Browsing by Author "Chen, Duojiao"
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Item A human skeletal muscle stem/myotube model reveals multiple signaling targets of cancer secretome in skeletal muscle(Elsevier, 2023-03-31) Wang, Ruizhong; Kumar, Brijesh; Bhat-Nakshatri, Poornima; Khatpe, Aditi S.; Murphy, Michael P.; Wanczyk, Kristen E.; Simpson, Edward; Chen, Duojiao; Gao, Hongyu; Liu, Yunlong; Doud, Emma H.; Mosley, Amber L.; Nakshatri, Harikrishna; Surgery, School of MedicineSkeletal muscle dysfunction or reprogramming due to the effects of the cancer secretome is observed in multiple malignancies. Although mouse models are routinely used to study skeletal muscle defects in cancer, because of species specificity of certain cytokines/chemokines in the secretome, a human model system is required. Here, we establish simplified multiple skeletal muscle stem cell lines (hMuSCs), which can be differentiated into myotubes. Using single nuclei ATAC-seq (snATAC-seq) and RNA-seq (snRNA-seq), we document chromatin accessibility and transcriptomic changes associated with the transition of hMuSCs to myotubes. Cancer secretome accelerated stem to myotube differentiation, altered the alternative splicing machinery and increased inflammatory, glucocorticoid receptor, and wound healing pathways in hMuSCs. Additionally, cancer secretome reduced metabolic and survival pathway associated miR-486, AKT, and p53 signaling in hMuSCs. hMuSCs underwent myotube differentiation when engrafted into NSG mice and thus providing a humanized in vivo skeletal muscle model system to study cancer cachexia.Item Author Correction: Lipid exposure activates gene expression changes associated with estrogen receptor negative breast cancer(Springer Nature, 2023-03-13) Yadav, Shivangi; Virk, Ranya; Chung, Carolina H.; Bustamante Eduardo, Mariana; VanDerway, David; Chen, Duojiao; Burdett, Kirsten; Gao, Hongyu; Zeng, Zexian; Ranjan, Manish; Cottone, Gannon; Xuei, Xiaoling; Chandrasekaran, Sriram; Backman, Vadim; Chatterton, Robert; Khan, Seema Ahsan; Clare, Susan E.; Medical and Molecular Genetics, School of MedicineCorrection to: npj Breast Cancer 10.1038/s41523-022-00422-0, published online 04 May 2022 In this article, funding from the National Institutes of Health (award number R01CA228272) was inadvertently omitted. The original article has been corrected.Item Cryopreservation Preserves Cell-Type Composition and Gene Expression Profiles in Bone Marrow Aspirates From Multiple Myeloma Patients(Frontiers Media, 2021-04-21) Chen, Duojiao; Abu Zaid, Mohammad I.; Reiter, Jill L.; Czader, Magdalena; Wang, Lin; McGuire, Patrick; Xuei, Xiaoling; Gao, Hongyu; Huang, Kun; Abonour, Rafat; Walker, Brian A.; Liu, Yunlong; Medical and Molecular Genetics, School of MedicineSingle-cell RNA sequencing reveals gene expression differences between individual cells and also identifies different cell populations that are present in the bulk starting material. To obtain an accurate assessment of patient samples, single-cell suspensions need to be generated as soon as possible once the tissue or sample has been collected. However, this requirement poses logistical challenges for experimental designs involving multiple samples from the same subject since these samples would ideally be processed at the same time to minimize technical variation in data analysis. Although cryopreservation has been shown to largely preserve the transcriptome, it is unclear whether the freeze-thaw process might alter gene expression profiles in a cell-type specific manner or whether changes in cell-type proportions might also occur. To address these questions in the context of multiple myeloma clinical studies, we performed single-cell RNA sequencing (scRNA-seq) to compare fresh and frozen cells isolated from bone marrow aspirates of six multiple myeloma patients, analyzing both myeloma cells (CD138+) and cells constituting the microenvironment (CD138-). We found that cryopreservation using 90% fetal calf serum and 10% dimethyl sulfoxide resulted in highly consistent gene expression profiles when comparing fresh and frozen samples from the same patient for both CD138+ myeloma cells (R ≥ 0.96) and for CD138- cells (R ≥ 0.9). We also demonstrate that CD138- cell-type proportions showed minimal alterations, which were mainly related to small differences in immune cell subtype sensitivity to the freeze-thaw procedures. Therefore, when processing fresh multiple myeloma samples is not feasible, cryopreservation is a useful option in single-cell profiling studies.Item Deciphering Gene Regulatory Mechanisms Through Multi-omics Integration(2022-09) Chen, Duojiao; Liu, Yunlong; Wan, Jun; Zhang, Chi; Yan, JingwenComplex biological systems are composed of many regulatory components, which can be measured with the advent of genomics technology. Each molecular assay is normally designed to interrogate one aspect of the cell state. However, a comprehensive understanding of the regulatory mechanism requires characterization from multiple levels such as genome, epigenome, and transcriptome. Integration of multi-omics data is urgently needed for understanding the global regulatory mechanism of gene expression. In recent years, single-cell technology offers unprecedented resolution for a deeper characterization of cellular diversity and states. High-quality single-cell suspensions from tissue biopsies are required for single-cell sequencing experiments. Tissue biopsies need to be processed as soon as being collected to avoid gene expression changes and RNA degradation. Although cryopreservation is a feasible solution to preserve freshly isolated samples, its effect on transcriptome profiles still needs to be investigated. Investigation of multi-omics data at the single-cell level can provide new insights into the biological process. In addition to the common method of integrating multi-omics data, it is also capable of simultaneously profiling the transcriptome and epigenome at single-cell resolution, enhancing the power of discovering new gene regulatory interactions. In this dissertation, we integrated bulk RNA-seq with ATAC-seq and several additional assays and revealed the complex mechanisms of ER–E2 interaction with nucleosomes. A comparison analysis was conducted for comparing fresh and frozen multiple myeloma single-cell RNA sequencing data and concluded that cryopreservation is a feasible protocol for preserving cells. We also analyzed the single-cell multiome data for mesenchymal stem cells. With the unified landscape from simultaneously profiling gene expression and chromatin accessibility, we discovered distinct osteogenic differentiation potential of mesenchymal stem cells and different associations with bone disease-related traits. We gained a deeper insight into the underlying gene regulatory mechanisms with this frontier single-cell mutliome sequencing technique.Item Lipid exposure activates gene expression changes associated with estrogen receptor negative breast cancer(Springer Nature, 2022-05-04) Yadav, Shivangi; Virk, Ranya; Chung, Carolina H.; Bustamante Eduardo, Mariana; VanDerway, David; Chen, Duojiao; Burdett, Kirsten; Gao, Hongyu; Zeng, Zexian; Ranjan, Manish; Cottone, Gannon; Xuei, Xiaoling; Chandrasekaran, Sriram; Backman, Vadim; Chatterton, Robert; Khan, Seema Ahsan; Clare, Susan E.; Medical and Molecular Genetics, School of MedicineImproved understanding of local breast biology that favors the development of estrogen receptor negative (ER-) breast cancer (BC) would foster better prevention strategies. We have previously shown that overexpression of specific lipid metabolism genes is associated with the development of ER- BC. We now report results of exposure of MCF-10A and MCF-12A cells, and mammary organoids to representative medium- and long-chain polyunsaturated fatty acids. This exposure caused a dynamic and profound change in gene expression, accompanied by changes in chromatin packing density, chromatin accessibility, and histone posttranslational modifications (PTMs). We identified 38 metabolic reactions that showed significantly increased activity, including reactions related to one-carbon metabolism. Among these reactions are those that produce S-adenosyl-L-methionine for histone PTMs. Utilizing both an in-vitro model and samples from women at high risk for ER- BC, we show that lipid exposure engenders gene expression, signaling pathway activation, and histone marks associated with the development of ER- BC.Item Loss of succinyl-CoA synthetase in mouse forebrain results in hypersuccinylation with perturbed neuronal transcription and metabolism(Elsevier, 2023) Lancaster, Makayla S.; Kim, Byungwook; Doud, Emma H.; Tate, Mason D.; Sharify, Ahmad D.; Gao, Hongyu; Chen, Duojiao; Simpson, Ed; Gillespie, Patrick; Chu, Xiaona; Miller, Marcus J.; Wang, Yue; Liu, Yunlong; Mosley, Amber L.; Kim, Jungsu; Graham, Brett H.; Medical and Molecular Genetics, School of MedicineLysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS complex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.Item Nonlinear relationship between chromatin accessibility and estradiol-regulated gene expression(Nature, 2021-02) Chen, Duojiao; Parker, Taylor M.; Bhat-Nakshatri, Poornima; Chu, Xiaona; Liu, Yunlong; Wang, Yue; Nakshatri, Harikrishna; Surgery, School of MedicineChromatin accessibility is central to basal and inducible gene expression. Through ATAC-seq experiments in estrogen receptor-positive (ER+) breast cancer cell line MCF-7 and integration with multi-omics data, we found estradiol (E2) induced chromatin accessibility changes in a small number of breast cancer-relevant E2-regulated genes. As expected, open chromatin regions associated with E2-inducible gene expression showed enrichment of estrogen response element (ERE) and those associated with E2-repressible gene expression were enriched for ERE, PBX1, and PBX3. While a significant number of open chromatin regions showed pioneer factor FOXA1 occupancy in the absence of E2, E2-treatment further enhanced FOXA1 occupancy suggesting that ER–E2 enhances chromatin occupancy of FOXA1 to a subset of E2-regulated genes. Surprisingly, promoters of 80% and enhancers of 60% of E2-inducible genes displayed closed chromatin configuration both in the absence and presence of E2. Integration of ATAC-seq data with ERα ChIP-seq data revealed that ~40% ERα binding sites in the genome are found in chromatin regions that are not accessible as per ATAC-seq. Such ERα binding regions were enriched for binding sites of multiple nuclear receptors including ER, ESRRB, ERRγ, COUP-TFII (NR2F2), RARα, EAR2 as well as traditional pioneer factors FOXA1 and GATA3. Similar data were also obtained when ERα ChIP-seq data were integrated with MNase-seq and DNase-seq data sets. In summation, our results reveal complex mechanisms of ER–E2 interaction with nucleosomes. Notably, “closed chromatin” configuration as defined by ATAC-seq or by other techniques is not necessarily associated with lack of gene expression and technical limitations may preclude ATAC-seq to demonstrate accessibility of chromatin regions that are bound by ERα.Item Osteogenic Differentiation Potential of Mesenchymal Stem Cells Using Single Cell Multiomic Analysis(MDPI, 2023-09-26) Chen, Duojiao; Liu, Sheng; Chu, Xiaona; Reiter, Jill; Gao, Hongyu; McGuire, Patrick; Yu, Xuhong; Xuei, Xiaoling; Liu, Yichen; Wan, Jun; Fang, Fang; Liu, Yunlong; Wang, Yue; Medical and Molecular Genetics, School of MedicineMesenchymal stem cells (MSC) are multipotent stem cells that can differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes. Osteoblast differentiation is reduced during osteoporosis development, resulting in reduced bone formation. Further, MSC isolated from different donors possess distinct osteogenic capacity. In this study, we used single-cell multiomic analysis to profile the transcriptome and epigenome of MSC from four healthy donors. Data were obtained from ~1300 to 1600 cells for each donor. These cells were clustered into four groups, indicating that MSC from different donors have distinct chromatin accessible regulatory elements for regulating gene expression. To investigate the mechanism by which MSC undergo osteogenic differentiation, we used the chromatin accessibility data from the single-cell multiome data to identify individual-specific enhancer–promoter pairs and evaluated the expression levels and activities of the transcriptional regulators. The MSC from four donors showed distinct differentiation potential into osteoblasts. MSC of donor 1 showed the largest average motif activities, indicating that MSC from donor 1 was most likely to differentiate into osteoblasts. The results of our validation experiments were consistent with the bioinformatics prediction. We also tested the enrichment of genome-wide association study (GWAS) signals of several musculoskeletal disease traits in the patient-specific chromatin accessible regions identified in the single-cell multiome data, including osteoporosis, osteopenia, and osteoarthritis. We found that osteoarthritis-associated variants were only enriched in the regions identified from donor 4. In contrast, osteoporosis and osteopenia variants were enriched in regions from donor 1 and least enriched in donor 4. Since osteoporosis and osteopenia are related to the density of bone cells, the enrichment of variants from these traits should be correlated with the osteogenic potential of MSC. In summary, this study provides large-scale data to link regulatory elements with their target genes to study the regulatory relationships during the differentiation of mesenchymal stem cells and provide a deeper insight into the gene regulatory mechanism.Item Signaling Pathway Alterations Driven by BRCA1 and BRCA2 Germline Mutations are Sufficient to Initiate Breast Tumorigenesis by the PIK3CAH1047R Oncogene(American Association for Cancer Research, 2024) Bhat-Nakshatri, Poornima; Khatpe, Aditi S.; Chen, Duojiao; Batic, Katie; Mang, Henry; Herodotou, Christopher; McGuire, Patrick C.; Xuei, Xiaoling; Erdogan, Cihat; Gao, Hongyu; Liu, Yunlong; Sandusky, George; Storniolo, Anna Maria; Nakshatri, Harikrishna; Surgery, School of MedicineSingle-cell transcriptomics studies have begun to identify breast epithelial cell and stromal cell specific transcriptome differences between BRCA1/2 mutation carriers and non-carriers. We generated a single-cell transcriptome atlas of breast tissues from BRCA1, BRCA2 mutation carriers and compared this single-cell atlas of mutation carriers with our previously described single-cell breast atlas of healthy non-carriers. We observed that BRCA1 but not BRCA2 mutations altered the ratio between basal (basal-myoepithelial), luminal progenitor (luminal adaptive secretory precursor, LASP), and mature luminal (luminal hormone sensing) cells in breast tissues. A unique subcluster of cells within LASP cells is underrepresented in case of BRCA1 and BRCA2 mutation carriers compared with non-carriers. Both BRCA1 and BRCA2 mutations specifically altered transcriptomes in epithelial cells which are an integral part of NFκB, LARP1, and MYC signaling. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included STAT3, BRD4, SMARCA4, HIF2A/EPAS1, and Inhibin A signaling. BRCA2 mutations were associated with upregulation of IL6, PDK1, FOXO3, and TNFSF11 signaling. These signaling pathway alterations are sufficient to alter sensitivity of BRCA1/BRCA2-mutant breast epithelial cells to transformation as epithelial cells from BRCA1 mutation carriers overexpressing hTERT + PIK3CAH1047R generated adenocarcinomas, whereas similarly modified mutant BRCA2 cells generated basal carcinomas in NSG mice. Thus, our studies provide a high-resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers and reveal their susceptibility to PIK3CA mutation-driven transformation. Significance: This study provides a single-cell atlas of breast tissues of BRCA1/2 mutation carriers and demonstrates that aberrant signaling due to BRCA1/2 mutations is sufficient to initiate breast cancer by mutant PIK3CA.Item TONSL is an immortalizing oncogene and a therapeutic target in breast cancer(American Association for Cancer Research, 2023) Khatpe, Aditi S.; Dirks, Rebecca; Bhat-Nakshatri, Poornima; Mang, Henry; Batic, Katie; Swiezy, Sarah; Olson, Jacob; Rao, Xi; Wang, Yue; Tanaka, Hiromi; Liu, Sheng; Wan, Jun; Chen, Duojiao; Liu, Yunlong; Fang, Fang; Althouse, Sandra; Hulsey, Emily; Granatir, Maggie M.; Addison, Rebekah; Temm, Constance J.; Sandusky, George; Lee-Gosselin, Audrey; Nephew, Kenneth; Miller, Kathy D.; Nakshatri, Harikrishna; Surgery, School of MedicineStudy of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to the lack of isogenic models. To address this, we utilized healthy primary breast luminal epithelial cells of different genetic ancestry and their hTERT-immortalized counterparts to identify transcriptomic changes associated with immortalization. Elevated expression of TONSL (Tonsoku Like, DNA Repair Protein) was identified as one of the earliest events during immortalization. TONSL, which is located on chromosome 8q24.3, was found to be amplified in ~20% of breast cancers. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity, but overexpression was insufficient for neoplastic transformation. However, TONSL-immortalized primary cells overexpressing defined oncogenes generated estrogen receptor-positive adenocarcinomas in mice. Analysis of a breast tumor microarray with ~600 tumors revealed poor overall and progression free survival of patients with TONSL overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors including NF-κB and limited access to the tumor suppressor p53. TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in the homologous recombination (HR) and Fanconi Anemia pathways. Consistent with these results, TONSL overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was essential for growth of TONSL-amplified breast cancer cell lines in vivo, and these cells were sensitive to TONSL-FACT complex inhibitor CBL0137. Together, these findings identify TONSL as a regulator of epithelial cell immortalization to facilitate cancer initiation and as a target for breast cancer therapy.