Michael L Whitfield

Hospital for Special Surgery, New York, New York, United States

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Publications (68)389.35 Total impact

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    ABSTRACT: Autoantibody profiles represent important patient stratification markers in systemic sclerosis (SSc). Here, we performed serum-immunoprecipitations with patient antibodies followed by mass spectrometry (LC-MS/MS) to obtain an unbiased view of all possible autoantibody targets and their associated molecular complexes recognized by SSc. HeLa whole cell lysates were immunoprecipitated (IP) using sera of patients with SSc clinically positive for autoantibodies against RNA polymerase III (RNAP3), topoisomerase 1 (TOP1), and centromere proteins (CENP). IP eluates were then analyzed by LC-MS/MS to identify novel proteins and complexes targeted in SSc. Target proteins were examined using a functional interaction network to identify major macromolecular complexes, with direct targets validated by IP-Western blots and immunofluorescence. A wide range of peptides were detected across patients in each clinical autoantibody group. Each group contained peptides representing a broad spectrum of proteins in large macromolecular complexes, with significant overlap between groups. Network analyses revealed significant enrichment for proteins in RNA processing bodies (PB) and cytosolic stress granules (SG) across all SSc subtypes, which were confirmed by both Western blot and immunofluorescence. While strong reactivity was observed against major SSc autoantigens, such as RNAP3 and TOP1, there was overlap between groups with widespread reactivity seen against multiple proteins. Identification of PB and SG as major targets of the humoral immune response represents a novel SSc autoantigen and suggests a model in which a combination of chronic and acute cellular stresses result in aberrant cell death, leading to autoantibody generation directed against macromolecular nucleic acid-protein complexes.
    Preview · Article · Dec 2016 · Arthritis research & therapy
  • Viktor Martyanov · Michael L Whitfield
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    ABSTRACT: Purpose of review: The goal of this review is to summarize recent advances into the pathogenesis and treatment of systemic sclerosis (SSc) from genomic and proteomic studies. Recent findings: Intrinsic gene expression-driven molecular subtypes of SSc are reproducible across three independent datasets. These subsets are a consistent feature of SSc and are found in multiple end-target tissues, such as skin and esophagus. Intrinsic subsets as well as baseline levels of molecular target pathways are potentially predictive of clinical response to specific therapeutics, based on three recent clinical trials. A gene expression-based biomarker of modified Rodnan skin score, a measure of SSc skin severity, can be used as a surrogate outcome metric and has been validated in a recent trial. Proteome analyses have identified novel biomarkers of SSc that correlate with SSc clinical phenotypes. Summary: Integrating intrinsic gene expression subset data, baseline molecular pathway information, and serum biomarkers along with surrogate measures of modified Rodnan skin score provides molecular context in SSc clinical trials. With validation, these approaches could be used to match patients with the therapies from which they are most likely to benefit and thus increase the likelihood of clinical improvement.
    No preview · Article · Nov 2015 · Current opinion in rheumatology
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    ABSTRACT: The animal replication-dependent (RD) histone mRNAs are coordinately regulated with chromosome replication. The RD-histone mRNAs are the only known cellular mRNAs that are not polyadenylated. Instead, the mature transcripts end in a conserved stem-loop (SL) structure. This SL structure interacts with the stem-loop binding protein (SLBP), which is involved in all aspects of RD-histone mRNA metabolism. We used several genomic methods, including high-throughput sequencing of cross-linked immunoprecipitate (HITS-CLIP) to analyze the RNA-binding landscape of SLBP. SLBP was not bound to any RNAs other than histone mRNAs. We performed bioinformatic analyses of the HITS-CLIP data that included (i) clustering genes by sequencing read coverage using CVCA, (ii) mapping the bound RNA fragment termini, and (iii) mapping cross-linking induced mutation sites (CIMS) using CLIP-PyL software. These analyses allowed us to identify specific sites of molecular contact between SLBP and its RD-histone mRNA ligands. We performed in vitro crosslinking assays to refine the CIMS mapping and found that uracils one and three in the loop of the histone mRNA SL preferentially crosslink to SLBP, whereas uracil two in the loop preferentially crosslinks to a separate component, likely the 3'hExo. We also performed a secondary analysis of an iCLIP data set to map UPF1 occupancy across the RD-histone mRNAs and found that UPF1 is bound adjacent to the SLBP-binding site. Multiple proteins likely bind the 3' end of RD-histone mRNAs together with SLBP.
    No preview · Article · Sep 2015 · RNA
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    ABSTRACT: The goal of this study was to define a pharmacodynamic biomarker based on gene expression in skin that would provide a biological measure of disease extent in patients with diffuse cutaneous systemic sclerosis (dcSSc) and that could be used to monitor skin disease longitudinally. Skin biopsies taken from a cohort of dcSSc patients that included longitudinal samples were analyzed by microarray. Expression of genes correlating with the modified Rodnan skin score (MRSS) were examined by nanostring for change over time, and a generalized estimating equation used to define and validate longitudinal, pharmacodynamic biomarkers composed of multiple genes. Microarray analysis of genes parsed to include only genes correlating with the MRSS revealed prominent clusters of profibrotic/TGFβ-regulated, IFN-regulated/proteasome, macrophage and vascular marker genes. Using genes changing longitudinally with the MRSS, two multigene, pharmacodynamic biomarkers were defined. The first was defined mathematically, applying a generalized estimating equation to longitudinal samples. This modeling method selected cross-sectional THBS1 and longitudinal THBS1 and MS4A4A genes. The second model was based on a weighted selection of genes, including additional genes with statistically significant change over time: CTGF, CD163, CCL2 and WIF1. Biomarker levels calculated using both models correlated highly with the MRSS in an independent validation dataset. Skin gene expression can be used effectively to monitor SSc skin disease change over time. We have implemented these relatively simple models on a nanostring platform permitting highly reproducible assays that can be applied directly to samples from patients or collected as part of clinical trials. This article is protected by copyright. All rights reserved. © 2015, American College of Rheumatology.
    No preview · Article · Aug 2015 · Arthritis and Rheumatology
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    ABSTRACT: Tyrosine kinase inhibitors (TKI) are medications of interest in the treatment of Systemic Sclerosis (SSc) because of their ability to inhibit pathways involved in fibrosis. In this open-label pilot trial, our objectives were to assess the safety, efficacy, and molecular change associated with treatment of patients with diffuse cutaneous (dc)SSc with the TKI nilotinib (Tasigna™). Ten adult patients with early dcSSc were treated with nilotinib. Primary endpoints were safety and change in modified Rodnan Skin Score (MRSS) after 6 months. Lesional skin biopsies at baseline, 6 and 12 months of treatment were assessed by histopathology, immunohistochemistry, and DNA microarray. Patients had early and active dcSSc with median disease duration of 0.7 years (range 0.5, 1.7) and increasing MRSS in the month prior to baseline (mean +2.9, p=0.02). Seven out of ten patients completed 6 and 12 months of treatment. Seventy-one adverse events (AEs) including 2 serious AEs were observed, and 92 % of AEs were grade 1-2. Two patients discontinued the medication due to mild QTc prolongation. MRSS improved by a mean of 4.2 points (16 %) at 6 months and by 6.3 points (23 %) at 12 months in the 7 completers, p=0.02 and 0.01, respectively. Patients with a decrease in MRSS >20 % from baseline at 12 months (classified as improvers) had significantly higher expression of transforming growth factor beta receptor (TGFBR) and platelet-derived growth factor receptor beta (PDGFRB) signaling genes at baseline than non-improvers, and the expression of these genes significantly decreased in improvers post-treatment. Nilotinib was well tolerated by the majority of patients in this study, with tolerability limited primarily by mild QTc-prolongation. Significant MRSS improvement was observed in these early, active patients, but is not conclusive of treatment effect given the open-label study-design and small number of patients in this pilot study. Improvers had higher levels of expression of genes associated with TGFBR and PDGFRB signaling at baseline, and a significant decrease in the expression of these genes occurred only in patients with higher MRSS improvement. The findings of this pilot study warrant more conclusive evaluation. Clinicaltrials.gov NCT01166139 , July 1, 2010.
    Full-text · Article · Aug 2015 · Arthritis research & therapy
  • Michael E Johnson · Patricia A Pioli · Michael L Whitfield
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    ABSTRACT: Systemic sclerosis (SSc) is characterized by inflammation, vascular dysfunction, and ultimately fibrosis. Progress in understanding disease pathogenesis and developing effective disease treatments has been hampered by an incomplete understanding of SSc heterogeneity. To clarify this, we have used genomic approaches to identify distinct patient subsets based on gene expression patterns in SSc skin and other end-target organs. Here, we review what is known about the gene expression-based subsets in SSc, currently defined as the inflammatory, fibroproliferative, limited, and normal-like subsets. The inflammatory subset of patients is characterized by infiltrating immune cells that include T cells, macrophages, and possibly dendritic cells, although little is known about the mediators these cells secrete and the pathways that govern cell activation. Prior studies have suggested a role for pathogens as a trigger of immune responses in SSc, and recent data have identified viral and mycobiome components as potential environmental triggers. We present a model based on analyses of gene expression data and a review of the literature, which suggests that the gene expression subsets observed in patients possibly represent distinct, interconnected molecular states of disease, to which an innate immune response is central that results in the generation of clinical disease.
    No preview · Article · Jul 2015 · Seminars in Immunopathology
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    ABSTRACT: Esophageal involvement in patients with systemic sclerosis (SSc) is common, but tissue-specific pathological mechanisms are poorly understood. There are no animal scleroderma esophagus models and esophageal smooth muscle cells dedifferentiate in culture prohibiting in vitro studies. Esophageal fibrosis is thought to disrupt smooth muscle function and lead to esophageal dilatation, but autopsy studies demonstrate esophageal smooth muscle atrophy and the absence of fibrosis in the majority of SSc cases. Herein, we perform a detailed characterization of SSc esophageal histopathology and molecular signatures at the level of gene expression. Esophageal biopsies were prospectively obtained during esophagogastroduodenoscopy in 16 consecutive SSc patients and 7 subjects without SSc. Upper and lower esophageal biopsies were evaluated for histopathology and gene expression. Individual patient's upper and lower esophageal biopsies showed nearly identical patterns of gene expression. Similar to skin, inflammatory and proliferative gene expression signatures were identified suggesting that molecular subsets are a universal feature of SSc end-target organ pathology. The inflammatory signature was present in biopsies without high numbers of infiltrating lymphocytes. Molecular classification of esophageal biopsies was independent of SSc skin subtype, serum autoantibodies and esophagitis. Proliferative and inflammatory molecular gene expression subsets in tissues from patients with SSc may be a conserved, reproducible component of SSc pathogenesis. The inflammatory signature is observed in biopsies that lack large inflammatory infiltrates suggesting that immune activation is a major driver of SSc esophageal pathogenesis.
    Full-text · Article · Jul 2015 · Arthritis research & therapy
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    ABSTRACT: BACKGROUND. TGF-beta has potent profibrotic activity in vitro and has long been implicated in systemic sclerosis (SSc), as expression of TGF-beta-regulated genes is increased in the skin and lungs of patients with SSc. Therefore, inhibition of TGF-beta may benefit these patients. METHODS. Patients with early, diffuse cutaneous SSc were enrolled in an open-label trial of fresolimumab, a high-affinity neutralizing antibody that targets all 3 TGF-beta isoforms. Seven patients received two 1 mg/kg doses of fresolimumab, and eight patients received one 5 mg/kg dose of fresolimumab. Serial mid-forearm skin biopsies, performed before and after treatment, were analyzed for expression of the TGF-beta-regulated biomarker genes thrombospondin-1 (THBS1) and cartilage oligomeric protein (COMP) and stained for myofibroblasts. Clinical skin disease was assessed using the modified Rodnan skin score (MRSS). RESULTS. In patient skin, THBS1 expression rapidly declined after fresolimumab treatment in both groups (P = 0.0313 at 7 weeks and P = 0.0156 at 3 weeks), and skin expression of COMP exhibited a strong downward trend in both groups. Clinical skin disease dramatically and rapidly decreased (P < 0.001 at all time points). Expression levels of other TGF-beta-regulated genes, including SERPINE1 and CTGF, declined (P = 0.049 and P. 0.012, respectively), and a 2-gene, longitudinal pharmacodynamic biomarker of SSc skin disease decreased after fresolimumab treatment (P = 0.0067). Dermal myofibroblast infiltration also declined in patient skin after fresolimumab (P < 0.05). Baseline levels of THBS1 were predictive of reduced THBS1 expression and improved MRSS after fresolimumab treatment. CONCLUSION. The rapid inhibition of TGF-beta-regulated gene expression in response to fresolimumab strongly implicates TGF-beta in the pathogenesis of fibrosis in SSc. Parallel improvement in the MRSS indicates that fresolimumab rapidly reverses markers of skin fibrosis.
    Full-text · Article · Jun 2015 · The Journal of clinical investigation
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    ABSTRACT: Systemic sclerosis (SSc) is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic sclerosis (dcSSc). Adult dcSSc patients were randomized in a 2:1 double-blinded fashion to receive abatacept or placebo over 24 weeks. Primary outcomes were safety and the change in modified Rodnan Skin Score (mRSS) at week 24 compared with baseline. Improvers were defined as patients with a decrease in mRSS of ≥30 % post-treatment compared to baseline. Skin biopsies were obtained for differential gene expression and pathway enrichment analyses and intrinsic gene expression subset assignment. 10 subjects were randomized to abatacept (n = 7) or placebo (n = 3). Disease duration from first non-Raynaud's symptom was significantly longer (8.8 ± 3.8 years vs. 2.4 ± 1.6 years, p = 0.004) and median mRSS was higher (30 vs. 22, p = 0.05) in the placebo compared to abatacept group. Adverse events were similar in the two groups. 5/7 patients (71 %) randomized to abatacept and 1/3 patients (33 %) randomized to placebo experienced ≥30 % improvement in skin score. Subjects receiving abatacept showed a trend toward improvement in mRSS at week 24 (-8.6 ± 7.5, p = 0.0625) while those in the placebo group did not (-2.3 ± 15, p = 0.75). After adjusting for disease duration, mRSS significantly improved in abatacept compared with placebo group (abatacept vs. placebo mRSS decrease estimate -9.8, 95 % confidence interval -16.7 to -3.0, p = 0.0114). In the abatacept group, the patients in the inflammatory intrinsic subset showed a trend toward greater improvement in skin score at 24 weeks compared with the patients in the normal-like intrinsic subset (-13.5 ± 3.1 vs. -4.5 ± 6.4, p = 0.067). Abatacept resulted in decreased CD28 co-stimulatory gene expression in improvers consistent with its mechanism of action. Improvers mapped to the inflammatory intrinsic subset and showed decreased gene expression in inflammatory pathways, while non-improver and placebos showed stable or reverse gene expression over 24 weeks. Clinical improvement following abatacept therapy was associated with modulation of inflammatory pathways in skin. ClinicalTrials.gov NCT00442611 . Registered March 1, 2007.
    Preview · Article · Jun 2015 · Arthritis research & therapy
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    ABSTRACT: Genome-wide expression profiling in systemic sclerosis (SSc) has identified four 'intrinsic' subsets of disease (fibroproliferative, inflammatory, limited, and normal-like), each of which shows deregulation of distinct signaling pathways; however, the full set of pathways contributing to this differential gene expression has not been fully elucidated. Here we examine experimentally derived gene expression signatures in dermal fibroblasts for thirteen different signaling pathways implicated in SSc pathogenesis. These data show distinct and overlapping sets of genes induced by each pathway, allowing for a better understanding of the molecular relationship between profibrotic and immune signaling networks. Pathway-specific gene signatures were analyzed across a compendium of microarray datasets consisting of skin biopsies from three independent cohorts representing 80 SSc patients, 4 morphea, and 26 controls. IFNα signaling showed a strong association with early disease, while TGFβ signaling spanned the fibroproliferative and inflammatory subsets, was associated with worse MRSS, and was higher in lesional than non-lesional skin. The fibroproliferative subset was most strongly associated with PDGF signaling, while the inflammatory subset demonstrated strong activation of innate immune pathways including TLR signaling upstream of NF-κB. The limited and normal-like subsets did not show associations with fibrotic and inflammatory mediators such as TGFβ and TNFα. The normal-like subset showed high expression of genes associated with lipid signaling, which was absent in the inflammatory and limited subsets. Together, these data suggest a model by which IFNα is involved in early disease pathology, and disease severity is associated with active TGFβ signaling.
    Full-text · Article · Jan 2015 · PLoS ONE
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    ABSTRACT: Systemic sclerosis (SSc) is a rare systemic autoimmune disease characterized by skin and organ fibrosis. The pathogenesis of SSc and its progression are poorly understood. The SSc intrinsic gene expression subsets (inflammatory, fibroproliferative, normal-like, and limited) are observed in multiple clinical cohorts of patients with SSc. Analysis of longitudinal skin biopsies suggests that a patient's subset assignment is stable over 6-12 months. Genetically, SSc is multi-factorial with many genetic risk loci for SSc generally and for specific clinical manifestations. Here we identify the genes consistently associated with the intrinsic subsets across three independent cohorts, show the relationship between these genes using a gene-gene interaction network, and place the genetic risk loci in the context of the intrinsic subsets. To identify gene expression modules common to three independent datasets from three different clinical centers, we developed a consensus clustering procedure based on mutual information of partitions, an information theory concept, and performed a meta-analysis of these genome-wide gene expression datasets. We created a gene-gene interaction network of the conserved molecular features across the intrinsic subsets and analyzed their connections with SSc-associated genetic polymorphisms. The network is composed of distinct, but interconnected, components related to interferon activation, M2 macrophages, adaptive immunity, extracellular matrix remodeling, and cell proliferation. The network shows extensive connections between the inflammatory- and fibroproliferative-specific genes. The network also shows connections between these subset-specific genes and 30 SSc-associated polymorphic genes including STAT4, BLK, IRF7, NOTCH4, PLAUR, CSK, IRAK1, and several human leukocyte antigen (HLA) genes. Our analyses suggest that the gene expression changes underlying the SSc subsets may be long-lived, but mechanistically interconnected and related to a patients underlying genetic risk.
    Preview · Article · Jan 2015 · PLoS Computational Biology
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    ABSTRACT: Objective Accumulation of myofibroblasts in fibrotic skin is a hallmark of systemic sclerosis (SSc; scleroderma), but the origins of these cells remain unknown. Because loss of intradermal adipose tissue is a consistent feature of cutaneous fibrosis, we sought to examine the hypothesis that myofibroblasts populating fibrotic dermis derive from adipocytic progenitors.Methods We performed genetic fate mapping studies to investigate the loss of intradermal adipose tissue and its potential role in fibrosis in mice with bleomycin-induced scleroderma. Modulation of adipocytic phenotypes ex vivo was investigated in adipose tissue-derived cells in culture.ResultsA striking loss of intradermal adipose tissue and its replacement with fibrous tissue were consistently observed in mice with bleomycin-induced fibrosis. Loss of adipose tissue and a decline in the expression of canonical adipogenic markers in lesional skin preceded the onset of dermal fibrosis and expression of fibrogenic markers. Ex vivo, subcutaneous adipocytes were driven by transforming growth factor β to preferentially undergo fibrogenic differentiation. Cell fate mapping studies in mice with the adiponectin promoter-driven Cre recombinase transgenic construct indicated that adiponectin-positive progenitors that are normally confined to the intradermal adipose tissue compartment were distributed throughout the lesional dermis over time, lost their adipocytic markers, and expressed myofibroblast markers in bleomycin-treated mice.Conclusion These observations establish a novel link between intradermal adipose tissue loss and dermal fibrosis and demonstrate that adiponectin-positive intradermal progenitors give rise to dermal myofibroblasts. Adipose tissue loss and adipocyte-myofibroblast transition might be primary events in the pathogenesis of cutaneous fibrosis that represent novel potential targets for therapeutic intervention.
    Full-text · Article · Dec 2014 · Arthritis and Rheumatology
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    ABSTRACT: Objectives To investigate the role of microRNA-193b-3p (miR-193b) in the vascular pathophysiology of systemic sclerosis (SSc). Methods Expression of miR-193b in skin biopsies and fibroblasts from patients with SSc and normal healthy (NH) controls were determined by real-time PCR. Transfection with miR-193b precursor and inhibitor were used to confirm targets of miR-193b. Proliferative effects of urokinase-type plasminogen activator (uPA) were determined by water-soluble tetrazolium salt-1 assay and by analysis of proliferating cell nuclear antigen expression. Fluorescence activated cell sorting analysis was performed to investigate the effect of uPA on apoptosis. For inhibition of the uPA-cellular receptor for uPA (uPAR) pathway, uPAR neutralising antibodies and low molecular weight uPA were used. Results We found that miR-193b was downregulated in SSc fibroblasts and skin sections as compared with NH controls. The expression of miR-193b was not affected by major profibrotic cytokines and hypoxia. Induction of miR-193b in SSc fibroblasts suppressed, and accordingly, knockdown of miR-193b increased the levels of messenger RNA and protein for uPA. uPA was found to be upregulated in SSc as compared with NH controls in a transforming growth factor-β dependent manner, and uPA was strongly expressed in vascular smooth muscle cells in SSc skin section. Interestingly, uPA induced cell proliferation and inhibited apoptosis of human pulmonary artery smooth muscle cells, and these effects were independent of uPAR signalling. Conclusions In SSc, the downregulation of miR-193b induces the expression of uPA, which increases the number of vascular smooth muscle cells in an uPAR-independent manner and thereby contributes to the proliferative vasculopathy with intimal hyperplasia characteristic for SSc.
    No preview · Article · Nov 2014 · Annals of the Rheumatic Diseases
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    ABSTRACT: Systemic sclerosis (SSc) is a polygenic, autoimmune disorder of unknown etiology, characterized by the excessive accumulation of extracellular matrix (ECM) proteins, vascular alterations, and autoantibodies. The tight skin (Tsk)2/+ mouse model of SSc demonstrates signs similar to SSc including tight skin and excessive deposition of dermal ECM proteins. By linkage analysis, we mapped the Tsk2 gene mutation to less than 3 megabases on chromosome 1. We performed both RNA sequencing of skin transcripts and genome capture DNA sequencing of the region spanning this interval in Tsk2/+ and wild-type littermates. A missense point mutation in the procollagen III amino terminal propeptide segment (PIIINP) of Col3a1 was found to be the best candidate for Tsk2, so both in vivo and in vitro genetic complementation tests were used to prove that this Col3a1 mutation is the Tsk2 gene. All previously documented mutations in the human Col3a1 gene are associated with Ehlers-Danlos syndrome, a connective tissue disorder that leads to a defect in type III collagen synthesis. To our knowledge, the Tsk2 point mutation is the first documented gain-of-function mutation associated with Col3a1, which leads instead to fibrosis. This discovery provides insight into the mechanism of skin fibrosis manifested by Tsk2/+ mice.Journal of Investigative Dermatology accepted article preview online, 20 October 2014. doi:10.1038/jid.2014.455.
    Full-text · Article · Oct 2014 · Journal of Investigative Dermatology
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    ABSTRACT: Previous studies have suggested a role for pathogens as a trigger of systemic sclerosis (SSc), though neither a pathogen nor a mechanism of pathogenesis is known. Here we show enrichment of Rhodotorula sequences in the skin of patients with early, diffuse SSc compared to normal controls. RNA-seq was performed on four SSc and four controls, to a depth of 200 million reads per patient. Data were analyzed to quantify the non-human sequence reads in each sample. We found little difference between bacterial microbiome and viral read counts, but found a significant difference between the read counts for a mycobiome component, R. glutinis. Normal samples contained almost no detected R. glutinis or other Rhodotorula sequence reads (mean score 0.021 for R. glutinis, 0.024 for all Rhodotorula). In contrast, SSc samples had a mean score of 5.039 for R. glutinis (5.232 for Rhodotorula). We were able to assemble the D1-D2 hypervariable region of the 28 S rRNA of R. glutinis from each of the SSc samples. Taken together, these results suggest R. glutinis may be present in the skin of early SSc patients at higher levels than normal skin, raising the possibility that it may be triggering the inflammatory response found in SSc.Journal of Investigative Dermatology accepted article preview online, 7 March 2014; doi:10.1038/jid.2014.127.
    Full-text · Article · Mar 2014 · Journal of Investigative Dermatology
  • Y. Nesbeth · T. Wood · M. Hinchcliff · S. Podlusky · J. Reder · M. Whitfield

    No preview · Article · Mar 2014 · Clinical and experimental rheumatology
  • Michael L. Whitfield

    No preview · Article · Jan 2014 · Arthritis and Rheumatology
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    ABSTRACT: We identified the cell cycle-regulated mRNA transcripts genome-wide in the osteosarcoma derived U2OS cell line. This resulted in 2140 transcripts mapping to 1871 unique cell cycle-regulated genes that show periodic oscillations across multiple synchronous cell cycles. We identified genomic loci bound by the G2/M transcription factor FOXM1 by Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) and associated these with cell cycle-regulated genes. FOXM1 was bound to cell cycle-regulated genes with peak expression in both S phase and G2/M phases. ChIP-seq genomic loci were shown to be responsive to FOXM1 using a real-time luciferase assay in live cells, showing that FOXM1 strongly activates promoters of G2/M phase genes and weakly activates those induced in S phase. Analysis of ChIP-seq data from a panel of cell cycle-transcription factors (E2F1, E2F4, E2F6, and GABPA) from ENCODE and ChIP-seq data for the DREAM complex, found that a set of core cell cycle genes regulated in both U2OS and HeLa cells are bound by multiple cell cycle transcription factors. These data identify the cell cycle-regulated genes in a second cancer derived cell line and provide a comprehensive picture of the transcriptional regulatory systems controlling periodic gene expression in the human cell division cycle.
    Preview · Article · Oct 2013 · Molecular biology of the cell
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    Chao Cheng · Matthew Ung · Gavin D Grant · Michael L Whitfield
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    ABSTRACT: Author Summary Cell cycle is a complex and highly supervised process that must proceed with regulatory precision to achieve successful cellular division. Microarray time course experiments have been successfully used to identify cell cycle regulated genes but with several limitations, e.g. less effective in identifying genes with low expression. We propose a computational approach to predict cell cycle genes based on TF binding data and motif information in their promoters. Specifically, we take advantage of ChIP-seq TF binding data generated by the ENCODE project and the TF binding motif information available from public databases. These data were processed and utilized as predictor for predicting cell cycle genes using the Random Forest method. Our results show that both the trans- TF features and the cis- motif features are predictive to cell cycle genes, and a combination of the two types features can further improve prediction accuracy. We apply our model to a complete list of GENCODE promoters to predict novel cell cycle driving promoters for both protein-coding genes and non-coding RNAs such as lincRNAs. We find that a similar percentage of lincRNAs are cell cycle regulated as protein-coding genes, suggesting the importance of non-coding RNAs in cell cycle division.
    Full-text · Article · Jul 2013 · PLoS Computational Biology
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    ABSTRACT: Objective Pulmonary arterial hypertension (PAH), a common complication of limited cutaneous systemic sclerosis (lcSSc), is associated with alterations of markers of inflammation and vascular damage in peripheral blood mononuclear cells (PBMCs). Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been implicated in autoimmune and inflammatory diseases. The goal of this study was to assess whether markers of ER stress and the UPR are present in PBMCs from lcSSc patients with PAH. MethodsPBMCs were purified from 36 healthy controls, 32 lcSSc patients with PAH, and 34 lcSSc patients without PAH. Gene expression in healthy control PBMCs stimulated with thapsigargin was analyzed by DNA microarray. Genes were validated by quantitative real-time reverse transcription–polymerase chain reaction in PBMCs from healthy controls and lcSSc patients. ResultsSeveral ER stress/UPR genes, including BiP, activating transcription factor 4 (ATF-4), ATF-6, and a spliced form of X-box binding protein 1, were up-regulated in PBMCs from lcSSc patients, with the highest levels in patients with PAH. Thapsigargin up-regulated heat-shock proteins (HSPs) and interferon (IFN)–regulated genes in PBMCs from healthy controls. Selected HSP genes (particularly DnaJB1) and IFN-related genes were also found at significantly elevated levels in PBMCs from lcSSc patients, while IFN regulatory factor 4 expression was significantly decreased. There was a positive correlation between DnaJB1 and severity of PAH (measured by pulmonary artery pressure) (r = 0.56, P < 0.05) and between ER stress markers and interleukin-6 levels (r = 0.53, P < 0.0001) in PBMCs from lcSSc patients. Conclusion This study demonstrates an association between select ER stress/UPR markers and lcSSc with PAH, suggesting that ER stress and the UPR may contribute to the altered function of circulating immune cells in lcSSc.
    Full-text · Article · May 2013 · Arthritis & Rheumatology

Publication Stats

4k Citations
389.35 Total Impact Points

Institutions

  • 2015
    • Hospital for Special Surgery
      New York, New York, United States
  • 2006-2015
    • Geisel School of Medicine at Dartmouth
      • Department of Genetics
      Hanover, New Hampshire, United States
    • Princeton University
      • Department of Molecular Biology
      Princeton, New Jersey, United States
  • 2010-2012
    • Dartmouth College
      • Department of Genetics
      Hanover, New Hampshire, United States
  • 2009
    • Dartmouth–Hitchcock Medical Center
      Lebanon, New Hampshire, United States
  • 2005
    • Stanford Medicine
      • Department of Urology
      Stanford, California, United States
  • 2004
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2002-2004
    • Stanford University
      • Department of Genetics
      Palo Alto, California, United States
  • 2000-2004
    • University of North Carolina at Chapel Hill
      • • Department of Biochemistry and Biophysics
      • • Lineberger Comprehensive Cancer Center
      North Carolina, United States