IU Indianapolis ScholarWorks :: Browsing by Author "Roh, Hyun Cheol"

Browsing by Author "Roh, Hyun Cheol"

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Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting
(MyJove Corporation, 2023-03-17) Kim, Kyungchan; Taleb, Solaema; So, Jisun; Wann, Jamie; Roh, Hyun Cheol; Biochemistry and Molecular Biology, School of Medicine
Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin accessibility profiling. This technique has been useful for understanding the regulatory mechanisms of gene expression in a range of biological processes. Although ATAC-seq has been modified for different types of samples, there have not been effective modifications of ATAC-seq methods for adipose tissues. Challenges with adipose tissues include the complex cellular heterogeneity, large lipid content, and high mitochondrial contamination. To overcome these problems, we have developed a protocol that allows adipocyte-specific ATAC-seq by employing fluorescence-activated nucleus sorting with adipose tissues from the transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mouse. This protocol produces high-quality data with minimal wasted sequencing reads while reducing the amount of nucleus input and reagents. This paper provides detailed step-by-step instructions for the ATAC-seq method validated for the use of adipocyte nuclei isolated from mouse adipose tissues. This protocol will aid in the investigation of chromatin dynamics in adipocytes upon diverse biological stimulations, which will allow for novel biological insights.
Adipocytes fail to maintain cellular identity during obesity due to reduced PPARγ activity and elevated TGFβ-SMAD signaling
(Elsevier, 2020-09-28) Roh, Hyun Cheol; Kumari, Manju; Taleb, Solaema; Tenen, Danielle; Jacobs, Christopher; Lyubetskaya, Anna; Tsai, Linus T. -Y.; Rosen, Evan D.; Biochemistry and Molecular Biology, School of Medicine
Objective Obesity due to overnutrition causes adipose tissue dysfunction, which is a critical pathological step on the road to type 2 diabetes (T2D) and other metabolic disorders. In this study, we conducted an unbiased investigation into the fundamental molecular mechanisms by which adipocytes transition to an unhealthy state during obesity. Methods We used nuclear tagging and translating ribosome affinity purification (NuTRAP) reporter mice crossed with Adipoq-Cre mice to determine adipocyte-specific 1) transcriptional profiles (RNA-seq), 2) promoter and enhancer activity (H3K27ac ChIP-seq), 3) and PPARγ cistrome (ChIP-seq) profiles in mice fed chow or a high-fat diet (HFD) for 10 weeks. We also assessed the impact of the PPARγ agonist rosiglitazone (Rosi) on gene expression and cellular state of adipocytes from the HFD-fed mice. We integrated these data to determine the transcription factors underlying adipocyte responses to HFD and conducted functional studies using shRNA-mediated loss-of-function approaches in 3T3-L1 adipocytes. Results Adipocytes from the HFD-fed mice exhibited reduced expression of adipocyte markers and metabolic genes and enhanced expression of myofibroblast marker genes involved in cytoskeletal organization, accompanied by the formation of actin filament structures within the cell. PPARγ binding was globally reduced in adipocytes after HFD feeding, and Rosi restored the molecular and cellular phenotypes of adipocytes associated with HFD feeding. We identified the TGFβ1 effector protein SMAD to be enriched at HFD-induced promoters and enhancers and associated with myofibroblast signature genes. TGFβ1 treatment of mature 3T3-L1 adipocytes induced gene expression and cellular changes similar to those seen after HFD in vivo, and knockdown of Smad3 blunted the effects of TGFβ1. Conclusions Our data demonstrate that adipocytes fail to maintain cellular identity after HFD feeding, acquiring characteristics of a myofibroblast-like cell type through reduced PPARγ activity and elevated TGFβ-SMAD signaling. This cellular identity crisis may be a fundamental mechanism that drives functional decline of adipose tissues during obesity.
Chronic cAMP activation induces adipocyte browning through discordant biphasic remodeling of transcriptome and chromatin accessibility
(Elsevier, 2022) So, Jisun; Taleb, Solaema; Wann, Jamie; Strobel, Olivia; Kim, Kyungchan; Roh, Hyun Cheol; Biochemistry and Molecular Biology, School of Medicine
Objective: Adipose tissue thermogenesis has been suggested as a new therapeutic target to promote energy metabolism for obesity and metabolic disease. Cold-inducible thermogenic adipocytes, called beige adipocytes, have attracted significant attention for their potent anti-obesity activity in adult humans. In this study, we identified the mechanisms underlying beige adipocyte recruitment, so-called adipocyte browning, by different stimuli. Methods: We generated a new adipocyte cell line with enhanced browning potentials and determined its transcriptomic and epigenomic responses following cAMP (forskolin, FSK) versus PPARγ activation (rosiglitazone). We performed time-course RNA-seq and compared the treatments and in vivo adipocyte browning. We also developed an improved protocol for Assay for Transposase Accessible Chromatin-sequencing (ATAC-seq) and defined changes in chromatin accessibility in a time course. The RNA-seq and ATAC-seq data were integrated to determine the kinetics of their coordinated regulation and to identify a transcription factor that drives these processes. We conducted functional studies using pharmacological and genetic approaches with specific inhibitors and shRNA-mediated knockdown, respectively. Results: FSK, not rosiglitazone, resulted in a biphasic transcriptomic response, resembling the kinetics of in vivo cold-induced browning. FSK promoted tissue remodeling first and subsequently shifted energy metabolism, concluding with a transcriptomic profile similar to that induced by rosiglitazone. The thermogenic effects of FSK were abolished by PPARγ antagonists, indicating PPARγ as a converging point. ATAC-seq uncovered that FSK leads to a significant chromatin remodeling that precedes or persists beyond transcriptomic changes, whereas rosiglitazone induces minimal changes. Motif analysis identified nuclear factor, interleukin 3 regulated (NFIL3) as a transcriptional regulator connecting the biphasic response of FSK-induced browning, as indicated by disrupted thermogenesis with NFIL3 knockdown. Conclusions: Our findings elucidated unique dynamics of the transcriptomic and epigenomic remodeling in adipocyte browning, providing new mechanistic insights into adipose thermogenesis and molecular targets for obesity treatment.
Deciphering the Role of Eukaryotic Initiation Factor 5A in Pancreatic Organogenesis
(2024-08) Rutan, Caleb D.; Mastracci, Teresa L.; Berbari, Nicolas F.; Balakrishnan, Lata N.; Roh, Hyun Cheol
The pancreas is composed of a variety of cell types such as acinar, endocrine, and ductal cells, as well as endothelial cells and adipocytes. Whereas we understand the distinct functions of each, there remains an incomplete understanding of the molecular pathways and communications that exist between these cells that may influence development, growth, and function. Given that diabetes is characterized by the destruction or dysfunction of the insulin-producing pancreatic beta cell, a better understanding of the mechanisms that influence cell growth and maintenance in the pancreas is of therapeutic interest. Genome-wide association studies identified eukaryotic initiation factor 5A (eIF5A) to be within a type 1 diabetes susceptibility locus, which also suggests this translation factor may play a role in maintaining beta cell health. EIF5A is active once post-translationally modified by the rate-limiting enzyme deoxyhypusine synthase (DHPS) in a process known as hypusination, producing hypusinated eIF5A (eIF5AHYP). The functional loss of eIF5AHYP via pancreas-specific genetic deletion of Dhps or Eif5a within multipotent pancreatic progenitor cells (MPPCs) results in an mRNA translation defect detectable at E14.5 causing the decreased expression of many proteins required for exocrine growth and function. Moreover, DHPSΔPANC mice die by 6 weeks-of-age; however, eIF5AΔPANC mice survive up to 2 years-of-age. The postnatal phenotype of the eIF5AΔPANC model was investigated in this thesis.
Endoplasmic Reticulum Calcium in the Pathogenesis of Type 1 Diabetes
(2023-12) Weaver, Staci Anne; Evans-Molina, Carmella; Roh, Hyun Cheol; Sims, Emily K.; Wek, Ronald C.
Type 1 diabetes (T1D) accounts for 5-10% of all diabetes cases and results from immune-mediated destruction of pancreatic β-cells. Individuals with Darier Disease, which is caused by loss of function germline mutation(s) in the sarcoendoplasmic reticulum Ca2+-ATPase pump (SERCA2) gene, have an elevated risk of being diagnosed with T1D (risk ratio, 1.74; 95% CI, 1.13-2.69), suggesting a potential mechanistic relationship between SERCA2 and T1D pathogenesis. To determine the impact of reduced SERCA2 expression on T1D pathogenesis, we generated SERCA2 haploinsufficient mice by backcrossing C57BL6/J-S2+/- mice onto the non-obese diabetic (NOD) background (NOD-S2+/- mice). Female NOD-S2+/- mice showed accelerated T1D onset (14wks vs. 18wks, p<0.0001), elevated circulating anti-insulin antibodies, and increased immune cell infiltration into the islets compared to NOD-WT mice. Single-cell RNA sequencing (scRNA-seq) on islets and spatial proteomics on pancreatic lymph node (PLN) and spleen at 6 wks of age revealed increased immune cell presence in islets and enhanced B and T cell activation in PLN and spleen of NOD-S2+/- mice. Furthermore, scRNA-seq on isolated islets revealed temporal alterations in pathways related to mitochondria function in β cells, and mechanistic studies revealed decreased glucose-stimulated ATP production, reduced mitochondrial membrane potential, decreased islet expression of ATP synthase/mitochondrial complex III, increased mitochondrial Ca2+, and altered mitochondrial ultrastructure in NOD-S2+/- islets at 10 wks of age. In co-culture experiments, NOD-S2+/- B cells showed increased activation and NOD-S2+/- T cells showed increased proliferation and activation when cultured with NOD-WT islets. Interestingly, NOD-S2+/- islets induced B and T cell proliferation and T cell activation when cultured with NOD-WT immune cells. Lastly, administration of a small molecule SERCA activator in NOD-S2+/- mice decreased immune cell infiltration into the islet and delayed T1D onset. In summary, our results demonstrate a novel pathway whereby modulation of SERCA2 impacts islet mitochondrial function, islet immunogenicity, and immune cell proliferation and activation which fuel progression to T1D.
In vivo pulse-chase in C. elegans reveals intestinal histone turnover changes upon starvation
(bioRxiv, 2025-02-16) Borchers, Christopher; Osburn, Kara; Roh, Hyun Cheol; Aoki, Scott T.; Biochemistry and Molecular Biology, School of Medicine
The ability to study protein dynamics and function in the authentic context of a multicellular organism is paramount to better understand biological phenomena in animal health and disease. Pulse-chase of self-labeling fusion protein tags provide the opportunity to label proteins of interest and track those proteins over time. There are currently several challenges associated with performing in vivo protein pulse-chase in animals, such as cost, reproducibility, and accurate detection methods. The C. elegans model organism has attributes that alleviate many of these challenges. This work tests the feasibility of applying the Halo modified enzyme (HaloTag) for in vivo protein pulse-chase in C. elegans. HaloTag intestinal histone reporters were created in the worm and used to demonstrate that reporter protein could be efficiently pulse-labeled by soaking animals in ligand. Labeled protein stability could be monitored over time by fluorescent confocal microscopy. Further investigation revealed reporter protein stability was dependent on the animal's nutritional state. Chromatin Immunoprecipitation and sequencing (ChIP-seq) of the reporters showed incorporation in chromatin with little change hours into starvation, implying a lack of chromatin regulation at the time point tested. Collectively, this work presents a straightforward method to label and track proteins of interest in C. elegans that can address a multitude of biological questions surrounding protein stability and dynamics in this animal model.
MicroRNA-150 Deletion from Adult Myofibroblasts Augments Maladaptive Cardiac Remodeling Following Chronic Myocardial Infarction
(MDPI, 2024-12-22) Kawaguchi, Satoshi; Sepúlveda, Marisa N.; Teoh, Jian-peng; Hayasaka, Taiki; Moukette, Bruno; Aonuma, Tatsuya; Roh, Hyun Cheol; Madhur, Meena S.; Kim, Il-man; Anatomy, Cell Biology and Physiology, School of Medicine
MicroRNA (miR: small noncoding RNA)-150 is evolutionarily conserved and is downregulated in patients with diverse forms of heart failure (HF) and in multiple mouse models of HF. Moreover, miR-150 is markedly correlated with the outcome of patients with HF. We previously reported that systemic or cardiomyocyte-derived miR-150 in mice elicited myocardial protection through the inhibition of cardiomyocyte death, without affecting neovascularization and T cell infiltration. Our mechanistic studies also showed that the protective roles of miR-150 in ischemic mouse hearts and human cardiac fibroblasts were, in part, attributed to the inhibition of fibroblast activation via the repression of multiple profibrotic genes. However, the extent to which miR-150 expression in adult myofibroblasts (MFs) modulates the response to myocardial infarction (MI) remains unknown. Here, we develop a novel 4-hydroxytamoxifen-inducible MF-specific miR-150 conditional knockout mouse model and demonstrate that the mouse line exhibits worse cardiac dysfunction after MI. Our studies further reveal that miR-150 ablation selectively in adult MFs exacerbates cardiac damage and apoptosis after chronic MI. Lastly, MF-specific miR-150 deletion in adult mice promotes the expression of proinflammatory and profibrotic genes as well as cardiac fibrosis following chronic MI. Our findings indicate a key protective role for MF-derived miR-150 in modulating post-MI responses.
Neurotensin-neurotensin receptor 2 signaling in adipocytes suppresses food intake through regulating ceramide metabolism
(Springer Nature, 2025) Fu, Wei; Lai, Yuanting; Li, Kexin; Yang, Yue; Guo, Xiao; Gong, Qifan; Zhou, Xiaofeng; Zhou, Liying; Liu, Cenxi; Zhang, Zhi; So, Jisun; Zhang, Yufeng; Huang, Lin; Lu, Guangxing; Yi, Chuanyou; Wang, Qichu; Fan, Chenyu; Liu, Chao; Wang, Jiaxing; Yu, Haiyi; Zhao, Yimin; Huang, Tao; Roh, Hyun Cheol; Liu, Tiemin; Tang, Huiru; Qi, Jianping; Xu, Ming; Zheng, Yan; Huang, He; Li, Jin; Biochemistry and Molecular Biology, School of Medicine
Neurotensin (NTS) is a secretory peptide produced by lymphatic endothelial cells. Our previous study revealed that NTS suppressed the activity of brown adipose tissue via interactions with NTSR2. In the current study, we found that the depletion of Ntsr2 in white adipocytes upregulated food intake, while the local treatment of NTS suppressed food intake. Our mechanistic study revealed that suppression of NTS-NTSR2 signaling enhanced the phosphorylation of ceramide synthetase 2, increased the abundance of its products ceramides C20-C24, and downregulated the production of GDF15 in white adipose tissues, which was responsible for the elevation of food intake. We discovered a potential causal and positive correlation between serum C20-C24 ceramide levels and human food intake in four populations with different ages and ethnic backgrounds. Together, our study shows that NTS-NTSR2 signaling in white adipocytes can regulate food intake via its direct control of lipid metabolism and production of GDF15. The ceramides C20-C24 are key factors regulating food intake in mammals.
Robust single nucleus RNA sequencing reveals depot-specific cell population dynamics in adipose tissue remodeling during obesity
(bioRxiv, 2024-04-08) So, Jisun; Strobel, Olivia; Wann, Jamie; Kim, Kyungchan; Paul, Avishek; Acri, Dominic J.; Dabin, Luke C.; Kim, Jungsu; Roh, Hyun Cheol; Biochemistry and Molecular Biology, School of Medicine
Single nucleus RNA sequencing (snRNA-seq), an alternative to single cell RNA sequencing (scRNA-seq), encounters technical challenges in obtaining high-quality nuclei and RNA, persistently hindering its applications. Here, we present a robust technique for isolating nuclei across various tissue types, remarkably enhancing snRNA-seq data quality. Employing this approach, we comprehensively characterize the depot-dependent cellular dynamics of various cell types underlying adipose tissue remodeling during obesity. By integrating bulk nuclear RNA-seq from adipocyte nuclei of different sizes, we identify distinct adipocyte subpopulations categorized by size and functionality. These subpopulations follow two divergent trajectories, adaptive and pathological, with their prevalence varying by depot. Specifically, we identify a key molecular feature of dysfunctional hypertrophic adipocytes, a global shutdown in gene expression, along with elevated stress and inflammatory responses. Furthermore, our differential gene expression analysis reveals distinct contributions of adipocyte subpopulations to the overall pathophysiology of adipose tissue. Our study establishes a robust snRNA-seq method, providing novel insights into the mechanisms orchestrating adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.
Robust single-nucleus RNA sequencing reveals depot-specific cell population dynamics in adipose tissue remodeling during obesity
(eLife Sciences, 2025-01-13) So, Jisun; Strobel, Olivia; Wann, Jamie; Kim, Kyungchan; Paul, Avishek; Acri, Dominic J.; Dabin, Luke C.; Kim, Jungsu; Peng, Gang; Roh, Hyun Cheol; Biochemistry and Molecular Biology, School of Medicine
Single-nucleus RNA sequencing (snRNA-seq), an alternative to single-cell RNA sequencing (scRNA-seq), encounters technical challenges in obtaining high-quality nuclei and RNA, persistently hindering its applications. Here, we present a robust technique for isolating nuclei across various tissue types, remarkably enhancing snRNA-seq data quality. Employing this approach, we comprehensively characterize the depot-dependent cellular dynamics of various cell types underlying mouse adipose tissue remodeling during obesity. By integrating bulk nuclear RNA-seq from adipocyte nuclei of different sizes, we identify distinct adipocyte subpopulations categorized by size and functionality. These subpopulations follow two divergent trajectories, adaptive and pathological, with their prevalence varying by depot. Specifically, we identify a key molecular feature of dysfunctional hypertrophic adipocytes, a global shutdown in gene expression, along with elevated stress and inflammatory responses. Furthermore, our differential gene expression analysis reveals distinct contributions of adipocyte subpopulations to the overall pathophysiology of adipose tissue. Our study establishes a robust snRNA-seq method, providing novel insights into the biological processes involved in adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.

Browsing by Author "Roh, Hyun Cheol"

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