Publications by authors named "Sunil K Raghav"

27 Publications

  • Page 1 of 1

Aurora Kinase B Expression, Its Regulation and Therapeutic Targeting in Human Retinoblastoma.

Invest Ophthalmol Vis Sci 2021 Mar;62(3):16

The Operation Eyesight Universal Institute for Eye Cancer, LV Prasad Eye Institute, Bhubaneswar, India.

Purpose: Aurora kinase B (AURKB) plays a pivotal role in the regulation of mitosis and is gaining prominence as a therapeutic target in cancers; however, the role of AURKB in retinoblastoma (RB) has not been studied. The purpose of this study was to determine if AURKB plays a role in RB, how its expression is regulated, and whether it could be specifically targeted.

Methods: The protein expression of AURKB was determined using immunohistochemistry in human RB patient specimens and immunoblotting in cell lines. Pharmacological inhibition and shRNA-mediated knockdown were used to understand the role of AURKB in cell viability, apoptosis, and cell cycle distribution. Cell viability in response to AURKB inhibition was also assessed in enucleated RB specimens. Immunoblotting was employed to determine the protein levels of phospho-histone H3, p53, p21, and MYCN. Chromatin immunoprecipitation-qPCR was performed to verify the binding of MYCN on the promoter region of AURKB.

Results: The expression of AURKB was found to be markedly elevated in human RB tissues, and the overexpression significantly correlated with optic nerve and anterior chamber invasion. Targeting AURKB with small-molecule inhibitors and shRNAs resulted in reduced cell survival and increased apoptosis and cell cycle arrest at the G2/M phase. More importantly, primary RB specimens showed decreased cell viability in response to pharmacological AURKB inhibition. Additional studies have demonstrated that the MYCN oncogene regulates the expression of AURKB in RB.

Conclusions: AURKB is overexpressed in RB, and targeting it could serve as a novel therapeutic strategy to restrict tumor cell growth.
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http://dx.doi.org/10.1167/iovs.62.3.16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960835PMC
March 2021

Increased Extracellular ATP in Plasma of Rheumatoid Arthritis Patients Activates CD8T Cells.

Arch Med Res 2021 May 1;52(4):423-433. Epub 2021 Feb 1.

Disease Biology Laboratory, School of Biotechnology, Kalinga Institute of Industrial Technology, Deemed to be University, Bhubaneswar, Odisha, India. Electronic address:

Background: Rheumatoid arthritis (RA) is an autoimmune disorder with genetic and environmental causes often linked with the disease etiology. A disrupted metabolism has often been a characteristic of RA and an altered metabolic state of immune cells has been associated with their phenotypic and functional changes. The energy in the form of ATP produced by the metabolically active cells may thus initiate a cascade of immune responses there by influencing the disease pathogenesis or progression.

Aim Of The Study: Through this study we have focused on determining the role of ATP in etiology of RA and aberrant cellular functions.

Methods: Blood samples of 80 healthy controls (HC) and 95 RA patients were screened for extracellular ATP concentration, transcriptome analyses, an inflammatory mediator and the results were statistically analysed.

Results: In this study, ATP is shown to be excessive in the plasma of RA patients (453.5 ± 16.09% in RA vs. 233.9 ± 10.07% in HC, p <0.0001) and significantly increases with the disease severity. The abundant extracellular ATP could activate circulating cytotoxic CD8T cells in RA patients to produce Granzyme B.

Conclusion: Plasma ATP is thus identified to have a significant potential in progression and prognosis of RA and may thus be studied further to design better therapeutic approaches for the disease.
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http://dx.doi.org/10.1016/j.arcmed.2020.12.010DOI Listing
May 2021

Zbtb10 transcription factor is crucial for murine cDC1 activation and cytokine secretion.

Eur J Immunol 2021 May 1;51(5):1126-1142. Epub 2021 Mar 1.

Immuno-genomics & Systems Biology group, Institute of Life Sciences, Bhubaneswar, India.

Dendritic cell (DC) activation and cytokine production is tightly regulated. In this study, we found that Zbtb10 expression is activation dependent and it is essential for the immunogenic function of cDC1. Zbtb10 knockdown (KD) significantly reduced the expression of co-stimulatory genes CD80 and CD86 along with cytokines including IL-12, IL-6, and IL-10, in activated cDC1 Mutu-DC line. Consequently, the clonal expansion of CD44 effector T cells in co-cultured CD4 T cells was drastically reduced owing to significantly reduced IL-2. At the same time, these CD44 effector T cells were unable to differentiate toward Tbet IFNγ Th1 subtype. Instead, an increased frequency of Th2 cells expressing GATA3 and IL-13 was observed. Interestingly, in Zbtb10 KD condition the co-cultured T cells depicted increased expression of PD1 and LAG3, the T-cell anergic markers. Moreover, the global transcriptome analysis identified that Zbtb10 is pertinent for DC activation and its depletion in cDC1 completely shuts down their immune responses. Mechanistic analysis revealed that Zbtb10 KD enhanced the expression of NKRF (NF-κB repressing factor) leading to drastic suppression of NF-κB related genes. Zbtb10 KD abrogated p65 and RelB nuclear translocation, thereby controlling the activation and maturation of cDC1 and the ensuing adaptive T cell responses.
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http://dx.doi.org/10.1002/eji.202048933DOI Listing
May 2021

Sequence analysis of Indian SARS-CoV-2 isolates shows a stronger interaction of mutant receptor-binding domain with ACE2.

Int J Infect Dis 2021 Mar 12;104:491-500. Epub 2021 Jan 12.

Indian Council of Medical Research Regional Medical Research Centre, Bhubaneswar, Odisha, India.

Objective: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected the whole world, including Odisha, a state in eastern India. Many people have migrated to the state from different countries as well as other states during this SARS-CoV-2 pandemic. The aim of this study was to analyse the receptor-binding domain (RBD) sequence of the spike protein from isolates collected from throat swab samples of COVID-19-positive patients and further to assess the RBD affinity for angiotensin-converting enzyme 2 (ACE2) of different species, including humans.

Methods: Whole-genome sequencing for 35 clinical SARS-CoV-2 isolates from COVID-19-positive patients was performed by ARTIC amplicon-based sequencing. Sequence analysis and phylogenetic analysis were performed for the spike region and the RBD region of all isolates. The interaction between the RBD and ACE2 of five different species was also analysed.

Results: The spike region of 32 isolates showed one or multiple alterations in nucleotide bases in comparison with the Wuhan reference strain. One of the identified mutations, at position 1204 (Ref A, RMRC 22 C), in the RBD coding region of the spike protein showed stronger binding affinity for human ACE2. Furthermore, RBDs of all the Indian isolates showed binding affinity for ACE2 of different species.

Conclusion: As mutant RBD showed stronger interaction with human ACE2, it could potentially result in higher infectivity. The binding affinity of the RBDs for ACE2 of all five species studied suggests that the virus can infect a wide variety of animals, which could also act as natural reservoir for SARS-CoV-2.
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http://dx.doi.org/10.1016/j.ijid.2021.01.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7833473PMC
March 2021

CMTM6 drives cisplatin resistance by regulating Wnt signaling through the ENO-1/AKT/GSK3β axis.

JCI Insight 2021 Feb 22;6(4). Epub 2021 Feb 22.

Institute of Life Sciences, Bhubaneswar, India.

Rewiring tumor cells to undergo drug-induced apoptosis is a promising way to overcome chemoresistance. Therefore, identifying causative factors for chemoresistance is of high importance. Unbiased global proteome profiling of sensitive, early, and late cisplatin-resistant oral squamous cell carcinoma (OSCC) lines identified CMTM6 as a top-ranked upregulated protein. Analyses of OSCC patient tumor samples demonstrated significantly higher CMTM6 expression in chemotherapy (CT) nonresponders as compared with CT responders. In addition, a significant association between higher CMTM6 expression and poorer relapse-free survival in esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, and lung squamous cell carcinoma was observed from Kaplan-Meier plot analysis. Stable knockdown (KD) of CMTM6 restored cisplatin-mediated cell death in chemoresistant OSCC lines. Upon CMTM6 overexpression in CMTM6-KD lines, the cisplatin-resistant phenotype was rescued. The patient-derived cell xenograft model of chemoresistant OSCC displaying CMTM6 depletion restored the cisplatin-induced cell death and tumor burden substantially. The transcriptome analysis of CMTM6-KD and control chemoresistant cells depicted enrichment of the Wnt signaling pathway. We demonstrated that CMTM6 interaction with membrane-bound Enolase-1 stabilized its expression, leading to activation of Wnt signaling mediated by AKT-glycogen synthase kinase-3β. CMTM6 has been identified as a stabilizer of programmed cell death ligand 1. Therefore, as CMTM6 facilitates tumor cells for immune evasion and mediates cisplatin resistance, it could be a promising therapeutic target for treating therapy-resistant OSCC.
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http://dx.doi.org/10.1172/jci.insight.143643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7934946PMC
February 2021

NCoR1 fine-tunes type-I IFN response in cDC1 dendritic cells by directly regulating Myd88-IRF7 axis under TLR9.

Eur J Immunol 2020 12 16;50(12):1959-1975. Epub 2020 Jul 16.

Immuno-genomics & Systems Biology Laboratory, Institute of Life Sciences (ILS), Bhubaneswar, India.

Plasmacytoid dendritic cells (DCs) are reported to induce robust type-I interferon (IFN) response, whereas cDC1 DCs develop moderate type-I IFN response upon TLR9 stimulation. It is very interesting to understand how this signaling under TLR9 is tightly regulated for the induction of type-I IFNs. Here, we report co-repressor protein NCoR1 as the major factor fine-tuning the signaling pathways regulating IFN-β expression under TLR9 in cDC1 DCs. We found that NCoR1 knockdown induced a robust IFN-β-mediated antiviral response upon TLR9 activation in cDC1 DCs. At the molecular level, we showed that NCoR1 directly repressed MyD88-IRF7 signaling axis in cDC1 cells. Therefore, NCoR1 depletion enhanced pIRF7 levels, IFN-β secretion, and downstream pSTAT1-pSTAT2 signaling, leading to sustained induction of IFN stimulatory genes. Integrative genomic analysis depicted strong enrichment of an antiviral gene-module in CpG-activated NCoR1 knockdown DCs upon TLR9 activation. Moreover, we confirmed our findings in primary DCs derived from splenocytes of WT and NCoR1 DC animals, which showed protection from Sendai and Vesicular Stomatitis viruses upon CpG activation. Ultimately, we identified that NCoR1-HDAC3 complex is involved in repressing the type-I IFN response in cDC1 DCs.
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http://dx.doi.org/10.1002/eji.202048566DOI Listing
December 2020

Identification and Characterization of Circular Intronic RNAs Derived from Insulin Gene.

Int J Mol Sci 2020 Jun 17;21(12). Epub 2020 Jun 17.

Institute of Life Sciences (ILS), Nalco Square, Bhubaneswar, Odisha 751023, India.

Circular RNAs (circRNAs) are a large family of noncoding RNAs that have emerged as novel regulators of gene expression. However, little is known about the function of circRNAs in pancreatic β-cells. Here, transcriptomic analysis of mice pancreatic islet RNA-sequencing data identified 77 differentially expressed circRNAs between mice fed with a normal diet and a high-fat diet. Surprisingly, multiple circRNAs were derived from the intron 2 of the preproinsulin 2 () gene and are termed as circular intronic (). The expression of transcripts in mouse pancreatic islets, and βTC6 cells were confirmed by reverse transcription PCR, DNA sequencing, and RNase R treatment experiments. The level of was altered in βTC6 cells upon exposure to elevated levels of palmitate and glucose. Computational analysis predicted the interaction of several RNA-binding proteins with and their flanking region, suggesting their role in the function or biogenesis. Additionally, bioinformatics analysis predicted the association of several microRNAs with . Gene ontology and pathway analysis of genes targeted by miRNAs associated with suggested the regulation of several key biological processes. Together, our findings indicate that differential expression of circRNAs, especially transcripts, may regulate β-cell function and may play a critical role in the development of diabetes.
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http://dx.doi.org/10.3390/ijms21124302DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352490PMC
June 2020

Altered mitochondrial proteome and functional dynamics in patients with rheumatoid arthritis.

Mitochondrion 2020 09 13;54:8-14. Epub 2020 Jun 13.

Disease Biology Laboratory, School of Biotechnology, Kalinga Institute of Industrial Technology (KIIT), Deemed to be University, Bhubaneswar, Odisha, India. Electronic address:

The autoimmune inflammatory disease, Rheumatoid arthritis (RA), has known imbalances in energy metabolism and superoxide levels thus may have an etiology associated with mitochondrial dysfunction. We thus evaluated the presence of a differential mitochondrial proteome as well as other characteristics including mitochondrial mass, membrane potential (Ψm), total cellular ATP and superoxide levels. Eighteen mitochondrial proteins were down-regulated while four were up-regulated in RA patients in comparison to the healthy controls (HC). A significant decrease in mitochondrial Ψm, superoxides and cellular ATP levels was observed in RA with constant mitochondrial mass suggesting mitochondrial dysfunction responsible for functional disparity in RA.
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http://dx.doi.org/10.1016/j.mito.2020.06.005DOI Listing
September 2020

ZFP30 promotes adipogenesis through the KAP1-mediated activation of a retrotransposon-derived Pparg2 enhancer.

Nat Commun 2019 04 18;10(1):1809. Epub 2019 Apr 18.

Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015, Lausanne, Switzerland.

Krüppel-associated box zinc finger proteins (KZFPs) constitute the largest family of mammalian transcription factors, but most remain completely uncharacterized. While initially proposed to primarily repress transposable elements, recent reports have revealed that KFZPs contribute to a wide variety of other biological processes. Using murine and human in vitro and in vivo models, we demonstrate here that one poorly studied KZFP, ZFP30, promotes adipogenesis by directly targeting and activating a retrotransposon-derived Pparg2 enhancer. Through mechanistic studies, we further show that ZFP30 recruits the co-regulator KRAB-associated protein 1 (KAP1), which, surprisingly, acts as a ZFP30 co-activator in this adipogenic context. Our findings provide an understanding of both adipogenic and KZFP-KAP1 complex-mediated gene regulation, showing that the KZFP-KAP1 axis can also function in a non-repressive manner.
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http://dx.doi.org/10.1038/s41467-019-09803-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472429PMC
April 2019

Importance of EMT Factor ZEB1 in cDC1 "MutuDC Line" Mediated Induction of Th1 Immune Response.

Front Immunol 2018 13;9:2604. Epub 2018 Nov 13.

Immuno-genomics and Systems Biology Laboratory, Institute of Life Sciences (ILS), Bhubaneswar, India.

The role of Epithelial to Mesenchymal Transition (EMT) factor Zeb1 is well defined in metastasis and cancer progression but it's importance in dendritic cells (DCs) is unexplored until now. For the first time we report here that Zeb1 controls immunogenic responses of CD8α conventional Type-I (cDC1) DCs. We found that ZEB1 expression increases significantly after TLR9 stimulation and its depletion impairs activation, co-stimulation and secretion of important cytokines like IL-6, IL-10 and IL-12 in cDC1 MutuDC line. We further confirmed our findings in primary cDC1 DCs derived from bone marrow. Co-culture of these Zeb1 knock down (KD) DCs with OT-II CD4 T helper cells skewed their differentiation toward Th2 subtype. Moreover, adoptive transfer of activated Zeb1 KD DCs cleared intestinal worms in helminth infected mice by increasing Th2 responses . Integrative genomic analysis showed Zeb1 as an activator of immune response genes in cDC1 MutuDCs as compared to other pathway genes. In addition, differentially regulated genes in Zeb1 KD RNA-seq showed significant enrichment of Th2 activation pathways supporting our findings. Mechanistically, we showed that decreased IL-12 secreted by Zeb1 KD DCs is the plausible mechanism for increased Th2 differentiation. Collectively our data demonstrate that Zeb1 could be targeted in DCs to modulate T-cell mediated adaptive immune responses.
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http://dx.doi.org/10.3389/fimmu.2018.02604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243008PMC
October 2019

TRIM16 controls assembly and degradation of protein aggregates by modulating the p62-NRF2 axis and autophagy.

EMBO J 2018 09 24;37(18). Epub 2018 Aug 24.

Cell Biology and Infectious Diseases Unit, Institute of Life Sciences, Bhubaneswar, India

Sequestration of protein aggregates in inclusion bodies and their subsequent degradation prevents proteostasis imbalance, cytotoxicity, and proteinopathies. The underlying molecular mechanisms controlling the turnover of protein aggregates are mostly uncharacterized. Herein, we show that a TRIM family protein, TRIM16, governs the process of stress-induced biogenesis and degradation of protein aggregates. TRIM16 facilitates protein aggregate formation by positively regulating the p62-NRF2 axis. We show that TRIM16 is an integral part of the p62-KEAP1-NRF2 complex and utilizes multiple mechanisms for stabilizing NRF2. Under oxidative and proteotoxic stress conditions, TRIM16 activates ubiquitin pathway genes and p62 via NRF2, leading to ubiquitination of misfolded proteins and formation of protein aggregates. We further show that TRIM16 acts as a scaffold protein and, by interacting with p62, ULK1, ATG16L1, and LC3B, facilitates autophagic degradation of protein aggregates. Thus, TRIM16 streamlines the process of stress-induced aggregate clearance and protects cells against oxidative/proteotoxic stress-induced toxicity and Taken together, this work identifies a new mechanism of protein aggregate turnover, which could be relevant in protein aggregation-associated diseases such as neurodegeneration.
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http://dx.doi.org/10.15252/embj.201798358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138442PMC
September 2018

Dissecting the brown adipogenic regulatory network using integrative genomics.

Sci Rep 2017 02 9;7:42130. Epub 2017 Feb 9.

Institute of Bioengineering, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.

Brown adipocytes regulate energy expenditure via mitochondrial uncoupling, which makes them attractive therapeutic targets to tackle obesity. However, the regulatory mechanisms underlying brown adipogenesis are still poorly understood. To address this, we profiled the transcriptome and chromatin state during mouse brown fat cell differentiation, revealing extensive gene expression changes and chromatin remodeling, especially during the first day post-differentiation. To identify putatively causal regulators, we performed transcription factor binding site overrepresentation analyses in active chromatin regions and prioritized factors based on their expression correlation with the bona-fide brown adipogenic marker Ucp1 across multiple mouse and human datasets. Using loss-of-function assays, we evaluated both the phenotypic effect as well as the transcriptomic impact of several putative regulators on the differentiation process, uncovering ZFP467, HOXA4 and Nuclear Factor I A (NFIA) as novel transcriptional regulators. Of these, NFIA emerged as the regulator yielding the strongest molecular and cellular phenotypes. To examine its regulatory function, we profiled the genomic localization of NFIA, identifying it as a key early regulator of terminal brown fat cell differentiation.
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http://dx.doi.org/10.1038/srep42130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5299609PMC
February 2017

Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

Cell 2015 Aug 20;162(5):1039-50. Epub 2015 Aug 20.

Swiss Institute of Bioinformatics, Lausanne 1015, Switzerland; Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva 1211, Switzerland; Institute of Genetics and Genomics in Geneva, University of Geneva, Geneva 1211, Switzerland. Electronic address:

Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.
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http://dx.doi.org/10.1016/j.cell.2015.08.001DOI Listing
August 2015

TLR4 activation by lipopolysaccharide confers survival advantage to growth factor deprived prostate cancer cells.

Prostate 2015 Jul 2;75(10):1020-33. Epub 2015 Apr 2.

Institute of Life Sciences, Bhubaneswar, Odisha, India.

Background: Prostate cancer (PCa) cells express Toll-like receptor-4 (TLR4), a known pro-tumorigenic molecule for different cancer cells. The cancer cells residing in the avascular region of the tumor confront various metabolic stresses and continuously adapt mechanisms to overcome them. We hypothesized that TLR4 activation might provide direct survival advantage to metabolically stressed PCa cells.

Methods: We first investigated the effect of LPS on survival of serum deprived PCa cells. To understand the molecular mechanisms involved in TLR4 mediated PCa survival, we next investigated change in expression of markers for apoptosis, senescence and autophagy. Ultimately, the effect of LPS on established prostate tumors was confirmed in vivo using a syngeneic rat model for PCa.

Results: Lipopolysaccharide (LPS)-mediated TLR4 activation significantly enhanced survival of serum deprived (SD) PC3, DU145 and MAT-LyLu PCa cells. TLR4 inhibition by a specific inhibitor resulted in rapid death of SD-PC3 cells, which was significantly suppressed by LPS. Interestingly, LPS treatment suppressed macroautophagy in SD-PC3 cells and increased expression of CCL2 (C-C motif ligand-2), a known autophagy inhibitor and pro-survival factor. Intra-tumor LPS injection resulted in increased tumor mass, induced TLR4 activation, suppressed autophagy, and increased the macrophage population in MAT-LyLu-tumors.

Conclusions: Our study reveals that bacterial LPS enhance survival of PCa cells under conditions of nutrient stress through TLR4 activation. Moreover, LPS induces overexpression of CCL2 involved in the suppression of starvation-induced macroautophagy in PCa cells, and enhanced macrophage population in prostate tumors in vivo. Taken together, the current study suggests the importance of bacterial infection or TLR4-activation in prostate cancer pathogenesis.
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http://dx.doi.org/10.1002/pros.22983DOI Listing
July 2015

Identification of the transcription factor ZEB1 as a central component of the adipogenic gene regulatory network.

Elife 2014 Aug 27;3:e03346. Epub 2014 Aug 27.

Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne and Swiss Institute of Bioinformatics, Lausanne, Switzerland.

Adipose tissue is a key determinant of whole body metabolism and energy homeostasis. Unraveling the regulatory mechanisms underlying adipogenesis is therefore highly relevant from a biomedical perspective. Our current understanding of fat cell differentiation is centered on the transcriptional cascades driven by the C/EBP protein family and the master regulator PPARγ. To elucidate further components of the adipogenic gene regulatory network, we performed a large-scale transcription factor (TF) screen overexpressing 734 TFs in mouse pre-adipocytes and probed their effect on differentiation. We identified 22 novel pro-adipogenic TFs and characterized the top ranking TF, ZEB1, as being essential for adipogenesis both in vitro and in vivo. Moreover, its expression levels correlate with fat cell differentiation potential in humans. Genomic profiling further revealed that this TF directly targets and controls the expression of most early and late adipogenic regulators, identifying ZEB1 as a central transcriptional component of fat cell differentiation.
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http://dx.doi.org/10.7554/eLife.03346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359378PMC
August 2014

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

Bioinformatics 2014 Jan 18;30(2):165-71. Epub 2013 Nov 18.

Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne, Swiss Institute of Bioinformatics, 1015, Lausanne, Switzerland, Department of Genetic Medicine and Development, University of Geneva Medical School, Institute of Genetics and Genomics in Geneva, University of Geneva, 1211, Geneva, Switzerland and Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, 1011, Lausanne, Switzerland.

Motivation: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts.

Results: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays.

Availability: The R package abs filter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter
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http://dx.doi.org/10.1093/bioinformatics/btt667DOI Listing
January 2014

Coordinated effects of sequence variation on DNA binding, chromatin structure, and transcription.

Science 2013 Nov 17;342(6159):744-7. Epub 2013 Oct 17.

Department of Genetic Medicine and Development, University of Geneva Medical School, 1211 Geneva, Switzerland.

DNA sequence variation has been associated with quantitative changes in molecular phenotypes such as gene expression, but its impact on chromatin states is poorly characterized. To understand the interplay between chromatin and genetic control of gene regulation, we quantified allelic variability in transcription factor binding, histone modifications, and gene expression within humans. We found abundant allelic specificity in chromatin and extensive local, short-range, and long-range allelic coordination among the studied molecular phenotypes. We observed genetic influence on most of these phenotypes, with histone modifications exhibiting strong context-dependent behavior. Our results implicate transcription factors as primary mediators of sequence-specific regulation of gene expression programs, with histone modifications frequently reflecting the primary regulatory event.
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http://dx.doi.org/10.1126/science.1242463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502466PMC
November 2013

A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks.

Mol Syst Biol 2013 Aug 6;9:682. Epub 2013 Aug 6.

Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.

The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein-DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF-DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.
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http://dx.doi.org/10.1038/msb.2013.38DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3779800PMC
August 2013

Global chromatin state analysis reveals lineage-specific enhancers during the initiation of human T helper 1 and T helper 2 cell polarization.

Immunity 2013 Jun 20;38(6):1271-84. Epub 2013 Jun 20.

Division of Medical Genetics and Department of Genome Sciences, Department of Medicine, University of Washington School of Medicine, Seattle, WA 98195, USA.

Naive CD4⁺ T cells can differentiate into specific helper and regulatory T cell lineages in order to combat infection and disease. The correct response to cytokines and a controlled balance of these populations is critical for the immune system and the avoidance of autoimmune disorders. To investigate how early cell-fate commitment is regulated, we generated the first human genome-wide maps of histone modifications that reveal enhancer elements after 72 hr of in vitro polarization toward T helper 1 (Th1) and T helper 2 (Th2) cell lineages. Our analysis indicated that even at this very early time point, cell-specific gene regulation and enhancers were at work directing lineage commitment. Further examination of lineage-specific enhancers identified transcription factors (TFs) with known and unknown T cell roles as putative drivers of lineage-specific gene expression. Lastly, an integrative analysis of immunopathogenic-associated SNPs suggests a role for distal regulatory elements in disease etiology.
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http://dx.doi.org/10.1016/j.immuni.2013.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4607036PMC
June 2013

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics.

Nat Methods 2013 Jun 14;10(6):570-6. Epub 2013 Apr 14.

Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ∼250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.
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http://dx.doi.org/10.1038/nmeth.2441DOI Listing
June 2013

Integrative genomics identifies the corepressor SMRT as a gatekeeper of adipogenesis through the transcription factors C/EBPβ and KAISO.

Mol Cell 2012 May 19;46(3):335-50. Epub 2012 Apr 19.

Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

The molecular role of corepressors is poorly understood. Here, we studied the transcriptional function of the corepressor SMRT during terminal adipogenesis. Genome-wide DNA-binding profiling revealed that this corepressor is predominantly located in active chromatin regions and that most distal SMRT binding events are lost after differentiation induction. Promoter-proximal tethering of SMRT in preadipocytes is primarily mediated by KAISO through the conserved TCTCGCGAGA motif. Further characterization revealed that KAISO, similar to SMRT, accelerates the cell cycle and increases fat accumulation upon knockdown, identifying KAISO as an adipogenic repressor that likely modulates the mitotic clonal expansion phase of this process. SMRT-bound promoter-distal sites tend to overlap with C/EBPβ-bound regions, which become occupied by proadipogenic transcription factors after SMRT clearance. This reveals a role for SMRT in masking enhancers from proadipogenic factors in preadipocytes. Finally, we identified SMRT as an adipogenic gatekeeper as it directly fine-tunes transcription of pro- and antiadipogenic genes.
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http://dx.doi.org/10.1016/j.molcel.2012.03.017DOI Listing
May 2012

Exhaustion of tumor-specific CD8⁺ T cells in metastases from melanoma patients.

J Clin Invest 2011 Jun 9;121(6):2350-60. Epub 2011 May 9.

Clinical Tumor Immune-Biology Unit, Ludwig Institute for Cancer Research, Lausanne, Switzerland.

In chronic viral infections, CD8⁺ T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8⁺ T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8⁺ T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy.
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http://dx.doi.org/10.1172/JCI46102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3104769PMC
June 2011

An insight into molecular mechanisms of human T helper cell differentiation.

Ann Med 2008 ;40(5):322-35

Turku Centre for Biotechnology, University of Turku and Abo Akademi University, Turku, Finland.

Selective activation of T helper (Th) cell subsets plays an important role in immune response to pathogens as well as in the pathogenesis of human allergy and inflammatory diseases. Th1 cells along with the recently discovered Th17 cells play a role in the pathogenesis of autoimmune diseases. Th2 cytokines lead to series of inflammatory processes characteristic for asthma and other atopic diseases. To understand the pathogenesis of immune-mediated diseases it is crucial to dissect pathways and regulatory networks leading to the development of distinct Th subsets. Such knowledge may lead to better strategies for developing diagnostics and therapies for these diseases. The differentiation of Th1, Th2, and Th17 effector cells is driven by signals originating from T cell and costimulatory receptors as well as cytokines in the surroundings of activated naive T helper cells. There are several proteins involved in the regulation of this differentiation process. Most of the data on T helper cell differentiation have been acquired using mouse. In this review, we have summarized what is known about human T helper differentiation. In addition, selected differences between human and mouse will be discussed.
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http://dx.doi.org/10.1080/07853890802068582DOI Listing
June 2008

S-nitrosylation of mannose binding lectin regulates its functional activities and the formation of autoantibody in rheumatoid arthritis.

Nitric Oxide 2008 Jun 29;18(4):266-73. Epub 2008 Feb 29.

Proteomics and Structural Biology Division, Institute of Genomics & Integrative Biology, Delhi University Campus, Mall Road, Delhi 110 007, India.

The possibility of post-translational modifications of mannose binding lectin (MBL) leading to functional impairment of the MBL pathway and the presence of anti-MBL autoantibodies were reported earlier in rheumatoid arthritis (RA). MBL was observed to be S-nitrosylated (S-nitrosated) in vitro. HepG2 cells were stimulated with 10% synovial fluid from RA patients to produce increased levels of MBL and nitric oxide. Under these experimental conditions MBL was observed to be S-nitrosated using biotin switch assay. The plasma of RA patients was also found to contain higher levels of S-nitrosylated MBL (SNO-MBL) in comparison to the healthy controls. Functional activities of SNO-MBL were compared with normal MBL. Mannan binding and C4 deposition ability of MBL was found to decrease after S-nitrosylation. It was also observed that S-nitrosylation of MBL leads to a decrease in the bacterial phagocytosis and apoptotic cell binding as measured by fluorescence microscopy and FACS analysis. These results indicate that the carbohydrate binding ability of MBL was affected by S-nitrosylation (S-nitrosation). High levels of anti-MBL autoantibodies were detected against SNO-MBL in plasma of RA patients in comparison to normal MBL suggesting a role of SNO-MBL in generation of autoantibodies in RA patients.
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http://dx.doi.org/10.1016/j.niox.2008.01.009DOI Listing
June 2008

Altered expression and glycosylation of plasma proteins in rheumatoid arthritis.

Glycoconj J 2006 May;23(3-4):167-73

Institute of Genomics & Integrative Biology, Delhi University Campus, Mall Road, Delhi, 110 007, India.

Altered glycosylation of plasma proteins has been directly implicated in the pathogenesis of rheumatoid arthritis (RA). The present study investigated the changes in the Concanavalin-A (Con-A)-bound plasma proteins in the RA patients in comparison to that of the healthy controls. Two proteins (MW approximately 32 kDa and approximately 62 kDa) showed an alteration in expression while an altered monosaccharide profile (high mannose) was observed in the approximately 62 kDa protein in the samples collected from RA patients. The 2-dimensional polyacrylamide gel electrophoresis analysis of the Con-A-bound plasma samples showed a large number of protein spots, a few of which were differentially expressed in the RA patients. Some unidentified proteins were detected in the RA patients which were absent in the control samples. The present study, therefore, enunciates the role of carbohydrates as well as that of the acute phase response in the disease pathogenesis.
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http://dx.doi.org/10.1007/s10719-006-7922-6DOI Listing
May 2006

Suppression of inducible nitric oxide synthase by 10-23 DNAzymes in murine macrophage.

FEBS Lett 2006 Apr 10;580(8):2046-52. Epub 2006 Mar 10.

Comparative Genomics Unit, Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi 110 007, India.

iNOS mRNA of J774 murine macrophage cells was cleaved by 10-23 DNAzymes. DNAzyme target site I or translation initiation site and site II have computer predicted (MFOLD) secondary structures but site III has no secondary structure. All the three DNAzymes cleaved the short transcripts generated from cloned DNA almost with equal efficiency while cleavage efficiency is higher at site III than the other two sites on isolated iNOS mRNA. Interestingly, at intracellular level, DNAzyme targeted at translation initiation codon (site I) having secondary structure cleaved iNOS mRNA, and suppressed its activity and protein expression more efficiently than that targeted at sites II and III.
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http://dx.doi.org/10.1016/j.febslet.2006.03.004DOI Listing
April 2006

Association of mannose-binding lectin gene (MBL2) polymorphisms with rheumatoid arthritis in an Indian cohort of case-control samples.

J Hum Genet 2005 12;50(11):583-591. Epub 2005 Oct 12.

Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi 110 007, India.

Single nucleotide polymorphisms in the mannose-binding lectin (MBL2) gene, as well as the serum MBL2 level, have been associated with various autoimmune diseases. We investigated whether such polymorphisms and/or the serum MBL2 level were associated with rheumatoid arthritis (RA) in an Indian population. The frequency of the B variant (codon 54) of the MBL2 gene was quite frequent in the healthy Indian population and was significantly (P=6.35x10(-6)) lower in RA patients. We replicated this association (P=1.78x10(-5)) in an independent cohort of control individuals. Promoter polymorphism at -550 nt showed a significant overrepresentation (P=0.003) of the minor allele G in severe RA patients compared with the less severe group. Haplotype LYA frequency was significantly (P=0.03) high in the less severe group, while the frequency of the HYA haplotype was significantly (P=0.04) increased in the severe RA patients. No statistically significant difference in serum MBL2 was observed as a whole, but the individuals homozygous for the LYA haplotype had significantly lower (P=0.017) serum MBL2 levels compared with individuals homozygous for the HYA haplotype. Therefore, the B variant of the MBL2 gene may be associated with protection from RA in our study population, and the promoter polymorphism (-550 nt) seems to have some role in disease progression.
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http://dx.doi.org/10.1007/s10038-005-0299-8DOI Listing
June 2006