AP-1/C-FOS and AP-1/FRA2 differentially regulate early and late adipogenic differentiation of mesenchymal stem cells.

Obesity is defined as an abnormal accumulation of adipose tissue in the body and is a major global health problem due to increased morbidity and mortality. Adipose tissue is made up of adipocytes, which are fat-storing cells, and the differentiation of these fat cells is known as adipogenesis. Several transcription factors (TFs) such as CEBPß, CEBPα, PPARγ, GATA, and KLF have been reported to play a key role in adipogenesis. In this study, we report one more TF AP-1, which is found to be involved in adipogenesis. Human mesenchymal stem cells  were differentiated into adipocytes, and the expression pattern of different subunits of AP-1 was examined during adipogenesis. It was observed that C-FOS was predominantly expressed at an early stage (Day 2), whereas FRA2 expression peaked at later stages (Days 6 and 8) of adipogenesis. Chromatin immunoprecipitation-sequencing analysis revealed that C-FOS binds mainly to the promoters of WNT1, miR-30a, and ANAPC7 and regulates their expression during mitotic clonal expansion. In contrast, FRA2 binds to the promoters of CIDEA, NOTCH1, ARAF, and MYLK, regulating their expression and lipid metabolism. Data obtained clearly indicate that the differential expression of C-FOS and FRA2 is crucial for different stages of adipogenesis. This also raises the possibility of considering AP-1 as a therapeutic target for treating obesity and related disorders.

Glutaredoxin 1 from Evolutionary Ancient Hydra: Characteristics of the Enzyme and Its Possible Functions in Cell.

Glutaredoxin (Grx) is an antioxidant redox protein that uses glutathione (GSH) as an electron donor. Grx plays a crucial role in various cellular processes, such as antioxidant defense, control of cellular redox state, redox control of transcription, reversible S-glutathionylation of specific proteins, apoptosis, cell differentiation, etc. In the current study, we have isolated and characterized dithiol glutaredoxin from Hydra vulgaris Ind-Pune (HvGrx1). Sequence analysis showed that HvGrx1 belongs to the Grx family with the classical Grx motif (CPYC). Phylogenetic analysis and homology modeling revealed that HvGrx1 is closely related to Grx2 from zebrafish. HvGrx1 gene was cloned and expressed in Escherichia coli cells; the purified protein had a molecular weight of 11.82 kDa. HvGrx1 efficiently reduced ß-hydroxyethyl disulfide (HED) with the temperature optimum of 25°C and pH optimum 8.0. HvGrx1 was ubiquitously expressed in all body parts of Hydra. Expression of HvGrx1 mRNA and enzymatic activity of HvGrx1 were significantly upregulated post H2O2 treatment. When expressed in human cells, HvGrx1 protected the cells from oxidative stress and enhanced cell proliferation and migration. Although Hydra is a simple invertebrate, HvGrx1 is evolutionary closer to its homologs from higher vertebrates (similar to many other Hydra proteins).

A novel thioredoxin glutathione reductase from evolutionary ancient metazoan Hydra.

Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.

Mapping of scaffold/matrix attachment regions in human genome: a data mining exercise.

Scaffold/matrix attachment regions (S/MARs) are DNA elements that serve to compartmentalize the chromatin into structural and functional domains. These elements are involved in control of gene expression which governs the phenotype and also plays role in disease biology. Therefore, genome-wide understanding of these elements holds great therapeutic promise. Several attempts have been made toward identification of S/MARs in genomes of various organisms including human. However, a comprehensive genome-wide map of human S/MARs is yet not available. Toward this objective, ChIP-Seq data of 14 S/MAR binding proteins were analyzed and the binding site coordinates of these proteins were used to prepare a non-redundant S/MAR dataset of human genome. Along with co-ordinate (location) details of S/MARs, the dataset also revealed details of S/MAR features, namely, length, inter-SMAR length (the chromatin loop size), nucleotide repeats, motif abundance, chromosomal distribution and genomic context. S/MARs identified in present study and their subsequent analysis also suggests that these elements act as hotspots for integration of retroviruses. Therefore, these data will help toward better understanding of genome functioning and designing effective anti-viral therapeutics. In order to facilitate user friendly browsing and retrieval of the data obtained in present study, a web interface, MARome (http://bioinfo.net.in/MARome), has been developed.

Andrographolide protects liver cells from H2O2 induced cell death by upregulation of Nrf-2/HO-1 mediated via adenosine A2a receptor signalling.

BACKGROUND: Andrographolide, principle constituent of Andrographis paniculata Nees is used in traditional medicine in Southeast Asia and is known to exhibit various biological activities. Its antioxidant activity is due to its ability to activate one of the antioxidant enzymes, heme oxygenase-1 (HO-1) which is regulated transcriptionally through Nrf-2. However, molecular mechanism underlying activation of Nrf-2/HO-1 has not yet been clearly understood. METHODS: Protective effect of andrographolide against H2O2 induced cell death, reactive oxygen species and lipid peroxidation was observed in HepG2 cells. Ability of andrographolide to modulate G-protein coupled receptor (GPCR) mediated signalling was determined using in silico docking and gene expression was analyzed by qRT-PCR, confocal microscopy and western blot analysis. RESULTS: We clearly show that andrographolide via adenosine A2A receptor signalling leads to activation of p38 MAP kinase, resulting in upregulation of Nrf-2, its translocation to nucleus and activation of HO-1. Additionally, it activates adenylate cyclase resulting in cAMP formation which in turn activates protein kinase A leading to inhibition of GSK-3ß by phosphorylation. Inactivated GSK-3ß leads to retention of Nrf-2 in the nucleus leading to sustained expression of HO-1 by binding to its antioxidant response element (ARE). CONCLUSIONS: Thus, andrographolide probably by binding to adenosine A2a receptor activates Nrf-2 transcription and also inhibits its exclusion from the nucleus by inactivating GSK-3ß, together resulting in activation of HO-1. GENERAL SIGNIFICANCE: We speculate that andrographolide can be used as a therapeutic drug to combat oxidative stress implicated in pathogenesis of various diseases such as diabetes, osteoporosis, neurodegenerative diseases etc.

Protective effect of geraniin against carbon tetrachloride induced acute hepatotoxicity in Swiss albino mice.

Geraniin is a hydrolysable tannin, widely present in many plant species, specifically used in traditional medicines. It has been shown to exhibit strong antioxidant activity in vitro. This study was performed to investigate hepatoprotective activity of geraniin against carbon tetrachloride (CCl4) induced damage in Swiss albino mice. Mice were treated with 30 and 60 mg/kg geraniin for 10 days followed by CCl4 administration for 24 h. Increase in Serum biochemical marker enzymes and histological deteriorative changes of liver tissue after CCl4 administration were attenuated by geraniin. Geraniin significantly reduced CCl4 induced lipid peroxidation, increase in amount of glutathione, glutathione reductase and Heme oxygenase-1 (HO-1). On the other hand it inhibited significant reduction in catalase activity and expression caused by CCl4 administration. Pre-treatment with geraniin reduced phosphorylation of translation initiation factor eIF2α, at serine 51, caused by CCl4 exposure and reduced elevated expression of its upstream kinase, Heme-regulated Inhibitor (HRI). These results clearly demonstrate hepatoprotective activity of geraniin against CCl4-induced acute hepatotoxicity via its free radical scavenging and antioxidant activities.

Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element.

How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti-apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1-PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage.

Identification and characterization of multidomain monothiol glutaredoxin 3 from diploblastic Hydra.

Intracellular antioxidant glutaredoxin controls cell proliferation and survival. Based on the active site, structure, and conserved domain motifs, it is classified into two classes. Class I contains dithiol Grxs with two cysteines in the consensus active site sequence CXXC, while class II has monothiol Grxs with one cysteine residue in the active site. Monothiol Grxs can also have an additional N-terminal thioredoxin (Trx)-like domain. Previously, we reported the characterization of Grx1 from Hydra vulgaris (HvGrx1), which is a dithiol isoform. Here, we report the molecular cloning, expression, analysis, and characterization of another isoform of Grx, which is the multidomain monothiol glutaredoxin-3 from Hydra vulgaris (HvGrx3). It encodes a protein with 303 amino acids and is significantly larger and more divergent than HvGrx1. In-silico analysis revealed that Grx1 and Grx3 have 22.5% and 9.9% identical nucleotide and amino acid sequences, respectively. HvGrx3 has two glutaredoxin domains and a thioredoxin-like domain at its amino terminus, unlike HvGrx1, which has a single glutaredoxin domain. Like other monothiol glutaredoxins, HvGrx3 failed to reduce glutathione-hydroxyethyl disulfide. In the whole Hydra, HvGrx3 was found to be expressed all over the body column, and treatment with H2O2 led to a significant upregulation of HvGrx3. When transfected in HCT116 (human colon cancer cells) cells, HvGrx3 enhanced cell proliferation and migration, indicating that this isoform could be involved in these cellular functions. These transfected cells also tolerate oxidative stress better.

SMAR1 and p53-regulated lncRNA RP11-431M3.1 enhances HIF1A translation via miR-138 in colorectal cancer cells under oxidative stress.

Eukaryotic cells respond to stress by altering coding and non-coding gene expression programs. Alongside many approaches and regulatory mechanisms, long non-coding RNAs (lncRNA) are finding a significant place in gene regulation, suggesting an involvement in various cellular processes and pathophysiology. LncRNAs are regulated by many transcription factors, including SMAR1 and p53, which are tumor suppressor genes. SMAR1 inhibits cancer cell metastasis and invasion and is also known to inhibit apoptosis during low-dose stress in coordination with p53. Data mining analysis suggested that these tumor suppressor genes might coregulate the lncRNA RP11-431M3.1 in colon cancer cells. Importantly, RP11-431M3.1 expression was found to be negatively correlated with patient survival rates in a number of cancers. Oxidative stress occurs when an imbalance in the body is caused by reactive oxygen species (ROS). This imbalance is known to be important in the development/pathogenesis of colon cancer. We are researching the role and control of this lncRNA in HCT116 cells under conditions of oxidative stress. We observed a dose-dependent differential expression of lncRNA upon H2O2 treatment and found that p53 and SMAR1 bind differentially to the promoter in response to the dose of stress inducer used. RP11-431M3.1 was observed to sponge miR-138 which has an important target gene, hypoxia-inducible factor (HIF1A). miR-138 was observed to bind differentially to RP11-431M3.1 and HIF1A RNA depending on the dose of oxidative stress. Furthermore, the knockdown of RP11-431M3.1 decreased the migration and proliferation of colon cancer cells. Our results suggest a previously undescribed regulatory mechanism through which RP11-431M3.1 is transcriptionally regulated by SMAR1 and p53, target HIF1A through miR-138, and highlight its potential as a therapeutic and diagnostic marker for cancer.

Andrographolide and pterostilbene inhibit adipocyte differentiation by downregulating PPARγ through different regulators.

Adipogenesis involves commitment of stem cells and their differentiation into mature adipocytes. It is tightly regulated by hormones, nutrients and adipokines. Many natural compounds are being tested for their anti-adipogenic activity which can be attributed to apoptosis induction in adipocytes, blocking adipocyte differentiation, or inhibiting intracellular triglyceride synthesis and accumulation. In this study, we have determined molecular mechanism of two phytocompounds: andrographolide (AN) and pterostilbene (PT) during differentiation of the human MSCs into adipocyte. Interestingly, AN upregulates miR27a, whereas, PT upregulated SIRT1 which inhibits the expression of PPARγ. Thus, our results clearly demonstrate that both AN and PT inhibited adipogenesis by blocking a surge of reactive oxygen species (ROS) during differentiation and inhibiting expression of crucial transcription factors like SREBP1c and PPARγ.

Characterization and Biological Studies of the Terpenoids from Ganoderma resinaceum and Serpula similis (Agaricomycetes).

Mycochemical properties and bioactivities of Ganoderma resinaceum and Serpula similis remain unexplored. The present study assessed antioxidant, cytotoxicity, and cell migration abilities of Ganoderma and Serpula extracts, followed by their phytochemical analyses. The MTT assay was conducted to determine the cytotoxicity along with the cell migration studies in human cancer cell lines. The antioxidant profiles were evaluated through DPPH and FRAP assays. Furthermore, LC-MS/MS analysis was performed to elucidate the phytochemicals responsible for anticancer and antioxidant activities. Significant concentration-dependent cytotoxicities of 12.7% and 13.7% were observed against HCT 116 cell lines at 1% and 5% concentrations of the G. resinaceum extract, respectively. Similarly, significant concentration-dependent cytotoxicities of 6.7% and 25.5% were observed at 1% and 5% concentrations of the S. similis extract, respectively. The extracts of G. resinaceum and S. similis both shows better anti-migration potential in lung cancer cells. Both extracts demonstrated good scavenging activity on DPPH and ferric ion free radicals. LC-MS analysis revealed 11 compounds from S. similis and 15 compounds from G. resinaceum fruiting bodies. Compounds such as terpenoids, alkaloids, cytotoxic peptides, and other metabolites were identified as major components in both extracts. These extracts exhibited cytotoxic activity against HCT 116 cancer cells, along with moderate antioxidant activity. This implies that the extracts might be used as bioactive natural sources in the pharmaceutical and food industries.

Cloning and characterization of Thioredoxin 1 from the Cnidarian Hydra.

Thioredoxins, small disulphide-containing redox proteins, play an important role in the regulation of cellular thiol redox balance through their disulfide reductase activity. In this study, we have identified, cloned, purified and characterized thioredoxin 1 (HvTrx1) from the Cnidarian Hydra vulgaris Ind-Pune. Bioinformatics analysis revealed that HvTrx1 contains an evolutionarily conserved catalytic active site Cys-Gly-Pro-Cys and shows a closer phylogenetic relationship with vertebrate Trx1. Optimum pH and temperature for enzyme activity of purified HvTrx1 was found to be pH 7.0 and 25°C, respectively. Enzyme activity decreased significantly at acidic or alkaline pH as well as at higher temperatures. HvTrx1 was found to be expressed ubiquitously in whole mount in situ hybridization. Treatment of Hydra with hydrogen peroxide (H2O2), a highly reactive oxidizing agent, led to a significant increase in gene expression and enzyme activity of Trx1. Further experiments using PX12, an inhibitor of Trx1, indicated that Trx1 plays an important role in regeneration in Hydra. Finally, by using growth assay in Escherichia coli and wound healing assay in human colon cancer cells, we demonstrate that HvTrx1 is functionally active in both prokaryotic and eukaryotic heterologous systems.

miR-3666 inhibits development of hepatic steatosis by negatively regulating PPARγ.

AIMS: PPARγ is a crucial transcription factor involved in development of hepatic steatosis, an early stage of NAFLD. PPARγ is tightly regulated through various positive and negative regulators including miRNAs. In this study, we report for the first time miR-3666 as a negative regulator of PPARγ and its involvement in development of hepatic steatosis. METHODS: Binding of miR-3666 to regulate PPARγ was checked by luciferase assay and was confirmed by mutating PPARγ 3'UTR. Regulation of PPARγ was determined by overexpression of miR-3666 in HepG2 cells. Hepatic steatotic state in HepG2 cells was developed by exposure to excess palmitic acid and expression of PPARγ, miR-3666 and some PPARγ target and non-target genes was checked. Involvement of mir-3666 by regulating PPARγ in hepatic steatosis was also examined in liver of HFD fed mice. RESULTS: On overexpression of miR-3666, PPARγ expression decreased significantly in a dose-dependent manner in HepG2 cells. Binding of miR-3666 to PPARγ was confirmed as the luciferase activity using pMIR-REPORT with PPARγ 3'UTR decreased in PA treated HepG2 cells overexpressing miR-3666 and remained unchanged when PPARγ 3'UTR was mutated. In PA treated HepG2 cells during development of hepatic steatosis PPARγ was significantly up-regulated concomitant with down-regulation of miR-3666. Overexpression of miR-3666 in these cells decreased the extent of hepatic steatosis. Significant up-regulation of PPARγ and down-regulation of miR-3666 was also observed in liver of HFD fed mice indicating that miR-3666 regulates PPARγ in vivo. CONCLUSIONS: miR-3666 negatively regulates PPARγ by binding to its 3'UTR during development of hepatic steatosis.

Andrographolide enhances redox status of liver cells by regulating microRNA expression.

Andrographis paniculata Nees and its principal compound andrographolide are well known for exerting beneficial effects by modulating signaling pathways in different biological systems. Our earlier studies have demonstrated the ability of andrographolide as well as andrographolide enriched extracts to activate Nrf2/HO-1 pathway through adenosine A2a receptor. Present study investigated ability of andrographolide to regulate Nrf2 induced antioxidant defense systems by miRNAs using HepG2 cells. Andrographolide strongly induced Nrf2 which in turn modulated enzymes of glutathione and thioredoxin antioxidant systems. It also regulated crucial transcription factors viz. hepatocyte nuclear factor alpha (HNF4A) and tumor suppressor protein 53 (p53). Downregulation of HNF4A by andrographolide led to decrease in miRNAs regulating Heme oxygenase-1 (miR-377) and glutathione cysteine ligase (miR-433). Upregulation of p53 on the other hand led to increase in miRNAs regulating thioredoxin interacting protein (miR-17, miR-224) and glutathione peroxidase (miR-181a). Involvement of p53 and HNF4A in modulation of these miRNAs was confirmed by chromatin immunoprecipitation assay. Overall, the work reveals that andrographolide through modulation of p53 and HNF4A, regulates miRNAs leading to upregulation of HO-1, glutathione and thioredoxin systems. Andrographolide thus, can play a beneficial role in modulating antioxidant defense in oxidative stress induced diseases such as diabetes, ageing etc.

Cytoprotective effect exerted by geraniin in HepG2 cells is through microRNA mediated regulation of BACH-1 and HO-1.

Geraniin, a hydrolysable tannin, used in traditional medicine in Southeast Asia, is known to exhibit various biological activities. As an antioxidant it is known to up-regulate phase II enzyme Heme oxygenase-1 (HO-1). However its mechanism is not clearly understood. Nuclear factor erythroid-derived 2 related factor 2 (Nrf-2) is transcriptionally up-regulated by Extracellular signal-regulated kinase (ERK) 1/2 and retained in nucleus due to inactivated Glycogen synthase kinase 3 beta (GSK-3ß). Geraniin additionally down-regulates expression of microRNA 217 and 377 (miR-217 and miR-377) which target HO-1 mRNA. Expression of BTB and CNC homolog 1 (BACH-1), another regulator of HO-1, is also down-regulated by up-regulating microRNA 98 (miR-98), a negative regulator of BACH-1. Thus, geraniin up-regulates HO-1 expression both through activating its positive regulator Nrf-2 and by down-regulating its negative regulator BACH-1. Up-regulation of HO-1 also confers protection to HepG2 cells from tertiary butyl hydroperoxide (TBH) induced cytotoxicity. [BMB Reports 2017; 50(11): 560-565].

Sequence data mining in search of hookworm (Necator americanus) microRNAs.

The new world hookworm, Necator americanus is a soil-transmitted nematode responsible for Necatoriasis (a type of helminthiasis) in hosts such as humans, dogs, and cats. N. americanus genome and transcriptome has been sequenced and a draft assembly analysis has been published highlighting protein coding genes and possible drug target proteins. Hookworm microRNA identification, annotations and their public release is yet to be attempted. The same is evident from lack of hookworm miRNA information in related popular public nucleotide sequence repositories such as miRBase, GenBank, WormBase etc. Therefore, in the present study we addressed these issues using EST and assembled transcript sequence information of hookworm. Using computational approaches, we identified three miRNAs precursor sequences and their mature forms. We also identified their potential targets from hookworm ESTs and transcripts, and from human transcriptome. Overall, the results indicate presence of nematode specific miRNA homologs in N. americanus and shades light on their putative targets in worm itself and the human host.

SMAR1 binds to T(C/G) repeat and inhibits tumor progression by regulating miR-371-373 cluster.

Chromatin architecture and dynamics are regulated by various histone and non-histone proteins. The matrix attachment region binding proteins (MARBPs) play a central role in chromatin organization and function through numerous regulatory proteins. In the present study, we demonstrate that nuclear matrix protein SMAR1 orchestrates global gene regulation as determined by massively parallel ChIP-sequencing. The study revealed that SMAR1 binds to T(C/G) repeat and targets genes involved in diverse biological pathways. We observe that SMAR1 binds and targets distinctly different genes based on the availability of p53. Our data suggest that SMAR1 binds and regulates one of the imperative microRNA clusters in cancer and metastasis, miR-371-373. It negatively regulates miR-371-373 transcription as confirmed by SMAR1 overexpression and knockdown studies. Further, deletion studies indicate that a ~200 bp region in the miR-371-373 promoter is necessary for SMAR1 binding and transcriptional repression. Recruitment of HDAC1/mSin3A complex by SMAR1, concomitant with alteration of histone marks results in downregulation of the miRNA cluster. The regulation of miR-371-373 by SMAR1 inhibits breast cancer tumorigenesis and metastasis as determined by in vivo experiments. Overall, our study highlights the binding of SMAR1 to T(C/G) repeat and its role in cancer through miR-371-373.

Dose-dependent differential response of mammalian cells to cytoplasmic stress is mediated through the heme-regulated eIF2α kinase.

The heme-regulated inhibitor (HRI), a regulator of translation initiation, is known to be activated and upregulated, and it acts as either a cytoprotective player promoting cell survival or as an inducer of apoptosis during stress. However, the exact role of HRI in these two responses has not been elucidated. In the present investigation, using human cell lines, we attempted to unravel the molecular mechanism(s) of HRI-mediated differential response and the involved signaling pathways. While during low dose (5 µM) lead acetate treatment, cells did not show any diminished cell survival, significant level of apoptosis was observed at high dose (100 µM) lead acetate. Based on the results of an interactome analysis, we determined the interaction of HRI with PI-3-Kca, only at a low dose stress, which is followed by phosphorylation and activation of its downstream target, AKT. Interestingly, such an interaction and AKT activation was not observed at a high dose stress. On the other hand, an increased level of APAF-1 and activation of caspases were observed. These results indicate a critical role of HRI in cell survival during low dose stress, and in apoptosis at high dose stress. Furthermore, HRI knockdown cells are sensitized even to 5 µM lead treatment leading to caspase activation and apoptosis. Our results taken together thus elucidate for the first time the molecular mechanism and the involved signaling pathways for dose-dependent differential response of mammalian cells to lead exposure. These findings thus suggest the possibility of using HRI downregulation as a therapeutic strategy to sensitize cancer cells subjected to apoptogenic drugs.

miR-320a regulates erythroid differentiation through MAR binding protein SMAR1.

Erythropoiesis is controlled by a complex interplay of several signaling pathways and key transcription factors, as well as microRNAs (miRNAs). MicroRNAs function as critical modulators of gene expression for cellular processes. In the present study, we found that miR-320a inhibits erythroid differentiation by targeting Matrix Attachment Region binding protein SMAR1. miR-320a negatively regulates the expression of SMAR1 by directly binding to its 3'UTR. In response to mild DNA damage, miR-320a expression is decreased resulting in enhanced expression of SMAR1 protein, which in turn, reduces its targets, Bax and Puma inhibiting apoptosis. Our data demonstrate that during hemin-induced erythroid differentiation, enhanced expression of SMAR1 negatively correlates with miR-320a expression. Further analysis reveals that SMAR1 regulates erythroid differentiation, by binding to the promoter of miR-221/222, which play a crucial role in early erythropoiesis. Overall, our studies provide an insight into the regulation of hemin mediated erythroid differentiation of K562 cells through post-transcriptional regulation of SMAR1.

Chromatin remodelling protein SMAR1 inhibits p53 dependent transactivation by regulating acetyl transferase p300.

Acetylation of p53 is indispensable for its transcriptional activities and induction of apoptosis upon DNA damage. Here, we show that chromatin remodelling protein SMAR1 inhibits p53 acetylation and p53 dependent apoptosis by repressing p300 expression in response to DNA damage. The repression of p300 expression by SMAR1 is relieved upon treatment with proteosomal inhibitors MG132 and Lactacystin. We demonstrate that SMAR1 interacts with p53-p300 transcriptional complex and SMAR1 overexpression antagonizes p300 interaction with p53 and suppresses activation of p53 apoptotic targets and p53 regulated miRNA miR-34a. Conversely, knockdown of SMAR1 promotes p300 accumulation and p53 acetylation while ectopic expression of p300 rescues SMAR1 inhibition on p53. Collectively, these results indicate that SMAR1 is an important player in p300-p53 regulated DNA damage signalling pathway and can exert its effect on apoptosis in a transcription independent manner.