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.
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.
•Maternal malnutrition downregulates rDNA transcription in fetal tissues.•Switch to normal diet permanently upregulates rDNA transcription compared to controls.•These changes are mediated by DNA methylation and Pol I transcription factor Rrn3.•This mechanism is activated before implantation.
rDNA transcription; Maternal diet; DNA methylation; RRN3; TIF-1A; Thrifty gene
Aberrant gene expression is a molecular hallmark of acute kidney injury (AKI). Since 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 twenty 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, but also had major ischemia/reperfusion vs. 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.
acute kidney injury; gene expression; ischemia reperfusion; sepsis
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.
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.
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.
Matrix-IP is time-saving, easy to use high throughput method adaptable for low sample amounts and automation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12953-015-0079-0) contains supplementary material, which is available to authorized users.
hnRNP K; Immuoprecipitation; Interactome; Post-translational modifications; Quantitative proteomics
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.
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.
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).
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.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s13054-015-0943-4) contains supplementary material, which is available to authorized users.
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 versus 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.
Diabetic Nephropathy; Angiotensin II; Losartan; epigenetics; gene regulation
Acute kidney injury (AKI) is a risk factor for chronic kidney disease and death. Despite progress made in understanding cellular and molecular basis of AKI pathogenesis there has been no improvement in the high mortality from this disease in decades. Epigenetics is one of the most intensively studied filed of biology today and represents a new paradigm for understanding the pathophysiology of disease. Although epigenetics of AKI is a nascent field the available information is already providing compelling evidence that chromatin biology plays a critical role in this disease. In this review article we explore what is known about contribution of epigenetic mechanisms to pathophysiology of AKI and how this knowledge is already guiding the development of new diagnostic tools and epigenetic therapies.
epigenetics; acute kidney injury; chromatin; histone modification; DNA methylation
Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy. A strategy to mediate such targeted integration involves site specific endonucleases. Two genomic sites within the MBS85 and CCR5 genes [AAVS1 and CCR5 zinc finger nuclease (CCR5-ZFN) site, respectively] have recently been suggested as potential target regions for integration as their disruption has no functional consequence. We hypothesized that efficient transgene integration maybe affected by DNA accessibility of endonucleases and therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent stem (iPS) cell lines and pooled CD34+ hematopoietic stem cells. Matrixchromatin immunoprecipitation (ChIP) assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration consistent with its transcriptionally inactivity in these cell types. In contrast, the AAVS1 site was located within a transcriptionally active region and exhibited an open chromatin configuration in both iPS cells and hematopoietic stem cells. To show that the AAVS1 site is readily amendable to genome modification, we expressed Rep78, an AAV2-derived protein with AAVS1-specific endonuclease activity, in iPS cells after adenoviral gene transfer. We showed that Rep78 efficiently associated with the AAVS1 site and triggered genome modifications within this site. On the other hand, binding to and modification of the CCR5-ZFN site by a zinc-finger nuclease was relatively inefficient. Our data suggest a critical influence of chromatin structure on efficacy of site-specific endonucleases used for genome editing.
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.
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.
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.
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/β-catenin pathway has been implicated in facilitating melanoma cell apoptosis, we investigated the effect of Wnt/β-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/β-catenin signaling pathway by demonstrating that siRNA-mediated knockdown of an intracellular β-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/β-catenin signaling, and suggest that strategies to enhance Wnt/β-catenin signaling in combination with TRAIL agonists warrant further investigation.
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.
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.
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.
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.
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.
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.
Random monoallelic expression contributes to phenotypic variation of cells and organisms. However, the epigenetic mechanisms by which individual alleles are randomly selected for expression are not known. Taking cues from chromatin signatures at imprinted gene loci such as the insulin-like growth factor 2 gene 2 (IGF2), we evaluated the contribution of CTCF, a zinc finger protein required for parent-of-origin-specific expression of the IGF2 gene, as well as a role for allele-specific association with DNA methylation, histone modification and RNA polymerase II.
Using array-based chromatin immunoprecipitation, we identified 293 genomic loci that are associated with both CTCF and histone H3 trimethylated at lysine 9 (H3K9me3). A comparison of their genomic positions with those of previously published monoallelically expressed genes revealed no significant overlap between allele-specifically expressed genes and colocalized CTCF/H3K9me3. To analyze the contributions of CTCF and H3K9me3 to gene regulation in more detail, we focused on the monoallelically expressed IGF2BP1 gene. In vitro binding assays using the CTCF target motif at the IGF2BP1 gene, as well as allele-specific analysis of cytosine methylation and CTCF binding, revealed that CTCF does not regulate mono- or biallelic IGF2BP1 expression. Surprisingly, we found that RNA polymerase II is detected on both the maternal and paternal alleles in B lymphoblasts that express IGF2BP1 primarily from one allele. Thus, allele-specific control of RNA polymerase II elongation regulates the allelic bias of IGF2BP1 gene expression.
Colocalization of CTCF and H3K9me3 does not represent a reliable chromatin signature indicative of monoallelic expression. Moreover, association of individual alleles with both active (H3K4me3) and silent (H3K27me3) chromatin modifications (allelic bivalent chromatin) or with RNA polymerase II also fails to identify monoallelically expressed gene loci. The selection of individual alleles for expression occurs in part during transcription elongation.
Aberrant activation of protein kinases is one of the essential oncogenic driving forces inherent to the process of tumorigenesis. The protein kinase CK2 plays an important role in diverse biological processes, including cell growth and proliferation as well as in the governing and transduction of prosurvival signals. Increased expression of CK2 is a hallmark of some cancers, hence its antiapoptotic properties may be relevant to cancer onset. Thus, the designing and synthesis of the CK2 inhibitors has become an important pursuit in the search for cancer therapies.
Using a high-throughput microarray approach, we demonstrate that two potent inhibitors of CK2, 4,5,6,7-tetrabromo-benzimidazole (TBBz) and 2-Dimethyloamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), blocked mitogen induced mRNA expression of immediate early genes. Given the impact of these inhibitors on the process of transcription, we investigated their effects on RNA Polymerase II (RNAPII) elongation along the mitogen inducible gene, EGR1 (early growth response 1), using chromatin immunoprecipitation (ChIP) assay. ChIP analysis demonstrated that both drugs arrest RNAPII elongation. Finally, we show that CDK9 kinase activity, essential for the triggering of RNAPII elongation, was blocked by TBBz and to lesser degree by DMAT.
Our approach revealed that small molecules derived from halogenated imidazole compounds may decrease cell proliferation, in part, by inhibiting pathways that regulate transcription elongation.
In recent years, experimental studies of acute renal failure (ARF) have been most widely conducted in young mice that are in a rapid growth phase. However, the impact of early growth and development on renal susceptibility to ARF has not been defined. The present study has begun to address this issue by testing the severity of endotoxemic-, glycerol-, and maleate-induced ARF in male CD-1 mice ranging from 3-16 weeks of age (17-45 gm). The potential for age to alter the expression of at least one injury modifier, cellular cholesterol, was also assessed. The severity of each ARF model (BUN / histology) directly correlated with mouse wt. and age (p <0.001). Progressive age- dependent reductions in renal cholesterol content were also observed. The latter were paralleled by stepwise decrements in mRNA levels of two key cholesterol homeostatic genes (HMG CoA reductase, HMGCR, and low density lipoprotein receptor, LDL-R). The mRNA reductions were paralleled by falling RNA polymerase II and transcription factor (SREBP 1 / 2) densities at the HMGCR and LDL-R genes. Hence, we conclude that: 1) the early phase of mouse growth can profoundly alter renal susceptibility to diverse forms of ARF; 2) these changes can reflect fundamental fluxes in select, and likely protean, biochemical and molecular changes at the whole tissue and genomic levels (cholesterol being one such example); 3) ChIP is a powerful tool for studying such changes; and 4) the early growth period needs to be carefully controlled for when conducting studies of experimental ARF.
Hyperglycemia may potentiate the adverse renal effects of angiotensin II (AII). In the kidney, the major target of AII action is the glomerular mesangial cell, where its hemodynamic and proinflammatory action contributes to renal injury. AII action is mediated by several types of cell receptors. Among those, the AT1 receptor has been best studied using specific AII receptor blockers (ARBs). These agents have emerged as major new modalities in the prevention and amelioration of renal disease where the ARB renoprotective anti-inflammatory properties could be more important than previously appreciated. Like the ARBs, statins may also modulate inflammatory responses that are renoprotective and complement their cholesterol-lowering effects.
The aim of this project was to (i) identify a repertoire of proinflammatory mesangial cell AII-inducible mRNAs; (ii) determine if the AII-induced proinflammatory mRNA responses depend on ambient glucose, and (iii) test the anti-inflammatory effectiveness of an ARB, valsartan, either alone or in combination with a statin, simvastatin.
Using high-density microarrays and real-time PCR we identified several AII-inducible proinflammatory mesangial genes that exhibited augmented mRNA responses in high-glucose milieu. Valsartan blocked the AII-induced mRNA expression of proinflammatory genes (i.e. MCP-1, LIF and COX-2) maintained in normal and high glucose. These observations add to the mounting evidence that ARBs have anti-inflammatory effects in the kidney, a beneficial effect that may be more important in protecting renal function in diabetic patients. While simvastatin inhibited expression of some mRNAs encoding chemokines/cytokines, it enhanced expression of mRNA encoding COX-2, a key mediator of inflammation. Thus, the non-cholesterol effects of statins on inflammatory responses appear complex.
Angiotensin II; Hyperglycemia; Glomerular mesangial cell; Proinflammatory action
In budding yeast, telomeres, the ribosomal DNA array, and HM loci are transcriptionally silenced by chromatin complexes containing Sir proteins. Hek2, a protein containing three evolutionary conserved RNA-binding K-homology domains, was identified as a suppressor of telomeric silencing [telomeric position effect (TPE)]. To explore the mechanisms of Hek2p action in gene silencing, we examined its relationship with Sir proteins. This search revealed an epistatic interaction between HEK2 and SIR1 at telomeres. Both single mutations, sir1Δ and hek2Δ, enhanced TPE, whereas the effect of double mutation, sir1Δ hek2Δ, did not exceed that of the single mutations. The results of chromatin immunoprecipitation analysis demonstrate that the TPE enhancement observed in these mutants is associated with increased binding of Sir2 protein to telomeres. At the HMR locus, hek2Δ rescues the silencing defect caused by sir1Δ mutation and reverses the loss of Sir2p and Sir3p. These data suggest that the epistatic interaction of HEK2 and SIR1 reflects competition between telomeres and HMR for Sir2/3 factors where HEK2 acts to suppress silencing. Because chromatin immunoprecipitation analysis reveals the presence of Hek2p at a subtelomeric region and HMR, its silencing effects at these loci are likely direct. These observations suggest that HEK2 regulates the composition of Sir complexes at HMR and telomeres.
Hek2; Gene silencing; Sir proteins; Telomeres; HMR
The chromatin immunoprecipitation (ChIP) assay is a major tool in the study of genomic processes in vivo. This and other methods are revealing that control of gene expression, cell division and DNA repair involves multiple proteins and great number of their modifications. ChIP assay is traditionally done in test tubes limiting the ability to study signaling of the complex genomic events. To increase the throughput and to simplify the assay we have developed a microplate-based ChIP (Matrix ChIP) method, where all steps from immunoprecipitation to DNA purification are done in microplate wells without sample transfers. This platform has several important advantages over the tube-based assay including very simple sample handling, high throughput, improved sensitivity and reproducibility, and potential for automation. 96 ChIP measurements including PCR can be done by one researcher in one day. We illustrate the power of Matrix ChIP by parallel profiling 80 different chromatin and transcription time-course events along an inducible gene including transient recruitment of kinases.
Chromatin immunoprecipitation (ChIP) is a widely used method to explore in vivo interactions between proteins and DNA. The ChIP assay takes several days to complete, involves several tube transfers and uses either phenol–chlorophorm or spin columns to purify DNA. The traditional ChIP method becomes a challenge when handling multiple samples. We have developed an efficient and rapid Chelex resin-based ChIP procedure that dramatically reduces time of the assay and uses only a single tube to isolate PCR-ready DNA. This method greatly facilitates the probing of chromatin changes over many time points with several antibodies in one experiment.
The heterogeneous nuclear ribonucleoprotein K protein is an RNA- and DNA-binding protein implicated in the regulation of multiple processes that comprise gene expression. We used chromatin immunoprecipitation (ChIP) assays to explore K protein interactions with serum-inducible, constitutively expressed and untranscribed gene loci in vivo. In the rat HTC-IR hepatoma cell line, serum treatment induced transient increases in the mRNA levels of two immediate-early genes, egr-1 and c-myc. ChIP analysis showed that the induction of egr-1 and c-myc genes was associated with a transient recruitment of K protein to multiple sites within each of these loci, including the promoter and transcribed regions. In contrast, recruitment of K protein to the constitutively transcribed β-actin locus and to randomly chosen non-transcribed loci was far weaker. In rat mesangial cells, c-myc was constitutively expressed while egr-1 remained serum responsive. In these cells, ChIP analysis showed serum-induced recruitment to the inducible egr-1 but not to the c-myc locus. Pre-treatment with the transcription inhibitor actinomycin D blocked the inducible but not the constitutive binding of K protein to these loci. Taken together, the results of this study suggest that the transient recruitment of K protein to serum-responsive loci depends on the inducible transcription of these immediate-early genes.
hnRNPK and hnRNP E1/E2 mediate translational silencing of cellular and viral mRNAs in a differentiation-dependent way by binding to specific regulatory sequences. The translation of 15-lipoxygenase (LOX) mRNA in erythroid precursor cells and of the L2 mRNA of human papilloma virus type 16 (HPV-16) in squamous epithelial cells is silenced when either of these cells is immature and is activated in maturing cells by unknown mechanisms. Here we address the question of how the silenced mRNA can be translationally activated. We show that hnRNP K and the c-Src kinase specifically interact with each other, leading to c-Src activation and tyrosine phosphorylation of hnRNP K in vivo and in vitro. c-Src-mediated phosphorylation reversibly inhibits the binding of hnRNP K to the differentiation control element (DICE) of the LOX mRNA 3′ untranslated region in vitro and specifically derepresses the translation of DICE-bearing mRNAs in vivo. Our results establish a novel role of c-Src kinase in translational gene regulation and reveal a mechanism by which silenced mRNAs can be translationally activated.
Laminin is a multifunctional heterotrimeric protein present in extracellular matrix where it regulates processes that compose tissue architecture including cell differentiation. Laminin γ1 is the most widely expressed laminin chain and its absence causes early lethality in mouse embryos. Laminin γ1 chain gene (LAMC1) promoter contains several GC/GT-rich motifs including the bcn-1 element. Using the bcn-1 element as a bait in the yeast one-hybrid screen, we cloned the gut-enriched Kruppel-like factor (GKLF or KLF4) from a rat mesangial cell library. We show that GKLF binds bcn-1, but this binding is not required for the GKLF-mediated activation of the LAMC1 promoter. The activity of GKLF is dependent on a synergism with another Kruppel-like factor, Sp1. The LAMC1 promoter appears to have multiple GKLF- and Sp1-responsive elements which may account for the synergistic activation. We provide evidence that the synergistic action of GKLF and Sp1 is dependent on the promoter context and the integrity of GKLF activation and DNA-binding domain. GKLF is thought to participate in the switch from cell proliferation to differentiation. Thus, the Sp1–GKLF synergistic activation of the LAMC1 promoter may be one of the avenues for expression of laminin γ1 chain when laminin is needed to regulate cell differentiation.
Mammalian heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA- and DNA-binding protein implicated in the regulation of gene expression processes. To better understand its function, we studied two Saccharomyces cerevisiae homologues of the human hnRNP K, PBP2 and HEK2 (heterogeneous nuclear RNP K-like gene). pbp2Δ and hek2Δ mutations inhibited expression of a marker gene that was inserted near telomere but not at internal chromosomal locations. The telomere proximal to the ectopic marker gene became longer, while most of the other telomeres were not altered in the double mutant cells. We provide evidence that telomere elongation might be the primary event that causes enhanced silencing of an adjacent reporter gene. The telomere lengthening could, in part, be explained by the inhibitory effect of hek2Δ mutation on the telomeric rapid deletion pathway. Hek2p was detected in a complex with chromosome regions proximal to the affected telomere, suggesting a direct involvement of this protein in telomere maintenance. These results identify a role for hnRNP K-like genes in the structural and functional organization of telomeric chromatin in yeast.