Browsing by Author "Qian, Jiang"
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Item An all-to-all approach to the identification of sequence-specific readers for epigenetic DNA modifications on cytosine(Springer Nature, 2021-02-04) Song, Guang; Wang, Guohua; Luo, Ximei; Cheng, Ying; Song, Qifeng; Wan, Jun; Moore, Cedric; Song, Hongjun; Jin, Peng; Qian, Jiang; Zhu, Heng; Medical and Molecular Genetics, School of MedicineEpigenetic modifications of DNA play important roles in many biological processes. Identifying readers of these epigenetic marks is a critical step towards understanding the underlying mechanisms. Here, we present an all-to-all approach, dubbed digital affinity profiling via proximity ligation (DAPPL), to simultaneously profile human TF-DNA interactions using mixtures of random DNA libraries carrying different epigenetic modifications (i.e., 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine) on CpG dinucleotides. Many proteins that recognize consensus sequences carrying these modifications in symmetric and/or hemi-modified forms are identified. We further demonstrate that the modifications in different sequence contexts could either enhance or suppress TF binding activity. Moreover, many modifications can affect TF binding specificity. Furthermore, symmetric modifications show a stronger effect in either enhancing or suppressing TF-DNA interactions than hemi-modifications. Finally, in vivo evidence suggests that USF1 and USF2 might regulate transcription via hydroxymethylcytosine-binding activity in weak enhancers in human embryonic stem cells.Item 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 MedicineRecent 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.Item Astrocyte Ensheathment of Calyx-Forming Axons of the Auditory Brainstem Precedes Accelerated Expression of Myelin Genes and Myelination by Oligodendrocytes(Wiley, 2024) Heller, Daniel T.; Kolson, Douglas R.; Brandebura, Ashley N.; Amick, Emily M.; Wan, Jun; Ramadan, Jad; Holcomb, Paul S.; Liu, Sheng; Deerinck, Thomas J.; Ellisman, Mark H.; Qian, Jiang; Mathers, Peter H.; Spirou, George A.; Medical and Molecular Genetics, School of MedicineEarly postnatal brain development involves complex interactions among maturing neurons and glial cells that drive tissue organization. We previously analyzed gene expression in tissue from the mouse medial nucleus of the trapezoid body (MNTB) during the first postnatal week to study changes that surround rapid growth of the large calyx of Held (CH) nerve terminal. Here, we present genes that show significant changes in gene expression level during the second postnatal week, a developmental timeframe that brackets the onset of airborne sound stimulation and the early stages of myelination. Gene Ontology analysis revealed that many of these genes are related to the myelination process. Further investigation of these genes using a previously published cell type-specific bulk RNA-Seq data set in cortex and our own single-cell RNA-Seq data set in the MNTB revealed enrichment of these genes in the oligodendrocyte lineage (OL) cells. Combining the postnatal day (P)6-P14 microarray gene expression data with the previously published P0-P6 data provided fine temporal resolution to investigate the initiation and subsequent waves of gene expression related to OL cell maturation and the process of myelination. Many genes showed increasing expression levels between P2 and P6 in patterns that reflect OL cell maturation. Correspondingly, the first myelin proteins were detected by P4. Using a complementary, developmental series of electron microscopy 3D image volumes, we analyzed the temporal progression of axon wrapping and myelination in the MNTB. By employing a combination of established ultrastructural criteria to classify reconstructed early postnatal glial cells in the 3D volumes, we demonstrated for the first time that astrocytes within the mouse MNTB extensively wrap the axons of the growing CH terminal prior to OL cell wrapping and compaction of myelin. Our data revealed significant expression of several myelin genes and enrichment of multiple genes associated with lipid metabolism in astrocytes, which may subserve axon wrapping in addition to myelin formation. The transition from axon wrapping by astrocytes to OL cells occurs rapidly between P4 and P9 and identifies a potential new role of astrocytes in priming calyceal axons for subsequent myelination.Item Author Correction: An all-to-all approach to the identification of sequence-specific readers for epigenetic DNA modifications on cytosine(Springer Nature, 2021-02-23) Song, Guang; Wang, Guohua; Luo, Ximei; Cheng, Ying; Song, Qifeng; Wan, Jun; Moore, Cedric; Song, Hongjun; Jin, Peng; Qian, Jiang; Zhu, Heng; Medical and Molecular Genetics, School of MedicineCorrection to: Nature Communications 10.1038/s41467-021-20950-w, published online 04 February 2021. In the original version of this Article, the “Methods” section “Genome-wide hmC profiling of human embryonic stem cell H1” incorrectly stated “"Human embryonic stem cell H1 was purchased from WiCell Research Institute (WiCell) and the ethics approval was obtained from the Robert-Koch Institute, Berlin, Germany.”. Ethical approval was not required for the use of hESC H1 cells purchased from WiCell Research Institute. The statement has been corrected to “Human embryonic stem cell H1 was purchased from WiCell Research Institute (WiCell).” This has been corrected in the HTML and PDF version of this Article.Item 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 MedicineRetinal 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.Item 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 MedicinePurpose: 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.Item 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 MedicineUnderstanding 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/.Item 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 MedicineSingle-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.Item 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 MedicineStress 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.Item 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 MedicinePurpose: 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.