Analysis of gliomas DNA methylation: Assessment of pre-analytical variables.

Precision oncology is driven by biomarkers. For glioblastoma multiforme (GBM), the most common malignant adult primary brain tumor, O6-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation is an important prognostic and treatment clinical biomarker. Time consuming pre-analytical steps such as biospecimen storage, fixation, sampling, and processing are sources of data irreproducibility, and all these pre-analytical variables are confounded by intratumor heterogeneity of MGMT promoter methylation. To assess the effect of pre-analytical variables on GBM DNA methylation, tissue storage/sampling (CryoGrid), sample preparation multi-sonicator (PIXUL), and 5-methylcytosine (5mC) DNA immunoprecipitation (Matrix MeDIP-qPCR/seq) platforms were used. MGMT promoter methylation status assayed by MeDIP-qPCR was validated with methylation specific PCR (MS-PCR). MGMT promoter methylation levels in frozen and formalin fixed paraffin embedded (FFPE) sample pairs were not statistically different, confirming reliability of FFPEs for MGMT promoter methylation analysis. Warm ex-vivo ischemia (up to 4hrs at 37oC) and 3 cycles of repeated sample thawing and freezing did not statistically impact 5mC at MGMT promoter, exon, and enhancer regions, indicating the resistance of DNA methylation to common variations in sample processing conditions that might be encountered in research and clinical settings. 26-34% of specimens exhibited intratumor heterogeneity in the MGMT DNA promoter methylation. These data demonstrate that variations in sample fixation, ischemia duration and temperature, and DNA methylation assay technique do not have a statistically significant impact on MGMT promoter methylation assessment. However, intratumor methylation heterogeneity underscores the value of multiple biopsies at different GBM geographic tumor sites in the evaluation of MGMT promoter methylation status. Matrix-MeDIP-seq analysis revealed that MGMT promoter methylation status clustered with other differentially methylated genomic loci (e.g. HOXA and lncRNAs) that are resilient to variation in the above pre-analytical conditions. These observations offer new opportunities to develop more granular data-based epigenetic GBM biomarkers. In this regard, the high throughput CryoGrid-PIXUL-Matrix toolbox could be useful.

Regulation of versican expression in macrophages is mediated by canonical type I interferon signaling via ISGF3.

Growing evidence supports a role for versican as an important component of the inflammatory response, with both pro- and anti-inflammatory roles depending on the specific context of the system or disease under investigation. Our goal is to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. In previous work, we showed that LPS triggers a signaling cascade involving Toll-like receptor (TLR)4, the Trif adaptor, type I interferons, and the type I interferon receptor, leading to increased versican expression by macrophages. In the present study, we used a combination of chromatin immunoprecipitation, siRNA, chemical inhibitors, and mouse model approaches to investigate the regulatory events downstream of the type I interferon receptor to better define the mechanism controlling versican expression. Results indicate that transcriptional regulation by canonical type I interferon signaling via interferon-stimulated gene factor 3 (ISGF3), the heterotrimeric transcription factor complex of Irf9, Stat1, and Stat2, controls versican expression in macrophages exposed to LPS. This pathway is not dependent on MAPK signaling, which has been shown to regulate versican expression in other cell types. The stability of versican mRNA may also contribute to prolonged versican expression in macrophages. These findings strongly support a role for macrophage-derived versican as a type I interferon-stimulated gene and further our understanding of versican's role in regulating inflammation.NEW & NOTEWORTHY We report the novel finding that versican expression is regulated by the interferon-stimulated gene factor 3 (ISGF3) arm of canonical type I Ifn signaling in LPS-stimulated macrophages. This pathway is distinct from mechanisms that control versican expression in other cell types. This suggests that macrophage-derived versican may play a role in limiting a potentially excessive inflammatory response. The detailed understanding of how versican expression is regulated in different cells could lead to unique approaches for enhancing its anti-inflammatory properties.

Analysis of gliomas DNA methylation: Assessment of pre-analytical variables.

Precision oncology is driven by molecular biomarkers. For glioblastoma multiforme (GBM), the most common malignant adult primary brain tumor, O6-methylguanine-DNA methyltransferase ( MGMT ) gene DNA promoter methylation is an important prognostic and treatment clinical biomarker. Time consuming pre-analytical steps such as biospecimen storage before fixing, sampling, and processing are major sources of errors and batch effects, that are further confounded by intra-tumor heterogeneity of MGMT promoter methylation. To assess the effect of pre-analytical variables on GBM DNA methylation, tissue storage/sampling (CryoGrid), sample preparation multi-sonicator (PIXUL) and 5-methylcytosine (5mC) DNA immunoprecipitation (Matrix MeDIP-qPCR/seq) platforms were used. MGMT promoter CpG methylation was examined in 173 surgical samples from 90 individuals, 50 of these were used for intra-tumor heterogeneity studies. MGMT promoter methylation levels in paired frozen and formalin fixed paraffin embedded (FFPE) samples were very close, confirming suitability of FFPE for MGMT promoter methylation analysis in clinical settings. Matrix MeDIP-qPCR yielded similar results to methylation specific PCR (MS-PCR). Warm ex-vivo ischemia (37°C up to 4hrs) and 3 cycles of repeated sample thawing and freezing did not alter 5mC levels at MGMT promoter, exon and upstream enhancer regions, demonstrating the resistance of DNA methylation to the most common variations in sample processing conditions that might be encountered in research and clinical settings. 20-30% of specimens exhibited intratumor heterogeneity in the MGMT DNA promoter methylation. Collectively these data demonstrate that variations in sample fixation, ischemia duration and temperature, and DNA methylation assay technique do not have significant impact on assessment of MGMT promoter methylation status. However, intratumor methylation heterogeneity underscores the need for histologic verification and value of multiple biopsies at different GBM geographic tumor sites in assessment of MGMT promoter methylation. Matrix-MeDIP-seq analysis revealed that MGMT promoter methylation status clustered with other differentially methylated genomic loci (e.g. HOXA and lncRNAs), that are likewise resilient to variation in above post-resection pre-analytical conditions. These MGMT -associated global DNA methylation patterns offer new opportunities to validate more granular data-based epigenetic GBM clinical biomarkers where the CryoGrid-PIXUL-Matrix toolbox could prove to be useful.

dCas9 fusion to computer-designed PRC2 inhibitor reveals functional TATA box in distal promoter region.

Bifurcation of cellular fates, a critical process in development, requires histone 3 lysine 27 methylation (H3K27me3) marks propagated by the polycomb repressive complex 2 (PRC2). However, precise chromatin loci of functional H3K27me3 marks are not yet known. Here, we identify critical PRC2 functional sites at high resolution. We fused a computationally designed protein, EED binder (EB), which competes with EZH2 and thereby inhibits PRC2 function, to dCas9 (EBdCas9) to allow for PRC2 inhibition at a precise locus using gRNA. Targeting EBdCas9 to four different genes (TBX18, p16, CDX2, and GATA3) results in precise H3K27me3 and EZH2 reduction, gene activation, and functional outcomes in the cell cycle (p16) or trophoblast transdifferentiation (CDX2 and GATA3). In the case of TBX18, we identify a PRC2-controlled, functional TATA box >500 bp upstream of the TBX18 transcription start site (TSS) using EBdCas9. Deletion of this TATA box eliminates EBdCas9-dependent TATA binding protein (TBP) recruitment and transcriptional activation. EBdCas9 technology may provide a broadly applicable tool for epigenomic control of gene regulation.

Selective Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibition by the SCH772984 Compound Attenuates In Vitro and In Vivo Inflammatory Responses and Prolongs Survival in Murine Sepsis Models.

Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.

Multi-Organ Transcriptome Dynamics in a Mouse Model of Cecal Ligation and Puncture-Induced Polymicrobial Sepsis.

PURPOSE: During sepsis, an excessive inflammatory immune reaction contributes to multi-organ dysfunction syndrome (MODS), a critical condition associated with high morbidity and mortality; however, the molecular mechanisms driving MODS remain elusive. METHODS: We used RNA sequencing to characterize transcriptional changes in the early phase of sepsis, at 6, 12, 24 hour time points in lung, kidney, liver, and heart tissues, in a cecal ligation and puncture (CLP)-induced polymicrobial sepsis murine model. RESULTS: The CLP surgery induced significant changes (adj. p-value<0.05) in expression of hundreds of transcripts in the four organs tested, with the highest number exceeding 2,000 differentially expressed genes (DEGs) in all organs at 12 hours post-CLP. Over-representation analysis by functional annotations of DEGs to the Reactome database revealed the immune system, hemostasis, lipid metabolism, signal transduction, and extracellular matrix remodeling biological processes as significantly altered in at least two organs, while metabolism of proteins and RNA were revelaed as being liver tissue specific in the early phase of sepsis. CONCLUSION: RNA sequencing across organs and time-points in the CLP murine model allowed us to study the trajectories of transcriptome changes demonstrating alterations common across multiple organs as well as biological pathways altered in an organ-specific manner. These findings could pave new directions in the research of sepsis-induced MODS and indicate new sepsis treatment strategies.

Engineering a multicellular vascular niche to model hematopoietic cell trafficking.

BACKGROUND: The marrow microenvironment and vasculature plays a critical role in regulating hematopoietic cell recruitment, residence, and maturation. Extensive in vitro and in vivo studies have aimed to understand the marrow cell types that contribute to hematopoiesis and the stem cell environment. Nonetheless, in vitro models are limited by a lack of complex multicellular interactions, and cellular interactions are not easily manipulated in vivo. Here, we develop an engineered human vascular marrow niche to examine the three-dimensional cell interactions that direct hematopoietic cell trafficking. METHODS: Using soft lithography and injection molding techniques, fully endothelialized vascular networks were fabricated in type I collagen matrix, and co-cultured under flow with embedded marrow fibroblast cells in the matrix. Marrow fibroblast (mesenchymal stem cells (MSCs), HS27a, or HS5) interactions with the endothelium were imaged via confocal microscopy and altered endothelial gene expression was analyzed with RT-PCR. Monocytes, hematopoietic progenitor cells, and leukemic cells were perfused through the network and their adhesion and migration was evaluated. RESULTS: HS27a cells and MSCs interact directly with the vessel wall more than HS5 cells, which are not seen to make contact with the endothelial cells. In both HS27a and HS5 co-cultures, endothelial expression of junctional markers was reduced. HS27a co-cultures promote perfused monocytes to adhere and migrate within the vessel network. Hematopoietic progenitors rely on monocyte-fibroblast crosstalk to facilitate preferential recruitment within HS27a co-cultured vessels. In contrast, leukemic cells sense fibroblast differences and are recruited preferentially to HS5 and HS27a co-cultures, but monocytes are able to block this sensitivity. CONCLUSIONS: We demonstrate the use of a microvascular platform that incorporates a tunable, multicellular composition to examine differences in hematopoietic cell trafficking. Differential recruitment of hematopoietic cell types to distinct fibroblast microenvironments highlights the complexity of cell-cell interactions within the marrow. This system allows for step-wise incorporation of cellular components to reveal the dynamic spatial and temporal interactions between endothelial cells, marrow-derived fibroblasts, and hematopoietic cells that comprise the marrow vascular niche. Furthermore, this platform has potential for use in testing therapeutics and personalized medicine in both normal and disease contexts.

Quadruplex formation by both G-rich and C-rich DNA strands of the C9orf72 (GGGGCC)8•(GGCCCC)8 repeat: effect of CpG methylation.

Unusual DNA/RNA structures of the C9orf72 repeat may participate in repeat expansions or pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded repeats are CpG methylated with unknown consequences. Typically, quadruplex structures form by G-rich but not complementary C-rich strands. Using CD, UV and electrophoresis, we characterized the structures formed by (GGGGCC)8 and (GGCCCC)8 strands with and without 5-methylcytosine (5mCpG) or 5-hydroxymethylcytosine (5hmCpG) methylation. All strands formed heterogenous mixtures of structures, with features of quadruplexes (at pH 7.5, in K(+), Na(+) or Li(+)), but no feature typical of i-motifs. C-rich strands formed quadruplexes, likely stabilized by G•C•G•C-tetrads and C•C•C•C-tetrads. Unlike G•G•G•G-tetrads, some G•C•G•C-tetrad conformations do not require the N7-Guanine position, hence C9orf72 quadruplexes still formed when N7-deazaGuanine replace all Guanines. 5mCpG and 5hmCpG increased and decreased the thermal stability of these structures. hnRNPK, through band-shift analysis, bound C-rich but not G-rich strands, with a binding preference of unmethylated > 5hmCpG > 5mCpG, where methylated DNA-protein complexes were retained in the wells, distinct from unmethylated complexes. Our findings suggest that for C-rich sequences interspersed with G-residues, one must consider quadruplex formation and that methylation of quadruplexes may affect epigenetic processes.

Beads-free protein immunoprecipitation for a mass spectrometry-based interactome and posttranslational modifications analysis.

BACKGROUND: Protein immunoprecipitation (IP) coupled with MS provides means to interrogate protein complexes and their posttranslational modifications (PTMs). In a typical protein IP assay antibodies are conjugated to protein A/G beads requiring large amounts of antibodies, tube transfers and centrifugations. RESULTS: As an alternative, we present Matrix-IP, beads-free microplate-based platform with surface-immobilized antibodies. Assay utilizes standard 96-well polypropylene PCR plates that are laboratory-fabricated with UV-C light and then protein A/G coated prior to IP reaction. We demonstrate application of Matrix-IP platform in MS analysis of heterogeneous nuclear ribonucleoprotein K (hnRNP K) interactome and PTMs. CONCLUSION: Matrix-IP is time-saving, easy to use high throughput method adaptable for low sample amounts and automation.

Temporal Dissection of Rate Limiting Transcriptional Events Using Pol II ChIP and RNA Analysis of Adrenergic Stress Gene Activation.

In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the generality of genetic control subsequent to RNA polymerase II (Pol II) recruitment. In this report, we use Pol II Chromatin Immunoprecipitation to investigate relationships between the mechanistic events controlling immediate early gene (IEG) activation following stimulation of the α1a-Adrenergic Receptor expressed in rat-1 fibroblasts. We validate our Pol II ChIP assay by comparison to major transcriptional events assessable by microarray and PCR analysis of precursor and mature mRNA. Temporal analysis of Pol II density suggests that reduced proximal pausing often enhances gene expression and was essential for Nr4a3 expression. Nevertheless, for Nr4a3 and several other genes, proximal pausing delayed the time required for initiation of productive elongation, consistent with a role in ensuring transcriptional fidelity. Arrival of Pol II at the 3' cleavage site usually correlated with increased polyadenylated mRNA; however, for Nfil3 and probably Gprc5a expression was delayed and accompanied by apparent pre-mRNA degradation. Intragenic pausing not associated with polyadenylation was also found to regulate and delay Gprc5a expression. Temporal analysis of Nr4a3, Dusp5 and Nfil3 shows that transcription of native IEG genes can proceed at velocities of 3.5 to 4 kilobases/min immediately after activation. Of note, all of the genes studied here also used increased Pol II recruitment as an important regulator of expression. Nevertheless, the generality of co-transcriptional regulation during IEG activation suggests temporal and integrated analysis will often be necessary to distinguish causative from potential rate limiting mechanisms.

Heterogeneity of epigenetic changes at ischemia/reperfusion- and endotoxin-induced acute kidney injury genes.

Aberrant gene expression is a molecular hallmark of acute kidney injury (AKI). As epigenetic processes control gene expression in a cell- and environment-defined manner, understanding the epigenetic pathways that regulate genes altered by AKI may open vital new insights into the complexities of disease pathogenesis and identify possible therapeutic targets. Here we used matrix chromatin immunoprecipitation and integrative analysis to study 20 key permissive and repressive epigenetic histone marks at transcriptionally induced Tnf, Ngal, Kim-1, and Icam-1 genes in mouse models of AKI; unilateral renal ischemia/reperfusion, lipopolysaccharide (LPS), and their synergistically injurious combination. Results revealed unexpected heterogeneity of transcriptional and epigenetic responses. Tnf and Ngal were transcriptionally upregulated in response to both treatments individually, and to combination treatment. Kim-1 was induced by ischemia/reperfusion and Icam-1 by LPS only. Epigenetic alterations at these genes exhibited distinct time-dependent changes that shared some similarities, such as reduction in repressive histone modifications, and also had major ischemia/reperfusion versus endotoxin differences. Thus, diversity of changes at AKI genes in response to different insults indicates involvement of several epigenetic pathways. This could be exploited pharmacologically through rational-drug design to alter the course and improve clinical outcomes of this syndrome.

M2 Macrophage Polarization Mediates Anti-inflammatory Effects of Endothelial Nitric Oxide Signaling.

Endothelial nitric oxide (NO) signaling plays a physiological role in limiting obesity-associated insulin resistance and inflammation. This study was undertaken to investigate whether this NO effect involves polarization of macrophages toward an anti-inflammatory M2 phenotype. Mice with transgenic endothelial NO synthase overexpression were protected against high-fat diet (HFD)-induced hepatic inflammation and insulin resistance, and this effect was associated with reduced proinflammatory M1 and increased anti-inflammatory M2 activation of Kupffer cells. In cell culture studies, exposure of macrophages to endothelial NO similarly reduced inflammatory (M1) and increased anti-inflammatory (M2) gene expression. Similar effects were induced by macrophage overexpression of vasodilator-stimulated phosphoprotein (VASP), a key downstream mediator of intracellular NO signaling. Conversely, VASP deficiency induced proinflammatory M1 macrophage activation, and the transplantation of bone marrow from VASP-deficient donor mice into normal recipients caused hepatic inflammation and insulin resistance resembling that induced in normal mice by consumption of an HFD. These data suggest that proinflammatory macrophage M1 activation and macrophage-mediated inflammation are tonically inhibited by NO → VASP signal transduction, and that reduced NO → VASP signaling is involved in the effect of HFD feeding to induce M1 activation of Kupffer cells and associated hepatic inflammation. Our data implicate endothelial NO → VASP signaling as a physiological determinant of macrophage polarization and show that signaling via this pathway is required to prevent hepatic inflammation and insulin resistance.

Synchronous recruitment of epigenetic modifiers to endotoxin synergistically activated Tnf-α gene in acute kidney injury.

BACKGROUND: As a consequence of acute kidney injury (AKI), proximal tubular cells hyperrespond to endotoxin (lipopolysaccharide, LPS) by exaggerated renal Tnf-α Production. This LPS hyperresponsiveness is transcriptionally mediated. The epigenetic pathways that control these responses are unknown. METHODS/FINDINGS: We applied multiplex chromatin immunoprecipitation platform (Matrix ChIP) to explore epigenetic pathways that underlie endotoxin hyperresponsiveness in the setting of preceding unilateral renal ischemia/reperfusion (I/R) in mouse AKI model. Endotoxin exposure after I/R resulted in enhanced transcription, manifested by hyperresponsive recruitment of RNA polymerase II (Pol II) at the Tnf-α gene. At this locus, LPS but not I/R increased levels of Pol II C-terminal domain (CTD) phosho-serine2 &5 and induced dephosphorylation of the transcription-repressive histone H4 phospho-serine-1. In contrast, I/R but not LPS increased the transcription-permissive histone phosphorylation (H3 phospho-serine-10, H3.3 phospho-serine-31) at the Tnf-α gene. In agreement with these observations, I/R but not LPS increased activity of cognate kinases (Erk1/2, Msk1/2 and Aurora A) at the Tnf-α locus. Cross-talk of histone phosphorylation and acetylation synergize to active gene expression. I/R and LPS increased histone acetylation. (H3K9/14Ac, H4K5/8/12/16Ac, H2KA5Ac, H2BK4/7Ac). Levels of some histone acetyltransferases at this gene (PCAF and MOF) were increased by I/R but not by LPS, while others were induced by either I/R or LPS and exhibited endotoxin hyperresponsive patterns (GCN5, CBP and p300). The adaptor protein 14-3-3 couples histone phosphorylation with acetylation, and tethers chromatin modifiers/transcription elongation factors to target genes. Both I/R and LPS increased levels of 14-3-3 and several chromatin/transcription modifiers (BRD4, BRG1, HP-1γ and IKKα) at the Tnf-α gene, all exhibiting endotoxin hyperresponsive recruitment patterns similar to Pol II. CONCLUSIONS: Our results suggest that I/R and LPS differentially trigger phosphorylation (Pol II and histone) and acetylation (histone) epigenetic pathways that interact at the Tnf-α gene to generate endotoxin hyperresponse in AKI.

Heterogeneous nuclear ribonucleoprotein (HnRNP) K genome-wide binding survey reveals its role in regulating 3'-end RNA processing and transcription termination at the early growth response 1 (EGR1) gene through XRN2 exonuclease.

The heterogeneous nuclear ribonucleoprotein K (hnRNPK) is a nucleic acid-binding protein that acts as a docking platform integrating signal transduction pathways to nucleic acid-related processes. Given that hnRNPK could be involved in other steps that compose gene expression the definition of its genome-wide occupancy is important to better understand its role in transcription and co-transcriptional processes. Here, we used chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) to analyze the genome-wide hnRNPK-DNA interaction in colon cancer cell line HCT116. 9.1/3.6 and 7.0/3.4 million tags were sequenced/mapped, then 1809 and 642 hnRNPK binding sites were detected in quiescent and 30-min serum-stimulated cells, respectively. The inspection of sequencing tracks revealed inducible hnRNPK recruitment along a number of immediate early gene loci, including EGR1 and ZFP36, with the highest densities present at the transcription termination sites. Strikingly, hnRNPK knockdown with siRNA resulted in increased pre-RNA levels transcribed downstream of the EGR1 polyadenylation (A) site suggesting altered 3'-end pre-RNA degradation. Further ChIP survey of hnRNPK knockdown uncovered decreased recruitment of the 5'-3' exonuclease XRN2 along EGR1 and downstream of the poly(A) signal without altering RNA polymerase II density at these sites. Immunoprecipitation of hnRNPK and XRN2 from intact and RNase A-treated nuclear extracts followed by shotgun mass spectrometry revealed the presence of hnRNPK and XRN2 in the same complexes along with other spliceosome-related proteins. Our data suggest that hnRNPK may play a role in recruitment of XRN2 to gene loci thus regulating coupling 3'-end pre-mRNA processing to transcription termination.

Activation of Wnt/ß-catenin signaling increases apoptosis in melanoma cells treated with trail.

While the TRAIL pathway represents a promising therapeutic target in melanoma, resistance to TRAIL-mediated apoptosis remains a barrier to its successful adoption. Since the Wnt/ß-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/ß-catenin signaling on regulating the responses of melanoma cells to TRAIL. Co-treatment of melanoma cell lines with WNT3A-conditioned media and recombinant TRAIL significantly enhanced apoptosis compared to treatment with TRAIL alone. This apoptosis correlates with increased abundance of the pro-apoptotic proteins BCL2L11 and BBC3, and with decreased abundance of the anti-apoptotic regulator Mcl1. We then confirmed the involvement of the Wnt/ß-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular ß-catenin antagonist, AXIN1, or treating cells with an inhibitor of GSK-3 also enhanced melanoma cell sensitivity to TRAIL. These studies describe a novel regulation of TRAIL sensitivity in melanoma by Wnt/ß-catenin signaling, and suggest that strategies to enhance Wnt/ß-catenin signaling in combination with TRAIL agonists warrant further investigation.

Acyl-CoA synthetase 1 is induced by Gram-negative bacteria and lipopolysaccharide and is required for phospholipid turnover in stimulated macrophages.

The enzyme acyl-CoA synthetase 1 (ACSL1) is induced by peroxisome proliferator-activated receptor α (PPARα) and PPARγ in insulin target tissues, such as skeletal muscle and adipose tissue, and plays an important role in ß-oxidation in these tissues. In macrophages, however, ACSL1 mediates inflammatory effects without significant effects on ß-oxidation. Thus, the function of ACSL1 varies in different tissues. We therefore investigated the signals and signal transduction pathways resulting in ACSL1 induction in macrophages as well as the consequences of ACSL1 deficiency for phospholipid turnover in LPS-activated macrophages. LPS, Gram-negative bacteria, IFN-γ, and TNFα all induce ACSL1 expression in macrophages, whereas PPAR agonists do not. LPS-induced ACSL1 expression is dependent on Toll-like receptor 4 (TLR4) and its adaptor protein TRIF (Toll-like receptor adaptor molecule 1) but does not require the MyD88 (myeloid differentiation primary response gene 88) arm of TLR4 signaling; nor does it require STAT1 (signal transducer and activator of transcription 1) for maximal induction. Furthermore, ACSL1 deletion attenuates phospholipid turnover in LPS-stimulated macrophages. Thus, the regulation and biological function of ACSL1 in macrophages differ markedly from that in insulin target tissues. These results suggest that ACSL1 may have an important role in the innate immune response. Further, these findings illustrate an interesting paradigm in which the same enzyme, ACSL1, confers distinct biological effects in different cell types, and these disparate functions are paralleled by differences in the pathways that regulate its expression.

Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays.

BACKGROUND: The processes that compose expression of a given gene are far more complex than previously thought presenting unprecedented conceptual and mechanistic challenges that require development of new tools. Chromatin structure, which is regulated by DNA methylation and histone modification, is at the center of gene regulation. Immunoprecipitations of chromatin (ChIP) and methylated DNA (MeDIP) represent a major achievement in this area that allow researchers to probe chromatin modifications as well as specific protein-DNA interactions in vivo and to estimate the density of proteins at specific sites genome-wide. Although a critical component of chromatin structure, DNA methylation has often been studied independently of other chromatin events and transcription. RESULTS: To allow simultaneous measurements of DNA methylation with other genomic processes, we developed and validated a simple and easy-to-use high throughput microplate-based platform for analysis of DNA methylation. Compared to the traditional beads-based MeDIP the microplate MeDIP was more sensitive and had lower non-specific binding. We integrated the MeDIP method with a microplate ChIP assay which allows measurements of both DNA methylation and histone marks at the same time, Matrix ChIP-MeDIP platform. We illustrated several applications of this platform to relate DNA methylation, with chromatin and transcription events at selected genes in cultured cells, human cancer and in a model of diabetic kidney disease. CONCLUSION: The high throughput capacity of Matrix ChIP-MeDIP to profile tens and potentially hundreds of different genomic events at the same time as DNA methylation represents a powerful platform to explore complex genomic mechanism at selected genes in cultured cells and in whole tissues. In this regard, Matrix ChIP-MeDIP should be useful to complement genome-wide studies where the rich chromatin and transcription database resources provide fruitful foundation to pursue mechanistic, functional and diagnostic information at genes of interest in health and disease.

Direct recruitment of ERK cascade components to inducible genes is regulated by heterogeneous nuclear ribonucleoprotein (hnRNP) K.

Components of the ERK cascade are recruited to genes, but it remains unknown how they are regulated at these sites. The RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) K interacts with kinases and is found along genes including the mitogen-inducible early response gene EGR-1. Here, we used chromatin immunoprecipitations to study co-recruitment of hnRNP K and ERK cascade activity along the EGR-1 gene. These measurements revealed that the spatiotemporal binding patterns of ERK cascade transducers (GRB2, SOS, B-Raf, MEK, and ERK) at the EGR-1 locus resemble both hnRNP K and RNA polymerase II (Pol II). Inhibition of EGR-1 transcription with either serum-responsive factor knockdown or 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole altered recruitment of all of the above ERK cascade components along this locus that mirrored the changes in Pol II and hnRNP K profiles. siRNA knockdown of hnRNP K decreased the levels of active MEK and ERK at the EGR-1, changes associated with decreased levels of elongating pre-mRNA and less efficient splicing. The hnRNP K dependence and pattern of ERK cascade activation at the c-MYC locus were different from at EGR-1. Ribonucleoprotein immunoprecipitations revealed that hnRNP K was associated with the EGR-1 but not c-MYC mRNAs. These data suggest a model where Pol II transcription-driven recruitment of hnRNP K along the EGR-1 locus compartmentalizes activation of the ERK cascade at these genes, events that regulate synthesis of mature mRNA.

Direct recruitment of insulin receptor and ERK signaling cascade to insulin-inducible gene loci.

OBJECTIVE: Insulin receptor (IR) translocates to the nucleus, but its recruitment to gene loci has not been demonstrated. Here, we tested the hypothesis that IR and its downstream mitogenic transducers are corecruited to two prototypic insulin-inducible genes: early growth response 1 (egr-1), involved in mitogenic response, and glucokinase (Gck), encoding a key metabolic enzyme. RESEARCH DESIGN AND METHODS: We used RNA and chromatin from insulin-treated rat hepatic tumor cell line expressing human insulin receptor (HTC-IR) and livers from lean and insulin-resistant ob/ob glucose-fed mice in quantitative RT-PCR and chromatin immunoprecipitation studies to determine gene expression levels and associated recruitment of RNA polymerase II (Pol II), insulin receptor, and cognate signaling proteins to gene loci, respectively. RESULTS: Insulin-induced egr-1 mRNA in HTC-IR cells was associated with corecruitment of IR signaling cascade (IR, SOS, Grb2, B-Raf, MEK, and ERK) to this gene. Recruitment profiles of phosphorylated IR, B-Raf, MEK, and Erk along egr-1 transcribed region were similar to those of elongating Pol II. Glucose-feeding increased Gck mRNA expression in livers of lean but not ob/ob mice. In lean mice, there was glucose feeding-induced recruitment of IR and its transducers to Gck gene synchronized with elongating Pol II. In sharp contrast, in glucose-fed ob/ob mice, the Gck recruitment patterns of active MEK/Erk, IR, and Pol II were asynchronous. CONCLUSIONS: IR and its signal transducers recruited to genes coupled to elongating Pol II may play a role in maintaining productive mRNA synthesis of target genes. These studies suggest a possibility that impaired Pol II processivity along genes bearing aberrant levels of IR/signal transducers is a previously unrecognized facet of insulin resistance.