Browsing by Author "Qian, Jiang"

Now showing 1 - 8 of 8
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    Analysis of KLF4 regulated genes in cancer cells reveals a role of DNA methylation in promoter- enhancer interactions
    (Taylor & Francis, 2018) Oyinlade, Olutobi; Wei, Shuang; Kammers, Kai; Liu, Sheng; Wang, Shuyan; Ma, Ding; Huang, Zhi-yong; Qian, Jiang; Zhu, Heng; Wan, Jun; Xia, Shuli; Medical and Molecular Genetics, School of Medicine
    Recent studies have revealed an unexpected role of DNA methylation at promoter regions in transcription activation. However, whether DNA methylation at enhancer regions activates gene expression and influences cellular functions remains to be determined. In this study, by employing the transcription factor krÜppel-like factor 4 (KLF4) that binds to methylated CpGs (mCpGs), we investigated the molecular outcomes of the recruitment of KLF4 to mCpGs at enhancer regions in human glioblastoma cells. First, by integrating KLF4 ChIP-seq, whole-genome bisulfite sequence, and H3K27ac ChIP-seq datasets, we found 1,299 highly methylated (β >0.5) KLF4 binding sites, three-quarters of which were located at putative enhancer regions, including gene bodies and intergenic regions. In the meantime, by proteomics, we identified 16 proteins as putative targets upregulated by KLF4-mCpG binding at enhancer regions. By chromosome conformation capture (3C) analysis, we demonstrated that KLF4 bound to methylated CpGs at the enhancer regions of the B-cell lymphocyte kinase (BLK) and Lim domain only protein 7 (LMO7) genes, and activated their expression via 3D chromatin loop formation with their promoter regions. Expression of mutant KLF4, which lacks KLF4 ability to bind methylated DNA, or removal of DNA methylation in enhancer regions by a DNA methyltransferase inhibitor abolished chromatin loop formation and gene expression, suggesting the essential role of DNA methylation in enhancer-promoter interactions. Finally, we performed functional assays and showed that BLK was involved in glioblastoma cell migration. Together, our study established the concept that DNA methylation at enhancer regions interacts with transcription factors to activate gene expression and influence cellular functions.
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    Epigenomic profiling of retinal progenitors reveals LHX2 is required for developmental regulation of open chromatin
    (Springer Nature, 2019-04-25) Zibetti, Cristina; Liu, Sheng; Wan, Jun; Qian, Jiang; Blackshaw, Seth; Medical and Molecular Genetics, School of Medicine
    Retinal neurogenesis occurs through partially overlapping temporal windows, driven by concerted actions of transcription factors which, in turn, may contribute to the establishment of divergent genetic programs in the developing retina by coordinating variations in chromatin landscapes. Here we comprehensively profile murine retinal progenitors by integrating next generation sequencing methods and interrogate changes in chromatin accessibility at embryonic and post-natal stages. An unbiased search for motifs in open chromatin regions identifies putative factors involved in the developmental progression of the epigenome in retinal progenitor cells. Among these factors, the transcription factor LHX2 exhibits a developmentally regulated cis-regulatory repertoire and stage-dependent motif instances. Using loss-of-function assays, we determine LHX2 coordinates variations in chromatin accessibility, by competition for nucleosome occupancy and secondary regulation of candidate pioneer factors.
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    Integrated Stress Response and Decreased ECM in Cultured Stromal Cells From Keratoconus Corneas
    (Association for Research in Vision and Ophthalmology, 2018-06-01) Foster, James W.; Shinde, Vishal; Soiberman, Uri S.; Sathe, Gajanan; Liu, Sheng; Wan, Julius; Qian, Jiang; Dauoud, Yassine; Pandey, Akhilesh; Jun, Albert S.; Chakravarti, Shukti; Medical and Molecular Genetics, School of Medicine
    Purpose: Keratoconus (KC) is a multifactorial disease where progressive thinning and weakening of the cornea leads to loss of visual acuity. Although the underlying etiology is poorly understood, a major endpoint is a dysfunctional stromal connective tissue matrix. Using multiple individual KC corneas, we determined that matrix production by keratocytes is severely impeded due to an altered stress response program. Methods: KC and donor (DN) stromal keratocytes were cultured in low glucose serum-free medium containing insulin, selenium and transferrin. Fibronectin, collagens and proteins related to their chaperone, processing and export, matrix metalloproteinase, and stress response related proteins were investigated by immunoblotting, immunocytochemistry, hydroxyproline quantification, and gelatin zymography. Multiplexed mass spectrometry was used for global proteomic profiling of 5 individual DN and KC cell culture. Transcription of selected proteins was assayed by qPCR. Results: DN and KC cells showed comparable survival and growth. However, immunoblotting of selected ECM proteins and global proteomics showed decreased fibronectin, collagens, PCOLCE, ADAMTS2, BMP1, HSP47, other structural and cytoskeletal proteins in KC. Phosphorylated (p) eIF2α, a translation regulator and its target, ATF4 were increased in KC cultured cells and corneal sections. Conclusions: The profound decrease in structural proteins in cultured KC cells and increase in the p-eIF2α, and ATF4, suggest a stress related blockade in structural proteins not immediately needed for cell survival. Therefore, this cell culture system reveals an intrinsic aggravated stress response with consequent decrease in ECM proteins as potential pathogenic underpinnings in KC.
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    MeDReaders: a database for transcription factors that bind to methylated DNA
    (Oxford Academic, 2018-01-04) Wang, Guohua; Luo, Ximei; Wang, Jianan; Wan, Jun; Xia, Shuli; Zhu, Heng; Qian, Jiang; Wang, Yadong; Medical and Molecular Genetics, School of Medicine
    Understanding the molecular principles governing interactions between transcription factors (TFs) and DNA targets is one of the main subjects for transcriptional regulation. Recently, emerging evidence demonstrated that some TFs could bind to DNA motifs containing highly methylated CpGs both in vitro and in vivo. Identification of such TFs and elucidation of their physiological roles now become an important stepping-stone toward understanding the mechanisms underlying the methylation-mediated biological processes, which have crucial implications for human disease and disease development. Hence, we constructed a database, named as MeDReaders, to collect information about methylated DNA binding activities. A total of 731 TFs, which could bind to methylated DNA sequences, were manually curated in human and mouse studies reported in the literature. In silico approaches were applied to predict methylated and unmethylated motifs of 292 TFs by integrating whole genome bisulfite sequencing (WGBS) and ChIP-Seq datasets in six human cell lines and one mouse cell line extracted from ENCODE and GEO database. MeDReaders database will provide a comprehensive resource for further studies and aid related experiment designs. The database implemented unified access for users to most TFs involved in such methylation-associated binding actives. The website is available at http://medreader.org/.
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    PanoView: An iterative clustering method for single-cell RNA sequencing data
    (PLOS, 2019-08-30) Hu, Ming-Wen; Kim, Dong Won; Liu, Sheng; Zack, Donald J.; Blackshaw, Seth; Qian, Jiang; Medical and Molecular Genetics, School of Medicine
    Single-cell RNA-sequencing (scRNA-seq) provides new opportunities to gain a mechanistic understanding of many biological processes. Current approaches for single cell clustering are often sensitive to the input parameters and have difficulty dealing with cell types with different densities. Here, we present Panoramic View (PanoView), an iterative method integrated with a novel density-based clustering, Ordering Local Maximum by Convex hull (OLMC), that uses a heuristic approach to estimate the required parameters based on the input data structures. In each iteration, PanoView will identify the most confident cell clusters and repeat the clustering with the remaining cells in a new PCA space. Without adjusting any parameter in PanoView, we demonstrated that PanoView was able to detect major and rare cell types simultaneously and outperformed other existing methods in both simulated datasets and published single-cell RNA-sequencing datasets. Finally, we conducted scRNA-Seq analysis of embryonic mouse hypothalamus, and PanoView was able to reveal known cell types and several rare cell subpopulations.
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    Proteome Landscape of Epithelial-to-Mesenchymal Transition (EMT) of Retinal Pigment Epithelium Shares Commonalities With Malignancy-Associated EMT
    (American Society for Biochemistry and Molecular Biology, 2021) Sripathi, Srinivasa R.; Hu, Ming-Wen; Turaga, Ravi Chakra; Mertz, Joseph; Liu, Melissa M.; Wan, Jun; Maruotti, Julien; Wahlin, Karl J.; Berlinicke, Cynthia A.; Qian, Jiang; Zack, Donald J.; Medical and Molecular Genetics, School of Medicine
    Stress and injury to the retinal pigment epithelium (RPE) often lead to dedifferentiation and epithelial-to-mesenchymal transition (EMT). These processes have been implicated in several retinal diseases, including proliferative vitreoretinopathy, diabetic retinopathy, and age-related macular degeneration. Despite the importance of RPE-EMT and the large body of data characterizing malignancy-related EMT, comprehensive proteomic studies to define the protein changes and pathways underlying RPE-EMT have not been reported. This study sought to investigate the temporal protein expression changes that occur in a human-induced pluripotent stem cell-based RPE-EMT model. We utilized multiplexed isobaric tandem mass tag labeling followed by high-resolution tandem MS for precise and in-depth quantification of the RPE-EMT proteome. We have identified and quantified 7937 protein groups in our tandem mass tag-based MS analysis. We observed a total of 532 proteins that are differentially regulated during RPE-EMT. Furthermore, we integrated our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into RPE-EMT mechanisms. To validate these results, we have performed a label-free single-shot data-independent acquisition MS study. Our integrated analysis indicates both the commonality and uniqueness of RPE-EMT compared with malignancy-associated EMT. Our comparative analysis also revealed that multiple age-related macular degeneration-associated risk factors are differentially regulated during RPE-EMT. Together, our integrated dataset provides a comprehensive RPE-EMT atlas and resource for understanding the molecular signaling events and associated biological pathways that underlie RPE-EMT onset. This resource has already facilitated the identification of chemical modulators that could inhibit RPE-EMT, and it will hopefully aid in ongoing efforts to develop EMT inhibition as an approach for the treatment of retinal disease.
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    Transcriptome Landscape of Epithelial to Mesenchymal Transition of Human Stem Cell–Derived RPE
    (Association for Research in Vision and Ophthalmology, 2021-04) Sripathi, Srinivasa R.; Hu, Ming-Wen; Liu, Melissa M.; Wan, Jun; Cheng, Jie; Duan, Yukan; Mertz, Joseph L.; Wahlin, Karl J.; Maruotti, Julien; Berlinicke, Cynthia A.; Qian, Jiang; Zack, Donald J.; Medical and Molecular Genetics, School of Medicine
    Purpose: RPE injury often induces epithelial to mesenchymal transition (EMT). Although RPE-EMT has been implicated in a variety of retinal diseases, including proliferative vitroretinopathy, neovascular and atrophic AMD, and diabetic retinopathy, it is not well-understood at the molecular level. To contribute to our understanding of EMT in human RPE, we performed a time-course transcriptomic analysis of human stem cell-derived RPE (hRPE) monolayers induced to undergo EMT using 2 independent, yet complementary, model systems. Methods: EMT of human stem cell-derived RPE monolayers was induced by either enzymatic dissociation or modulation of TGF-β signaling. Transcriptomic analysis of cells at different stages of EMT was performed by RNA-sequencing, and select findings were confirmed by reverse transcription quantitative PCR and immunostaining. An ingenuity pathway analysis (IPA) was performed to identify signaling pathways and regulatory networks associated with EMT. Results: Proteocollagenolytic enzymatic dissociation and cotreatment with TGF-β and TNF-α both induce EMT in human stem cell-derived RPE monolayers, leading to an increased expression of mesenchymal factors and a decreased expression of RPE differentiation-associated factors. Ingenuity pathway analysis identified the upstream regulators of the RPE-EMT regulatory networks and identified master switches and nodes during RPE-EMT. Of particular interest was the identification of widespread dysregulation of axon guidance molecules during RPE-EMT progression. Conclusions: The temporal transcriptome profiles described here provide a comprehensive resource of the dynamic signaling events and the associated biological pathways that underlie RPE-EMT onset. The pathways defined by these studies may help to identify targets for the development of novel therapeutic targets for the treatment of retinal disease.
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    Whole Exome Sequencing Identifies PHF14 Mutations in Neurocytoma and Predicts Responsivity to the PDGFR Inhibitor Sunitinib
    (MDPI, 2022-11-08) Zhang, Dongyun; Yong, William H.; Movassaghi, Masoud; Rodriguez, Fausto J.; Yang, Issac; McKeever, Paul; Qian, Jiang; Li, Jian Yi; Mao, Qinwen; Newell, Kathy L.; Green, Richard M.; Welsh, Cynthia T.; Heaney, Anthony P.; Pathology and Laboratory Medicine, School of Medicine
    Neurocytomas are rare low-grade brain tumors predominantly affecting young adults, but their cellular origin and molecular pathogenesis is largely unknown. We previously reported a sellar neurocytoma that secreted excess arginine vasopressin causing syndrome of inappropriate anti-diuretic hormone (SIADH). Whole exome sequencing in 21 neurocytoma tumor tissues identified somatic mutations in the plant homeodomain finger protein 14 (PHF14) in 3/21 (14%) tumors. Of these mutations, two were missense mutations and 4 caused splicing site losses, resulting in PHF14 dysfunction. Employing shRNA-mediated knockdown and CRISPR/Cas9-based knockout approaches, we demonstrated that loss of PHF14 increased proliferation and colony formation in five different human, mouse and rat mesenchymal and differentiated cell lines. Additionally, we demonstrated that PHF14 depletion resulted in upregulation of platelet derived growth factor receptor-alpha (PDGFRα) mRNA and protein in neuroblastoma SHSY-5Y cells and led to increased sensitivity to treatment with the PDGFR inhibitor Sunitinib. Furthermore, in a neurocytoma primary culture harboring splicing loss PHF14 mutations, overexpression of wild-type PHF14 and sunitinib treatment inhibited cell proliferation. Nude mice, inoculated with PHF14 knockout SHSY-5Y cells developed earlier and larger tumors than control cell-inoculated mice and Sunitinib administration caused greater tumor suppression in mice harboring PHF-14 knockout than control SHSY-5Y cells. Altogether our studies identified mutations of PHF14 in 14% of neurocytomas, demonstrate it can serve as an alternative pathway for certain cancerous behavior, and suggest a potential role for Sunitinib treatment in some patients with residual/recurrent neurocytoma.