Transcriptional Elements Research Articles

Differential Gene Expression Profile Induced by Valproic Acid (VPA) in Pediatric Epileptic Patients.

Epilepsy is a neuronal disease that affects up to 70 million people worldwide. The development of effective therapies to combat childhood epilepsy requires early biomarkers. Here, we performed a whole-genome microarray analysis in blood cells to identify genes differentially expressed between epileptic and epileptic valproic acid (VPA)-treated children versus normal children to obtain information about the gene expression to help us to understand genetic aspects of this disease. We found that the most significant differentially expressed genes were related to the transcriptional factor cAMP-response element binding protein (CREB) that is overexpressed in children with epilepsy compared with normal children, and 6 and 12 months of VPA treatment reversed several of these changes. Interestingly, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), a type I transmembrane glycoprotein that binds collagen proteins and contains CREB binding sites, was one of the more up-regulated genes in epileptic patients, and treatment with VPA strongly reversed its up-regulation. CREB up-regulates genes related to epilepsy; here, we suggest that LAIR1 could activate CREB, and together, they trigger epilepsy. After VPA treatment, LAIR1 repressed genes by disrupting the functional LAIR1–CREB complex, resulting in successful treatment. A functional microarray analysis offers new information that could open novel avenues of research in biomarker discovery, which may be useful for the early identification of children with a predisposition to epilepsy.

The GntR family transcriptional regulator PMM1637 regulates the highly conserved cyanobacterial sRNA Yfr2 in marine picocyanobacteria.

Prochlorococcus is a marine picocyanobacterium with a streamlined genome that is adapted to different ecological niches in the oligotrophic oceans. There are currently >20 regulatory small RNAs (sRNAs) that have been identified in the model strain Prochlorococcus MED4. While most of these sRNAs are ecotype-specific, sRNA homologs of Yfr1 and of the Yfr2 family are widely found throughout the cyanobacterial phylum. Although they were identified 13 yrs ago, the functions of Yfr1 and Yfr2 have remained unknown. We observed a strong induction of two Yfr2 sRNA homologs of Prochlorococcus MED4 during high light stress and nitrogen starvation. Several Prochlorococcus and marine Synechococcus yfr2 promoter regions contain a conserved motif we named CGRE1 (cyanobacterial GntR family transcriptional regulator responsive element 1). Using the conserved promoter region as bait in a DNA affinity pull-down assay we identified the GntR family transcriptional regulator PMM1637 as a binding partner. Similar to Yfr2, homologs of PMM1637 are universally and exclusively found in cyanobacteria. We suggest that PMM1637 governs the induction of gene expression of Yfr2 homologs containing CGRE1 in their promoters under nitrogen-depleted and high-light stress conditions.

Properties of gene expression and chromatin structure with mechanically regulated elongation.

In recent years, physical elements of transcription have emerged as central in our understanding of gene expression. Recent work has been done introducing a simple description of the basic physical elements of transcription where RNA elongation, RNA polymerase (RNAP) rotation and DNA supercoiling are coupled (1). Here we generalize this framework to accommodate the behavior of many RNAPs operating on multiple genes on a shared piece of DNA. The resulting framework is combined with well-established stochastic processes of transcription resulting in a model which characterizes the impact of the mechanical properties of transcription on gene expression and DNA structure. Transcriptional bursting readily emerges as a common phenomenon with origins in the geometric nature of the genetic system and results in the bounding of gene expression statistics. Properties of a multiple gene system are examined with special attention paid to the role that genome composition (gene orientation, size and intergenic distance) plays in the ability of genes to transcribe. The role of transcription in shaping DNA structure is examined and the possibility of transcription driven domain formation is discussed.

Analysis of psoriasis-relevant gene expression and exon usage alterations after silencing of SR-rich splicing regulators.

In our recent cDNA microarray experiment, three SR-rich splicing factors-SFRS18, PPIG and LUC7L3-were shown to exert altered responsiveness upon T-lymphokine stimulation of psoriatic non-involved and healthy epidermis samples. We have also demonstrated that double silencing LUC7L3 and SFRS18 efficiently decreased production of the psoriasis-associated EDA+ fibronectin isoform. These findings prompted the further investigation of signalling pathways affected by LUC7L3 and SFRS18. To detect gene expression and splicing pattern alterations upon double silencing of LUC7L3 and SFRS18 in an HPV-immortalised keratinocyte cell culture, paired-end RNA sequencing was carried out. Marked changes in exon usage were revealed, in contrast to the modest alterations detected in gene expression, providing a closer delineation of the potential targets of the examined splicing factors. The most prominent gene expression change was detected for IFI6, an interferon-inducible gene highly expressed in psoriasis. Interacting partners of IFI6 and certain psoriasis-associated transcripts also exhibited significantly increased expression upon silencing. In addition to elevated abundance of the EDA+ fibronectin interactor ITGA5, we confirmed decreased EDA domain inclusion, which agrees well with our prior experimental data. Furthermore, differential exon usage was established for the transcription element CREB1, along with HERC6 and CUL1, which are implicated in ubiquitination. Although immortalised keratinocytes express low levels of TINCR, a long non-coding RNA involved in terminal differentiation of keratinocytes, splicing alterations were successfully demonstrated for this RNA as well. We believe that the targeted investigation of mRNA maturation disturbances may help us gain deeper insight into the molecular pathogenesis of psoriasis.

Serum response factor mediates nociceptor inflammatory pain plasticity.

Chronic metabotropic glutamate receptor activation in nociceptive afferents may upregulate A-Kinase Anchoring Protein 150 (AKAP150) expression and/or function. To quantify transcriptional changes in AKAP150 expression and/or function after long-term mGluR5 agonist exposure, and identify transcriptional elements responsible. Dorsal root ganglia (DRG) were dissected from Sprague-Dawley rats and cultured for biochemical analysis of AKAP150 expression after prolonged mGluR5 agonist exposure. Serum response factor (SRF) expression was knocked down through siRNA in cultures to demonstrate significance to AKAP150 upregulation. Serum response factor was also knocked down in vivo through intrathecal injections of specifically targeted oligonucleotides to demonstrate significance to hyperalgesic priming behavior in persistent mechanical hypersensitivity. Serum response factor and AKAP150 are coexpressed in TRPV1(+) DRG neurons in intact DRG. Prolonged mGluR5 agonist exposure increases SRF-dependent transcription and AKAP150 expression in a manner sensitive to protein kinase C inhibition and SRF knock down. Serum response factor in vivo knock down reduces mechanical hyperalgesic priming. Serum response factor transcription plays an important role in transcriptional upregulation of AKAP and hyperalgesic priming behavior, and may contribute to the increased role of AKAP150 in the transition from acute to chronic pain.

Human-relevant potency threshold (HRPT) for ER\u03b1 agonism

The European Commission has recently proposed draft criteria for the identification of endocrine disrupting chemicals (EDCs) that pose a significant hazard to humans or the environment. Identifying and characterizing toxic hazards based on the manner by which adverse effects are produced rather than on the nature of those adverse effects departs from traditional practice and requires a proper interpretation of the evidence regarding the chemical’s ability to produce physiological effect(s) via a specific mode of action (MoA). The ability of any chemical to produce a physiological effect depends on its pharmacokinetics and the potency by which it acts via the various MoAs that can lead to the particular effect. A chemical’s potency for a specific MoA—its mechanistic potency—is determined by two properties: (1) its affinity for the functional components that comprise the MoA, i.e., its specific receptors, enzymes, transporters, transcriptional elements, etc., and (2) its ability to alter the functional state of those components (activity). Using the agonist MoA via estrogen receptor alpha, we illustrate an empirical method for determining a human-relevant potency threshold (HRPT), defined as the minimum level of mechanistic potency necessary for a chemical to be able to act via a particular MoA in humans. One important use for an HRPT is to distinguish between chemicals that may be capable of, versus those likely to be incapable of, producing adverse effects in humans via the specified MoA. The method involves comparing chemicals that have different ERα agonist potencies with the ability of those chemicals to produce ERα-mediated agonist responses in human clinical trials. Based on this approach, we propose an HRPT for ERα agonism of 1E-04 relative to the potency of the endogenous estrogenic hormone 17β-estradiol or the pharmaceutical estrogen, 17α-ethinylestradiol. This approach provides a practical way to address Hazard Identification according to the draft criteria for identification of EDCs recently proposed by the European Commission.

Profile of apalutamide in the treatment of metastatic castration-resistant prostate cancer: evidence to date.

Advances in therapies have led to the approval of six therapeutic agents since 2004, each demonstrating overall survival benefit in randomized studies, and these have significantly improved the outlook for men facing metastatic castration-resistant prostate cancer (CRPC). More recently, efforts have been directed at trying to effect change at earlier phases of the disease. Apalutamide (ARN-509), a second-generation androgen receptor antagonist, recently received approval in the nonmetastatic (M0) CRPC space. Similar to enzalutamide, apalutamide inhibits the binding of androgen to androgen receptor (AR), nuclear translocation of the androgen–AR complex, and binding of AR transcription complex to DNA-binding sites and transcription elements. Phase I and II trial experience demonstrates the safety and tolerability of apalutamide, as well as its efficacy in effecting prostate-specific antigen response and radiographic-free survival in CRPC. US Food and Drug Administration approval in M0 CRPC was granted following positive results from the phase III SPARTAN study, where apalutamide demonstrated significant improvements in metastasis-free survival and time to symptomatic progression as compared to placebo.

Identification of Vasopressin‐Responsive Regulators of Aqp2 Gene Transcription

The peptide hormone vasopressin regulates water excretion largely through control of the water channel protein, aquaporin‐2 (AQP2). Vasopressin regulates AQP2 in part by increasing Aqp2 gene transcription in collecting duct principal cells. Dysregulation of Aqp2 gene transcription has been implicated in a number of water balance disorders. However, how vasopressin regulates Aqp2 gene transcription is unclear. Here, we used next generation sequencing (NGS) techniques as well as prior published data to identify vasopressin‐responsive transcriptional regulators for Aqp2 gene transcription.CTCF is a DNA‐binding protein that homodimerizes with upstream or downstream DNA‐bound CTCF molecules to create ‘insulated loops'. Genes in these insulated loops are regulated mainly via enhancer or repressor interactions within the same loop. From the CTCF ChIP‐Seq database in mouse ENCODE, we found that three genes (Faim2, Aqp2 and Aqp5) are within the same insulated loop (99 kb‐length) defined by two strong CTCF‐binding sites. To identify regulatory elements for Aqp2 gene transcription in mpkCCD cells, we carried out ATAC‐Seq to identify genomic regions accessible to Tn5 transposase (analogous to DNase sensitivity footprinting). We identified 12 transposase‐accessible regions within the Faim2‐Aqp2‐Aqp5 insulated loop, which mark likely locations of enhancer or repressor elements controlling Aqp2 gene transcription.ChIP‐Seq was carried out for two candidate transcription factors, namely cyclic AMP‐responsive element‐binding protein (CREB) and CCAAT/Enhancer‐binding protein β (C/EBPβ) in mpkCCD cells. Vasopressin resulted in increased phosphorylation of CREB at Ser133, confirming a physiological response. Surprisingly, over multiple replicates, ChIP‐Seq identified no CREB‐binding sites within the Faim2‐Aqp2‐Aqp5 insulated loop, while CREB‐binding sites were found in the promoters of 29 transcription factors including AP‐1 transcription factors (Fos and Junb). The latter are recognized to play roles in regulation of Aqp2 gene transcription. In contrast, a strong C/EBPβ‐binding site was found just downstream from the Aqp2 gene within one of the transposase‐accessible regions identified by ATAC‐Seq. Vasopressin treatment increased C/EBPβ‐binding at this and other sites. Overall, the data are consistent with roles of C/EBPβ and CREB in regulation of Aqp2 gene transcription, although CREB effects are likely to be indirect through regulation of other transcription factor genes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Elevated Thrombospondin‐2 Contributes to Delayed Wound Healing in Diabetes

Impaired wound healing is a major complication of diabetes and can lead to the development of chronic wounds in a significant portion of diabetes patients. Despite the risks posed by impaired healing, treatment strategies for diabetic wounds remain limited due to an incomplete understanding of the underlying pathological mechanisms. Previous studies have demonstrated that overexpression of thrombospondin‐2 (TSP2), a matricellular protein released after tissue injury, is associated with significant delays in dermal healing in various mouse models. Consistently, wounds lacking TSP2 (TSP2 KO) heal faster than wounds in wild‐type mice or mice with elevated TSP2. Thus, the present study aimed to examine the role of TSP2 in delayed healing in diabetes.First, we evaluated TSP2 expression in human wounds, and found that TSP2 is elevated in wounds from diabetes patients. Then, to determine TSP2's contribution to impaired healing in diabetes, we developed a novel diabetic TSP2 KO mouse model. Though these db/db TSP2 KO mice develop obesity and hyperglycemia comparable to db/db mice, db/db TSP2 KO mice exhibit significantly improved healing. Moreover, primary fibroblasts isolated from db/db TSP2 KO mice exhibit improved migration, providing mechanistic insight into the accelerated healing in these mice. We also studied TSP2 expression in fibroblasts, the major source of TSP2 in the wound, to explore the mechanisms leading to its overexpression in diabetes. Our results showed that TSP2 expression is increased in hyperglycemia, through increased activation of the hexosamine biosynthesis pathway and subsequent O‐GlcNAc modification of transcriptional elements. Overall, the results of this study indicate for the first time that: 1) TSP2 expression is elevated in diabetes and hyperglycemia and 2) TSP2 contributes to impaired healing in diabetes. These results represent a novel role for TSP2 in diabetes complications and a novel target for improving wound healing in diabetes.Support or Funding InformationNIH HL107205, NIH GM 072194, Gruber Science FellowshipThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differentiation Therapy Targeting the β-Catenin/CBP Interaction in Pancreatic Cancer.

Background: Although canonical Wnt signaling is known to promote tumorigenesis in pancreatic ductal adenocarcinoma (PDAC), a cancer driven principally by mutant K-Ras, the detailed molecular mechanisms by which the Wnt effector β-catenin regulates such tumorigenesis are largely unknown. We have previously demonstrated that β-catenin’s differential usage of the Kat3 transcriptional coactivator cyclic AMP-response element binding protein-binding protein (CBP) over its highly homologous coactivator p300 increases self-renewal and suppresses differentiation in other types of cancer. Aim/methods: To investigate Wnt-mediated carcinogenesis in PDAC, we have used the specific small molecule CBP/β-catenin antagonist, ICG-001, which our lab identified and has extensively characterized, to examine its effects in human pancreatic cancer cells and in both an orthotopic mouse model and a human patient-derived xenograft (PDX) model of PDAC. Results/conclusion: We report for the first time that K-Ras activation increases the CBP/β-catenin interaction in pancreatic cancer; and that ICG-001 specific antagonism of the CBP/β-catenin interaction sensitizes pancreatic cancer cells and tumors to gemcitabine treatment. These effects were associated with increases in the expression of let-7a microRNA; suppression of K-Ras and survivin; and the elimination of drug-resistant cancer stem/tumor-initiating cells.

SP1 upregulated FoxO3a promotes tumor progression in colorectal cancer.

FoxO transcription factors are important regulators of cell survival in response to a variety of stress stimuli and play vital functions in tumor progression. However, the functions and underlying regulators of FoxO3a in colorectal cancer (CRC) have not been fully elucidated. The aim of the current study was to identify the functions of FoxO3a in CRC and characterize the transcription elements within the promoter region of FoxO3a. The expression of FoxO3a was upregulated in response to hypoxic and oxidative stress stimuli. Furthermore, knockdown of FoxO3a significantly reduced cell proliferation and migration ability, while it promoted the response to cetuximab treatment. In addition, it was revealed that knockdown of FoxO3a reduced tumor progression invivo. A mechanistic study found that plenty of putative SP1 sites were identified in the FoxO3a promoter. Luciferase reporter assay revealed that a region corresponding to the SP1 binding sites located between ‑2,000 and ‑1,037bp of FoxO3a promoter was essential for the transcriptional activity. Co-transfection of a SP1 expression vector with the reporter constructs markedly increased luciferase activity. Collectively, these results indicated that SP1‑dependent promoter elements drive FoxO3a gene transcription in colorectal CRC, and indicated that SP1 upregulated FoxO3a is critical for CRC progression.

A strong promoter of a non-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis.

Bacillus thuringiensis bacteria show insecticidal activities that rely upon the production of insecticidal crystal proteins, which are encoded by cry or cyt genes and can target a variety of insect pests. It has been shown that cry1Ac is the only cry gene in B. thuringiensis subsp. kurstaki HD73 (B. thuringiensis HD73) and its expression is controlled by both σE and σK. Here, we report a novel σE-dependent strong promoter of a non-cry gene (HD73_5014), which can direct strong cry1Ac gene expression in B. thuringiensis HD73. We constructed an E. coli-B. thuringiensis shuttle vector (pHT315-P 5014 -1Ac) for cry1Ac gene expression, using the HD73_5014 gene promoter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis showed that expression of the cry1Ac gene directed by the HD73_5014 gene promoter was at the same level as that directed by the previously known strongest cry promoter, P cry8E . However, this strain did not form typical bipyramidal crystals in mother cells, as observed by transmission electron microscopy and atomic force microscope. The strain with Cry1Ac protein expression under the control of the HD73_5014 gene promoter (P 5014 -cry1Ac) showed insecticidal activity against Plutella xylostella similar to that under the control of the orf1cry8E gene promoter (P cry8E -cry1Ac). Collectively, these results suggest that the HD73_5014 gene promoter, as a non-cry gene promoter, would be an efficient transcriptional element for cry gene expression. These data also show the possibility for improving Cry production by searching for transcriptional elements in not only cry genes, but also non-cry genes.

Systemic inflammation without gliosis mediates cognitive deficits through impaired BDNF expression in bile duct ligation model of hepatic encephalopathy

Chronic liver disease per se induces neuroinflammation that contributes to cognitive deficits in hepatic encephalopathy (HE). However, the processes by which pro-inflammatory molecules result in cognitive impairment still remains unclear. In the present study, a significant increase in the activity of liver function enzymes viz. alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) was observed along with increase in plasma ammonia levels after four weeks of bile duct ligation (BDL) in rats suggesting hepatocellular damage. A significant increase was observed in mRNA expression of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in brain regions and liver of BDL rats. Concomitantly, IL-6, TNF-α and MCP-1 protein levels were also increased in brain regions, liver and serum of BDL rats suggesting the involvement of blood-brain-axis in inflammatory response. However, a significant decrease was observed in glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor molecule-1 (Iba-1) expression at transcriptional and translation level in brain of BDL rats. Immunohistochemical and flowcytometric analysis revealed reduced number of GFAP-immunopositive astrocytes and Iba1-immunopositive microglia in the brain regions of BDL rats. Further, a significant decline was observed in cognitive functions in BDL rats assessed using Morris water maze and novel object recognition tests. Expression of pro and mature form of brain derived neurotrophic factor (BDNF) and its upstream transcription element showed significant reduction in brain of BDL rats. Taken together, the results of the present study suggest that systemic inflammation and reduced expression of BDNF and its upstream transcription factor plays a key role in cognitive decline in HE.

A Systems Biology Study in Tomato Fruit Reveals Correlations between the Ascorbate Pool and Genes Involved in Ribosome Biogenesis, Translation, and the Heat-Shock Response.

Changing the balance between ascorbate, monodehydroascorbate, and dehydroascorbate in plant cells by manipulating the activity of enzymes involved in ascorbate synthesis or recycling of oxidized and reduced forms leads to multiple phenotypes. A systems biology approach including network analysis of the transcriptome, proteome and metabolites of RNAi lines for ascorbate oxidase, monodehydroascorbate reductase and galactonolactone dehydrogenase has been carried out in orange fruit pericarp of tomato (Solanum lycopersicum). The transcriptome of the RNAi ascorbate oxidase lines is inversed compared to the monodehydroascorbate reductase and galactonolactone dehydrogenase lines. Differentially expressed genes are involved in ribosome biogenesis and translation. This transcriptome inversion is also seen in response to different stresses in Arabidopsis. The transcriptome response is not well correlated with the proteome which, with the metabolites, are correlated to the activity of the ascorbate redox enzymes—ascorbate oxidase and monodehydroascorbate reductase. Differentially accumulated proteins include metacaspase, protein disulphide isomerase, chaperone DnaK and carbonic anhydrase and the metabolites chlorogenic acid, dehydroascorbate and alanine. The hub genes identified from the network analysis are involved in signaling, the heat-shock response and ribosome biogenesis. The results from this study therefore reveal one or several putative signals from the ascorbate pool which modify the transcriptional response and elements downstream.

Association of the SOD2 (rs2758339 and rs5746136) polymorphisms with the risk of heroin dependency and the SOD2 expression levels

BackgroundSuperoxide dismutase-2 (SOD2, OMIM: 147460) is involved in the detoxification of superoxide anions. The SOD2 mRNA level is down-regulated in cells exposed to morphine. Oxidative stress plays an important role in drug dependency. The rs2758339 (A/C) is located in the vicinity of SP1 and NF-κB transcription element sequences and the rs5746136 (A/G) creates a new glucocorticoid receptor binding site. Taken together, it is hypothesized that these polymorphisms might be associated with the risk of heroin dependency (HD) and the SOD2 expression level. MethodsTo investigate the association between the SOD2 polymorphisms and the risk of HD, 442 heroin dependent persons and 799 healthy controls were included. This study also, consisted of 77 healthy students of Shiraz University (Iran) to investigate the association between the SOD2 polymorphisms and the gene expression level. ResultsThe AC (OR = 0.72, 95% CI = 0.56–0.93, P = 0.013) and CC (OR = 0.64, 95% CI = 0.45–0.92, P = 0.015) genotypes of the rs2758339 were negatively associated with the risk of HD. The AA genotype of the rs5746136 increased the risk of HD (OR = 1.46, 95% CI = 1.03–2.07, P = 0.031). The AA haplotype was associated with the risk of HD (OR = 1.31, 95% CI = 1.09–1.58, P = 0.004). There was no relationship between the study genotypes (or diplotypes) and the SOD2 expression level. ConclusionThe rs2758339 and rs5746136 polymorphisms of the SOD2 are associated with the risk of HD but not associated with the SOD2 expression level.

Superior HIV-1 TAR Binders with Conformationally Constrained R52 Arginine Mimics in the Tat(48-57) Peptide.

We report a 100-fold increase in binding affinity of the Tat(48-57) peptide to HIV-1 transcriptional activator-responsive element (TAR) RNA by replacing Arg52, an essential and critical residue for Tat's specific binding, with (2S,4S)-4-guanidinoproline. The resulting αTat1M peptide is a far superior binder than γTat1M, a peptide containing another conformationally constrained arginine mimic, (2S,4S)-4-amino-N-(3-guanidinopropyl)proline, or even the control Tat peptide (CtrlTat) itself. Our observations are supported by circular dichroism (CD), isothermal titration calorimetry (ITC), gel electrophoresis and UV spectroscopy studies. Molecular dynamics simulations suggest increased interactions between the more compact αTat1M and TAR RNA, relative to CtrlTat. The CD signature of the RNA itself remains largely unchanged upon binding of the peptides. The Tat mimetics further have better cell uptake properties than the control Tat peptide, thus increasing their potential application as specific TAR-binding molecules.

The remote transcriptional control of Hox genes.

Since the discovery by Ed Lewis that the order of Hox genes on the chromosome reflects the partitioning of their patterning function along the anterior-posterior axis of the developing fruit fly embryo, extensive efforts have been dedicated to uncovering the regulatory events underlying the collinear expression of Hox genes. These studies have revealed various aspects of Hox regulation, including short-range and long-range transcriptional enhancers, insulator elements and non-coding RNAs. With the development of technologies allowing for high resolution probing of chromatin architecture, notably Chromosome Conformation Capture (3C)-based techniques, a clear relationship is emerging between long-range regulation of Hox genes and the three-dimensional organization of the genome. Here, we provide an overview of these studies and in particular we discuss the functional relevance of genome compartmentalization, CTCF- mediated insulation and the Polycomb Repressive Complexes in the remote control of Hox genes.

Dissimilar pigment regulation in Serpula lacrymans and Paxillus involutus during inter-kingdom interactions.

Production of basidiomycete atromentin-derived pigments like variegatic acid (pulvinic acid-type) and involutin (diarylcyclopentenone) from the brown-rotter Serpula lacrymans and the ectomycorrhiza-forming Paxillus involutus, respectively, is induced by complex nutrition, and in the case of S. lacrymans, bacteria. Pigmentation in S. lacrymans was stimulated by 13 different bacteria and cell-wall-damaging enzymes (lytic enzymes and proteases), but not by lysozyme or mechanical damage. The use of protease inhibitors with Bacillus subtilis or heat-killed bacteria during co-culturing with S. lacrymans significantly reduced pigmentation indicating that enzymatic hyphal damage and/or released peptides, rather than mechanical injury, was the major cause of systemic pigment induction. Conversely, no significant pigmentation by bacteria was observed from P. involutus. We found additional putative transcriptional composite elements of atromentin synthetase genes in P. involutus and other ectomycorrhiza-forming species that were absent from S. lacrymans and other brown-rotters. Variegatic and its precursor xerocomic acid, but not involutin, in return inhibited swarming and colony biofilm spreading of Bacillus subtilis, but did not kill B. subtilis. We suggest that dissimilar pigment regulation by fungal lifestyle was a consequence of pigment bioactivity and additional promoter motifs. The focus on basidiomycete natural product gene induction and regulation will assist in future studies to determine global regulators, signalling pathways and associated transcription factors of basidiomycetes.

In Situ Capture of the Molecular Composition of Erythroid Transcriptional Enhancers

Elucidating mechanisms that regulate gene transcription during erythropoiesis is critical for identifying fundamental principles of cellular differentiation, and for developing new therapies for blood disorders. Transcriptional enhancers determine cell identity by directing spatiotemporal gene expression, yet the molecular processes controlling enhancer activation and deactivation during lineage differentiation remain largely unknown. Recently, we and others have identified human erythroid cell-specific enhancers by mapping histone modifications, transcription factor binding and transcriptomic changes in primary fetal and adult hematopoietic stem/progenitor cells and lineage-committed erythroid progenitors. While these studies establish a comprehensive catalog of erythroid transcriptional enhancers, the molecular composition and in vivo function of the vast majority of these enhancers remain unknown. This is a major impediment for understanding the coordinated control of gene transcription in normal and diseased erythroid cells.Given that enhancers are frequently targeted by disease-associated genetic variations, there is a critical need to correct this gap in knowledge. Identifying the complete composition of a specific enhancer within its native chromatin can provide unprecedented insight into the molecular mechanisms regulating its activity. To facilitate the molecular characterization of enhancers within their native chromatin environment, we have developed a new method, CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements), to isolate enhancer-associated molecular interactions by repurposing the CRISPR/Cas9 system. By using the endonuclease-deficient Cas9 (dCas9) and enhancer-targeting guide RNA (sgRNA), the enhancer-associated protein, DNA and RNA complexes are isolated by affinity purification and identified by high-throughput sequencing and proteomics, respectively. Using these methods, we isolated chromatin-regulating protein complexes associated with several critical cis -regulatory elements within the human β-globin cluster such as the locus control region (LCR) in human erythroid cells, and identified many known factors such as GATA1, TAL1, NFE2, LDB1 and the SWI/SNF chromatin remodeling complexes involved in globin gene regulation. More importantly, we also identified several new factors, such as the nuclear pore proteins NUP98 and NUP153, which have not previously been implicated in globin gene regulation. Subsequent studies of these factors established their roles in the developmental regulation of enhancer activities for globin gene transcription. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes also establishes evidence for composition-based hierarchical organization of enhancer structure. Furthermore, unbiased analysis of cis -element-mediated long-range chromatin interactions at disease-associated non-coding elements and developmentally controlled super-enhancers reveals spatial features causally regulate gene transcription. Therefore, the dCas9-mediated affinity purification has the potential to advance the characterization of chromatin-templated hierarchical events by providing a method to examine the complete molecular composition of transcriptional enhancers and how composition changes in cellular differentiation. Our studies not only help elucidate the underlying principles regulating lineage-defining enhancer elements, but also establish new approaches for in situ molecular dissection of disease-associated cis -regulatory elements in globin gene transcription and erythropoiesis. Disclosures No relevant conflicts of interest to declare.

SNAIL1-mediated downregulation of FOXA proteins facilitates the inactivation of transcriptional enhancer elements at key epithelial genes in colorectal cancer cells.

Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of SNAIL1 and FOXA genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, SNAIL1 targets transcription factors at strategically important positions in gene-regulatory hierarchies, which may facilitate transcriptional reprogramming during EMT.