A High-Throughput PIXUL-Matrix-Based Toolbox to Profile Frozen and Formalin-Fixed Paraffin-Embedded Tissues Multiomes.

Large-scale high-dimensional multiomics studies are essential to unravel molecular complexity in health and disease. We developed an integrated system for tissue sampling (CryoGrid), analytes preparation (PIXUL), and downstream multiomic analysis in a 96-well plate format (Matrix), MultiomicsTracks96, which we used to interrogate matched frozen and formalin-fixed paraffin-embedded (FFPE) mouse organs. Using this system, we generated 8-dimensional omics data sets encompassing 4 molecular layers of intracellular organization: epigenome (H3K27Ac, H3K4m3, RNA polymerase II, and 5mC levels), transcriptome (messenger RNA levels), epitranscriptome (m6A levels), and proteome (protein levels) in brain, heart, kidney, and liver. There was a high correlation between data from matched frozen and FFPE organs. The Segway genome segmentation algorithm applied to epigenomic profiles confirmed known organ-specific superenhancers in both FFPE and frozen samples. Linear regression analysis showed that proteomic profiles, known to be poorly correlated with transcriptomic data, can be more accurately predicted by the full suite of multiomics data, compared with using epigenomic, transcriptomic, or epitranscriptomic measurements individually.

CryoGrid-PIXUL-RNA: high throughput RNA isolation platform for tissue transcript analysis.

BACKGROUND: Disease molecular complexity requires high throughput workflows to map disease pathways through analysis of vast tissue repositories. Great progress has been made in tissue multiomics analytical technologies. To match the high throughput of these advanced analytical platforms, we have previously developed a multipurpose 96-well microplate sonicator, PIXUL, that can be used in multiple workflows to extract analytes from cultured cells and tissue fragments for various downstream molecular assays. And yet, the sample preparation devices, such as PIXUL, along with the downstream multiomics analytical capabilities have not been fully exploited to interrogate tissues because storing and sampling of such biospecimens remain, in comparison, inefficient. RESULTS: To mitigate this tissue interrogation bottleneck, we have developed a low-cost user-friendly system, CryoGrid, to catalog, cryostore and sample tissue fragments. TRIzol is widely used to isolate RNA but it is labor-intensive, hazardous, requires fume-hoods, and is an expensive reagent. Columns are also commonly used to extract RNA but they involve many steps, are prone to human errors, and are also expensive. Both TRIzol and column protocols use test tubes. We developed a microplate PIXUL-based TRIzol-free and column-free RNA isolation protocol that uses a buffer containing proteinase K (PK buffer). We have integrated the CryoGrid system with PIXUL-based PK buffer, TRIzol, and PureLink column methods to isolate RNA for gene-specific qPCR and genome-wide transcript analyses. CryoGrid-PIXUL, when integrated with either PK buffer, TRIzol or PureLink column RNA isolation protocols, yielded similar transcript profiles in frozen organs (brain, heart, kidney and liver) from a mouse model of sepsis. CONCLUSIONS: RNA isolation using the CryoGrid-PIXUL system combined with the 96-well microplate PK buffer method offers an inexpensive user-friendly high throughput workflow to study transcriptional responses in tissues in health and disease as well as in therapeutic interventions.

PIXUL-ChIP: integrated high-throughput sample preparation and analytical platform for epigenetic studies.

Chromatin immunoprecipitation (ChIP) is the most widely used approach for identification of genome-associated proteins and their modifications. We have previously introduced a microplate-based ChIP platform, Matrix ChIP, where the entire ChIP procedure is done on the same plate without sample transfers. Compared to conventional ChIP protocols, the Matrix ChIP assay is faster and has increased throughput. However, even with microplate ChIP assays, sample preparation and chromatin fragmentation (which is required to map genomic locations) remains a major bottleneck. We have developed a novel technology (termed 'PIXUL') utilizing an array of ultrasound transducers for simultaneous shearing of samples in standard 96-well microplates. We integrated PIXUL with Matrix ChIP ('PIXUL-ChIP'), that allows for fast, reproducible, low-cost and high-throughput sample preparation and ChIP analysis of 96 samples (cell culture or tissues) in one day. Further, we demonstrated that chromatin prepared using PIXUL can be used in an existing ChIP-seq workflow. Thus, the high-throughput capacity of PIXUL-ChIP provides the means to carry out ChIP-qPCR or ChIP-seq experiments involving dozens of samples. Given the complexity of epigenetic processes, the use of PIXUL-ChIP will advance our understanding of these processes in health and disease, as well as facilitate screening of epigenetic drugs.

Regulation of ribosomal RNA expression across the lifespan is fine-tuned by maternal diet before implantation.

Cells and organisms respond to nutrient deprivation by decreasing global rates of transcription, translation and DNA replication. To what extent such changes can be reversed is largely unknown. We examined the effect of maternal dietary restriction on RNA synthesis in the offspring. Low protein diet fed either throughout gestation or for the preimplantation period alone reduced cellular RNA content across fetal somatic tissues during challenge and increased it beyond controls in fetal and adult tissues after challenge release. Changes in transcription of ribosomal RNA, the major component of cellular RNA, were responsible for this phenotype as evidenced by matching alterations in RNA polymerase I density and DNA methylation at ribosomal DNA loci. Cellular levels of the ribosomal transcription factor Rrn3 mirrored the rRNA expression pattern. In cell culture experiments, Rrn3 overexpression reduced rDNA methylation and increased rRNA expression; the converse occurred after inhibition of Rrn3 activity. These observations define novel mechanism where poor nutrition before implantation irreversibly alters basal rates of rRNA transcription thereafter in a process mediated by rDNA methylation and Rrn3 factor.

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.

Experimental acute lung injury induces multi-organ epigenetic modifications in key angiogenic genes implicated in sepsis-associated endothelial dysfunction.

INTRODUCTION: The Tie2/angiopoietin (Tie2/Ang) and vascular endothelial growth factor receptor-ligand systems (VEGFR/VEGF) are recognized to play important roles in the regulation of microvascular endothelial function. Downregulation of these genes during sepsis has been implicated in the pathogenesis of sepsis-related microvascular leak and multiple organ dysfunction syndrome. Mechanisms responsible for dysregulation of angiogenic genes in sepsis are poorly defined. METHODS: Western blot, reverse transcription-polymerase chain reaction, and multiplex chromatin immunoprecipitation platform (Matrix ChIP) were used to investigate serum albumin leak, changes in gene expression, and associated epigenetic alterations in a murine model of acute lung injury-induced sepsis (ALI-sepsis). RESULTS: Experimental ALI-sepsis induced microvascular leak and downregulation of expression of Angpt1 (Ang1), Tek (Tie2), and Kdr (Vegfr2 or Flk-1) genes in the lung, kidney, and liver. These changes correlate with a decrease in RNA polymerase II density at these genes, and the greatest response was observed in the lung. ALI-sepsis reduced levels of transcription-permissive histone H3 lysine acetylation (H3KAc) at these loci in all examined tissues. Decreases in permissive H3K4m3 and H3Km2 marks were detected only in the lung. In contrast, only minimal alterations in transcription-repressive histone modifications (H3K27m3, H3K9m2, H3K9m3, and H4K20m3) were observed in all tissues. CONCLUSIONS: Our results demonstrate that decreases in transcription-permissive, but not increases in transcription-repressive, histone modifications at Angpt1, Tek, and Kdr are a systemic, rather than a lung-restricted, response, involving key end-organs in experimental ALI-sepsis. Given that ventilator-associated pneumonia is a major cause of sepsis in critically ill patients, elucidation of mechanisms mediating epigenetic alterations during sepsis provides fundamental new insights into the pathogenesis of sepsis-induced microvascular leak and subsequent end-organ injury/dysfunction.

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.

Losartan reverses permissive epigenetic changes in renal glomeruli of diabetic db/db mice.

Epigenetic mechanisms such as chromatin histone H3 lysine methylation and acetylation have been implicated in diabetic vascular complications. However, histone modification profiles at pathologic genes associated with diabetic nephropathy in vivo and their regulation by the angiotensin II type 1 receptor (AT1R) are not clear. Here we tested whether treatment of type 2 diabetic db/db mice with the AT1R blocker losartan not only ameliorates diabetic nephropathy, but also reverses epigenetic changes. As expected, the db/db mice had increased blood pressure, mesangial hypertrophy, proteinuria, and glomerular expression of RAGE and PAI-1 vs. control db/+ mice. This was associated with increased RNA polymerase II recruitment and permissive histone marks as well as decreased repressive histone marks at these genes, and altered expression of relevant histone modification enzymes. Increased MCP-1 mRNA levels were not associated with such epigenetic changes, suggesting post-transcriptional regulation. Losartan attenuated key parameters of diabetic nephropathy and gene expression, and reversed some but not all the epigenetic changes in db/db mice. Losartan also attenuated increased H3K9/14Ac at RAGE, PAI-1, and MCP-1 promoters in mesangial cells cultured under diabetic conditions. Our results provide novel information about the chromatin state at key pathologic genes in vivo in diabetic nephropathy mediated in part by AT1R. Thus, combination therapies targeting epigenetic regulators and AT1R could be evaluated for more effective treatment of diabetic nephropathy.

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.

High incidence and geographic distribution of cleft palate cases in Finland are associated with a regulatory variant in IRF6.

In Finland the frequency of isolated cleft palate (CP) is higher than that of isolated cleft lip with or without cleft palate (CL/P). This trend contrasts to that in other European countries but its genetic underpinnings are unknown. We performed a genome-wide association study for orofacial clefts, which include CL/P and CP, in the Finnish population. We identified rs570516915, a single nucleotide polymorphism that is highly enriched in Finns and Estonians, as being strongly associated with CP ( P = 5.25 × 10 -34 , OR = 8.65, 95% CI 6.11-12.25), but not with CL/P ( P = 7.2 × 10 -5 ), with genome-wide significance. The risk allele frequency of rs570516915 parallels the regional variation of CP prevalence in Finland, and the association was replicated in independent cohorts of CP cases from Finland ( P = 8.82 × 10 -28 ) and Estonia ( P = 1.25 × 10 -5 ). The risk allele of rs570516915 disrupts a conserved binding site for the transcription factor IRF6 within a previously characterized enhancer upstream of the IRF6 gene. Through reporter assay experiments we found that the risk allele of rs570516915 diminishes the enhancer activity. Oral epithelial cells derived from CRISPR-Cas9 edited induced pluripotent stem cells demonstrate that the CP-associated allele of rs570516915 concomitantly decreases the binding of IRF6 and the expression level of IRF6 , suggesting impaired IRF6 autoregulation as a molecular mechanism underlying the risk for CP.

Epigenetic changes in renal genes dysregulated in mouse and rat models of type 1 diabetes.

Epigenetic processes are increasingly being recognized as factors in the pathophysiology of diabetes complications, but few chromatin studies have been done in diabetic nephropathy (DN). We hypothesized that changes in mRNA expression of DN-related genes are associated with epigenetic alterations and aberrant expression of histone-modifying enzymes. RT-PCR and a matrix-chromatin immunoprecipitation platform were used to examine renal mRNA expression, RNA polymerase II (Pol II) recruitment, and epigenetic marks at DN-related genes in the mouse (OVE26) and streptozotocin-induced rat models of type 1 diabetes. Diabetes induced renal expression of Cox2, S100A4/FSP-1, and vimentin genes in both the mouse and the rat models of DN. Mcp-1 and laminin γ1 (Lamc1) expression were increased in diabetic mice but not in rats. Comparison of mRNA and Pol II levels suggested that the diabetes-induced expression of these transcripts is mediated by transcriptional and posttranscriptional processes. Decreases in histone H3 lysine 27 tri-methylation (H3K27m3, silencing mark) and increases in H3 lysine 4 di-methylation (H3K4m2, activating mark) levels were the most consistent epigenetic alterations in the tested genes. In agreement with these results, immunoblot analysis showed increased protein abundance of renal H3K27m2/3 demethylase KDM6A, but no changes in cognate methyltransferase Ezh2 in kidneys of the OVE26 mice compared with controls. In diabetic rats, Ezh2 expression was higher without changes in KDM6A, demonstrating that mechanisms of DN-induced H3K27m3 loss could be species specific. In summary, we show that altered mRNA expression of some DN-related genes is associated with changes in Pol II recruitment and a corresponding decrease in repressive H3K27m3 at the selected loci, and at least in mice with equivalent changes in renal expression of cognate histone-modifying enzymes. This pattern could contribute to diabetes-mediated transitions in chromatin that facilitate transcriptional changes in the diabetic kidney.

MultiomicsTracks96: A high throughput PIXUL-Matrix-based toolbox to profile frozen and FFPE tissues multiomes.

Background: The multiome is an integrated assembly of distinct classes of molecules and molecular properties, or "omes," measured in the same biospecimen. Freezing and formalin-fixed paraffin-embedding (FFPE) are two common ways to store tissues, and these practices have generated vast biospecimen repositories. However, these biospecimens have been underutilized for multi-omic analysis due to the low throughput of current analytical technologies that impede large-scale studies. Methods: Tissue sampling, preparation, and downstream analysis were integrated into a 96-well format multi-omics workflow, MultiomicsTracks96. Frozen mouse organs were sampled using the CryoGrid system, and matched FFPE samples were processed using a microtome. The 96-well format sonicator, PIXUL, was adapted to extract DNA, RNA, chromatin, and protein from tissues. The 96-well format analytical platform, Matrix, was used for chromatin immunoprecipitation (ChIP), methylated DNA immunoprecipitation (MeDIP), methylated RNA immunoprecipitation (MeRIP), and RNA reverse transcription (RT) assays followed by qPCR and sequencing. LC-MS/MS was used for protein analysis. The Segway genome segmentation algorithm was used to identify functional genomic regions, and linear regressors based on the multi-omics data were trained to predict protein expression. Results: MultiomicsTracks96 was used to generate 8-dimensional datasets including RNA-seq measurements of mRNA expression; MeRIP-seq measurements of m6A and m5C; ChIP-seq measurements of H3K27Ac, H3K4m3, and Pol II; MeDIP-seq measurements of 5mC; and LC-MS/MS measurements of proteins. We observed high correlation between data from matched frozen and FFPE organs. The Segway genome segmentation algorithm applied to epigenomic profiles (ChIP-seq: H3K27Ac, H3K4m3, Pol II; MeDIP-seq: 5mC) was able to recapitulate and predict organ-specific super-enhancers in both FFPE and frozen samples. Linear regression analysis showed that proteomic expression profiles can be more accurately predicted by the full suite of multi-omics data, compared to using epigenomic, transcriptomic, or epitranscriptomic measurements individually. Conclusions: The MultiomicsTracks96 workflow is well suited for high dimensional multi-omics studies - for instance, multiorgan animal models of disease, drug toxicities, environmental exposure, and aging as well as large-scale clinical investigations involving the use of biospecimens from existing tissue repositories.

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.

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.

Increased apolipoprotein C3 drives cardiovascular risk in type 1 diabetes.

Type 1 diabetes mellitus (T1DM) increases the risk of atherosclerotic cardiovascular disease (CVD) in humans by poorly understood mechanisms. Using mouse models of T1DM-accelerated atherosclerosis, we found that relative insulin deficiency rather than hyperglycemia elevated levels of apolipoprotein C3 (APOC3), an apolipoprotein that prevents clearance of triglyceride-rich lipoproteins (TRLs) and their remnants. We then showed that serum APOC3 levels predict incident CVD events in subjects with T1DM in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. To explore underlying mechanisms, we investigated the impact of Apoc3 antisense oligonucleotides (ASOs) on lipoprotein metabolism and atherosclerosis in a mouse model of T1DM. Apoc3 ASO treatment abolished the increased hepatic Apoc3 expression in diabetic mice - resulting in lower levels of TRLs - without improving glycemic control. APOC3 suppression also prevented arterial accumulation of APOC3-containing lipoprotein particles, macrophage foam cell formation, and the accelerated atherosclerosis in diabetic mice. Our observations demonstrate that relative insulin deficiency increases APOC3 and that this results in elevated levels of TRLs and accelerated atherosclerosis in a mouse model of T1DM. Because serum levels of APOC3 predicted incident CVD events in the CACTI study, inhibiting APOC3 might reduce CVD risk in T1DM patients.

Chromatin changes trigger laminin genes dysregulation in aging kidneys.

Dysregulation of gene expression is a hallmark of aging. We examined epigenetic mechanisms that mediate aberrant expression of laminin genes in aging rat kidneys. In old animals, no alterations were found in the levels of abundant laminin mRNAs, whereas Lama3, b3, and c2 transcripts were increased compared to young animals. Lamc2 showed the strongest changes at the mRNA and protein levels. Lamc2 upregulation was transcriptional, as indicated by the elevated RNA polymerase II density at the gene. Furthermore, aging is associated with the loss of H3K27m3 and 5mC silencing modifications at the Lamc2 gene. Western blot analysis revealed no changes in cellular levels of H3K27m3 and cognate enzyme Ezh2 in old kidneys. Thus, the decrease in H3K27m3 at Lamc2 resulted from the re-distribution of this mark among genomic sites. Studies in kidney cells in vitro showed that reducing H3K27m3 density with Ezh2 inhibitor had no effect on Lamc2 expression, suggesting that this modification plays little role in gene upregulation in aging kidney. In contrast, treatment with DNA methylation inhibitor 2'-deoxy-5-azacytidine was sufficient to upregulate Lamc2 gene. We suggest that the loss of 5mC at silenced laminin genes drives their de-repression during aging, contributing to the age-related decline in renal function.

SYSTEM-WIDE MAPPING OF ACTIVATED CIRCUITRY IN EXPERIMENTAL SYSTEMIC INFLAMMATORY RESPONSE SYNDROME.

Sepsis-induced multiple organ dysfunction syndrome (MODS) is a major cause of morbidity and mortality in critically ill patients and remains impervious to most therapeutic interventions. We utilized a clinically relevant murine model of systemic inflammatory response syndrome (SIRS) during early MODS induced by ventilator-associated pneumonia to systematically delineate pathways dysregulated in lung, liver, and kidney. We focused on processes commonly activated across at-risk organs and constructed an SIRS-associated network based on connectivity among the gene members of these functionally coherent pathways. Our analyses led to the identification of several putative drivers of early MODS whose expression was regulated by epidermal growth factor receptor. Our unbiased, integrative method is a promising approach to unravel mechanisms in system-wide disorders afflicting multiple compartments such as sepsis-induced MODS, and identify putative therapeutic targets.

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.