolism, transcription factor carbohydrate response element binding protein (ChREBP) senses and responds to dietary glucose levels by stimulating the transcription of glycolytic and lipogenic enzymes. Genetic depletion of ChREBP rescues ß-cell dysfunction arising from high glucose levels, suggesting that inhibiting ChREBP might represent an attractive therapeutic approach to manage diabetes and other metabolic diseases. However, the molecular mechanisms governing ChREBP activation are poorly understood and chemical tools to probe the cellular activity of ChREBP are lacking. Here, we report a high-throughput pharmacological screen in INS-1E ß-cells that identified novel inhibitors of ChREBP-driven transcription at carbohydrate response element sites, including three putative covalent inhibitors and two likely non-covalent chemical scaffolds. This work affords a pharmacological toolkit to help uncover the signaling logic controlling ChREBP activation and may ultimately reveal potential therapeutic approaches for treating metabolic disease.

Histone deacetylase 9 plays a role in sevoflurane-induced neuronal differentiation inhibition by inactivating cAMP-response element binding protein transcription and inhibiting the expression of neurotrophin-3.

Postoperative cognitive decline (POCD) is a common and serious complication following anesthesia and surgery; however, the precise mechanisms of POCD remain unclear. Our previous research showed that sevoflurane impairs adult hippocampal neurogenesis (AHN) and thus cognitive function in the aged brain by affecting neurotrophin-3 (NT-3) expression; however, the signaling mechanism involved remains unexplored. In this study, we found a dramatic decrease in the proportion of differentiated neurons with increasing concentrations of sevoflurane, and the inhibition of neural stem cell differentiation was partially reversed after the administration of exogenous NT-3. Understanding the molecular underpinnings by which sevoflurane affects NT-3 is key to counteracting cognitive dysfunction. Here, we report that sevoflurane administration for 2 days resulted in upregulation of histone deacetylase 9 (HDAC9) expression, which led to transcriptional inactivation of cAMP-response element binding protein (CREB). Due to the colocalization of HDAC9 and CREB within cells, this may be related to the interaction between HDAC9 and CREB. Anyway, this ultimately led to reduced NT-3 expression and inhibition of neural stem cell differentiation. Furthermore, knockdown of HDAC9 rescued the transcriptional activation of CREB after sevoflurane exposure, while reversing the downregulation of NT-3 expression and inhibition of neural stem cell differentiation. In summary, this study identifies a unique mechanism by which sevoflurane can inhibit CREB transcription through HDAC9, and this process reduces NT-3 levels and ultimately inhibits neuronal differentiation. This finding may reveal a new strategy to prevent sevoflurane-induced neuronal dysfunction.

Therapeutic implications of phosphorylation- and dephosphorylation-dependent factors of cAMP-response element-binding protein (CREB) in neurodegeneration.

Neurodegeneration is a condition of the central nervous system (CNS) characterized by loss of neural structures and function. The most common neurodegenerative disorders (NDDs) include Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis (MS), motor neuron disorders, psychological disorders, dementia with vascular dementia (VaD), Lewy body dementia (DLB), epilepsy, cerebral ischemia, mental illness, and behavioral disorders. CREB (cAMP-response element-binding protein) represent a nuclear protein that regulates gene transcriptional activity. The primary focus of the review pertains to the exploration of CREB expression and activation within the context of neurodegenerative diseases, specifically in relation to the phosphorylation and dephosphorylation events that occur within the CREB signaling pathway under normal physiological conditions. The findings mentioned have contributed to the elucidation of the regulatory mechanisms governing CREB activity. Additionally, they have provided valuable insights into the potential mediation of diverse biological processes, such as memory consolidation and neuroprotective effects, by various related studies. The promotion of synaptic plasticity and neurodevelopment in the central nervous system through the targeting of CREB proteins has the potential to contribute to the prevention or delay of the onset of neurodegenerative disorders. Multiple drugs have been found to initiate downstream signaling pathways, leading to neuroprotective advantages in both animal model studies and clinical trials. The clinical importance of the cAMP-response element-binding protein (CREB) is examined in this article, encompassing its utility as both a predictive/prognostic marker and a target for therapeutic interventions.

Novel Aspects of cAMP-Response Element Modulator (CREM) Role in Spermatogenesis and Male Fertility.

Spermatogenesis is a very complex process with an intricate transcriptional regulation. The transition from the diploid to the haploid state requires the involvement of specialized genes in meiosis, among other specific functions for the formation of the spermatozoon. The transcription factor cAMP-response element modulator (CREM) is a key modulator that triggers the differentiation of the germ cell into the spermatozoon through the modification of gene expression. CREM has multiple repressor and activator isoforms whose expression is tissue-cell-type specific and tightly regulated by various factors at the transcriptional, post-transcriptional and post-translational level. The activator isoform CREMτ controls the expression of several relevant genes in post-meiotic stages of spermatogenesis. In addition, exposure to xenobiotics negatively affects CREMτ expression, which is linked to male infertility. On the other hand, antioxidants could have a positive effect on CREMτ expression and improve sperm parameters in idiopathically infertile men. Therefore, CREM expression could be used as a biomarker to detect and even counteract male infertility. This review examines the importance of CREM as a transcription factor for sperm production and its relevance in male fertility, infertility and the response to environmental xenobiotics that may affect CREMτ expression and the downstream regulation that alters male fertility. Also, some health disorders in which CREM expression is altered are discussed.

Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements.

The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Despite its biological importance, the mechanism by which p53 binds REs remains unclear. To address this, we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of full-length human p53 to five native REs in real time under equilibrium conditions. Our approach enabled us to quantify the oligomeric state of DNA-bound p53. We found little evidence that dimer/DNA complexes form as intermediates en route to binding or dissociation of p53 tetramer/DNA complexes. Interestingly, however, at some REs dimers can rapidly exchange from tetramer/DNA complexes. Real time kinetic measurements enabled us to determine rate constants for association and dissociation at all five REs, which revealed two kinetically distinct populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing that the more favorable binding sequences form the least kinetically stable complexes. Together our single molecule measurements provide new insight into mechanisms by which tetrameric p53 forms complexes on different native REs.

Comprehensive genome-wide analysis of the DREB gene family in Moso bamboo (Phyllostachys edulis): evidence for the role of PeDREB28 in plant abiotic stress response.

Dehydration response element binding (DREB) proteins are vital for plant abiotic stress responses, but the understanding of DREBs in bamboo, an important sustainable non-timber forest product, is limited. Here we conducted a comprehensive genome-wide analysis of the DREB gene family in Moso bamboo, representing the most important running bamboo species in Asia. In total, 44 PeDREBs were identified, and information on their gene structures, protein motifs, phylogenetic relationships, and stress-related cis-regulatory elements (CREs) was provided. Based on the bioinformatical analysis, we further analyzed PeDREBs from the A5 group and found that four of five PeDREB transcripts were induced by salt, drought, and cold stresses, and their proteins could bind to stress-related CREs. Among these, PeDREB28 was selected as a promising candidate for further functional characterization. PeDREB28 is localized in nucleus, has transcriptional activation activity, and could bind to the DRE- and coupling element 1- (CE1) CREs. Overexpression of PeDREB28 in Arabidopsis and bamboo improved plant abiotic stress tolerance. Transcriptomic analysis showed that broad changes due to the overexpression of PeDREB28. Furthermore, 628 genes that may act as the direct PeDREB28 downstream genes were identified by combining DAP-seq and RNA-seq analysis. Moreover, we confirmed that PeDREB28 could bind to the promoter of pyrabactin-resistance-like gene (DlaPYL3), which is a homolog of abscisic acid receptor in Arabidopsis, and activates its expression. In summary, our study provides important insights into the DREB gene family in Moso bamboo, and contributes to their functional verification and genetic engineering applications in the future.

Genistein-mediated thermogenesis and white-to-beige adipocyte differentiation involve transcriptional activation of cAMP response elements in the Ucp1 promoter.

Genistein is an isoflavone present in soybeans and is considered a bioactive compound due to its widely reported biological activity. We have previously shown that intraperitoneal genistein administration and diet supplementation activates the thermogenic program in rats and mice subcutaneous white adipose tissue (scWAT) under multiple environmental cues, including cold exposure and high-fat diet feeding. However, the mechanistic insights of this process were not previously unveiled. Uncoupling protein 1 (UCP1), a mitochondrial membrane polypeptide responsible for dissipating energy into heat, is considered the most relevant thermogenic marker; thus, we aimed to evaluate whether genistein regulates UCP1 transcription. Here we show that genistein administration to thermoneutral-housed mice leads to the appearance of beige adipocyte markers, including a sharp upregulation of UCP1 expression and protein abundance in scWAT. Reporter assays showed an increase in UCP1 promoter activity after genistein stimulation, and in silico analysis revealed the presence of estrogen (ERE) and cAMP (CRE) response elements as putative candidates of genistein activation. Mutation of the CRE but not the ERE reduced genistein-induced promoter activity by 51%. Additionally, in vitro and in vivo ChIP assays demonstrated the binding of CREB to the UCP1 promoter after acute genistein administration. Taken together, these data elucidate the mechanism of genistein-mediated UCP1 induction and confirm its potential applications in managing metabolic disorders.

Functional implications and therapeutic targeting of androgen response elements in prostate cancer.

The androgen receptor (AR) plays an essential role in the growth and progression of prostate cancer (CaP). Ligand-activated AR inside the nucleus binds to the androgen response element (ARE) of the target genes in dimeric form and recruits transcriptional machinery to facilitate gene transcription. Pharmacological compounds that inhibit the AR action either bind to the ligand binding domain (LBD) or interfere with the interactions of AR with other co-regulatory proteins, slowing the progression of the disease. However, the emergence of resistance to conventional treatment makes clinical management of CaP difficult. Resistance has been associated with activation of androgen/AR axis that restores AR transcriptional activity. Activated AR signaling in resistance cases can be mediated by several mechanisms including AR amplification, gain-of-function AR mutations, androgen receptor variant (ARVs), intracrine androgen production, and overexpression of AR coactivators. Importantly, in castration resistant prostate cancer, ARVs lacking the LBD become constitutively active and promote hormone-independent development, underlining the need to concentrate on the other domain or the AR-DNA interface for the identification of novel actionable targets. In this review, we highlight the plasticity of AR-DNA binding and explain how fine-tuning AR's cooperative interactions with DNA translate into developing an alternative strategy to antagonize AR activity.