Alpha TC1 and Beta-TC-6 genomic profiling uncovers both shared and distinct transcriptional regulatory features with their primary islet counterparts

Lawlor, Nathan; Youn, Ahrim; Kursawe, Romy; Ucar, Duygu; Stitzel, Michael L.

doi:10.1038/s41598-017-12335-1

Cited by 28 publications

(20 citation statements)

References 110 publications

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“…Although there was a statistically significant response to KCl, αTC1-6 cells as a rule do not show robust secretory responses to KCl, glucose or other secretagogues. In general, αTC1-6 cells differ in their complement of transcriptional and epigenetic factors from mouse primary alpha cells, which may explain the relatively blunted secretory response seen in this cell line (44). The reduced response to glucose in particular could be due to a low efficiency in coupling between the glycolytic and TCA pathways, as has been shown in pancreatic islets (45).…”

Section: Discussionmentioning

confidence: 85%

Stathmin-2 Mediates Glucagon Secretion From Pancreatic α-Cells

Asadi

Dhanvantari

2020

Front. Endocrinol.

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Inhibition of glucagon hypersecretion from pancreatic α-cells is an appealing strategy for the treatment of diabetes. Our hypothesis is that proteins that associate with glucagon within alpha cell secretory granules will regulate glucagon secretion, and may provide druggable targets for controlling abnormal glucagon secretion in diabetes. Recently, we identified a dynamic glucagon interactome within the secretory granules of the α cell line, αTC1-6, and showed that select proteins within the interactome could modulate glucagon secretion. In the present study, we show that one of these interactome proteins, the neuronal protein stathmin-2, is expressed in αTC1-6 cells and in mouse pancreatic alpha cells, and is a novel regulator of glucagon secretion. The secretion of both glucagon and Stmn2 was significantly enhanced in response to 55 mM K + , and immunofluorescence confocal microscopy showed co-localization of stathmin-2 with glucagon and the secretory granule markers chromogranin A and VAMP-2 in αTC1-6 cells. In mouse pancreatic islets, Stathmin-2 co-localized with glucagon, but not with insulin, and co-localized with secretory pathway markers. To show a function for stathmin-2 in regulating glucagon secretion, we showed that siRNA-mediated depletion of stathmin-2 in αTC1-6 cells caused glucagon secretion to become constitutive without any effect on proglucagon mRNA levels, while overexpression of stathmin-2 completely abolished both basal and K + -stimulated glucagon secretion. Overexpression of stathmin-2 increased the localization of glucagon into the endosomal-lysosomal compartment, while depletion of stathmin-2 reduced the endosomal localization of glucagon. Therefore, we describe stathmin-2 as having a novel role as an alpha cell secretory granule protein that modulates glucagon secretion via trafficking through the endosomal-lysosomal system. These findings describe a potential new pathway for the regulation of glucagon secretion, and may have implications for controlling glucagon hypersecretion in diabetes.

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Section: Discussionmentioning

confidence: 85%

Stathmin-2 Mediates Glucagon Secretion From Pancreatic α-Cells

Asadi

Dhanvantari

2020

Front. Endocrinol.

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“…To determine whether the functions we identified for the accessibility/expression groups can be generalized to other cell types, we ran our analysis pipeline on data from eight other mouse cell types: alpha cells [59], beta cells [59], embryonic stem cells (ESC) [60, 61], hematopoietic stem cells (HSC) [62], neuronal cells extracted from the dentate gyrus [63], retinal rods [64], lymphocytes from the spleen [65], and trophoblast stem cells (TSC) [66, 67]. First, we checked the correlation between the ATAC-seq promoter accessibility and RNA-seq TPM, which ranged from 0.658 to 0.799 (Additional file 1: Figure S5).…”

Section: Resultsmentioning

confidence: 99%

“…Data supporting the conclusion of this article are available in the GEO repository, under the data accession GSE120599. Publicly available ATAC-seq and RNA-seq datasets used in this analysis can be accessed from GEO [24, 59–67], detailed in Additional file 2: Tables S5, S6.…”

Section: Authors’ Contributionsmentioning

confidence: 99%

Combined analysis of dissimilar promoter accessibility and gene expression profiles identifies tissue-specific genes and actively repressed networks

Starks

Biswas

Jain

et al. 2019

Epigenetics & Chromatin

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Background The assay for transposase-accessible chromatin (ATAC-seq) is a powerful method to examine chromatin accessibility. While many studies have reported a positive correlation between gene expression and promoter accessibility, few have investigated the genes that deviate from this trend. In this study, we aimed to understand the relationship between gene expression and promoter accessibility in multiple cell types while also identifying gene regulatory networks in the placenta, an understudied organ that is critical for a successful pregnancy. Results We started by assaying the open chromatin landscape in the mid-gestation placenta, when the fetal vasculature has started developing. After incorporating transcriptomic data generated in the placenta at the same time point, we grouped genes based on their expression levels and ATAC-seq promoter coverage. We found that the genes with the strongest correlation (high expression and high coverage) are likely involved in housekeeping functions, whereas tissue-specific genes were highly expressed and had only medium–low coverage. We also predicted that genes with medium–low expression and high promoter coverage were actively repressed. Within this group, we extracted a protein–protein interaction network enriched for neuronal functions, likely preventing the cells from adopting a neuronal fate. We further confirmed that a repressive histone mark is bound to the promoters of genes in this network. Finally, we ran our pipeline using ATAC-seq and RNA-seq data generated in ten additional cell types. We again found that genes with the strongest correlation are enriched for housekeeping functions and that genes with medium–low promoter coverage and high expression are more likely to be tissue-specific. These results demonstrate that only two data types, both of which require relatively low starting material to generate and are becoming more commonly available, can be integrated to understand multiple aspects of gene regulation. Conclusions Within the placenta, we identified an active placenta-specific gene network as well as a repressed neuronal network. Beyond the placenta, we demonstrate that ATAC-seq data and RNA-seq data can be integrated to identify tissue-specific genes and actively repressed gene networks in multiple cell types. Electronic supplementary material The online version of this article (10.1186/s13072-019-0260-2) contains supplementary material, which is available to authorized users.

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“…When we compared our described glucagon interactome with the transcriptomic profile of mouse alpha cells (56), and human α-cells (57), there were some differences in the protein profiles. Additionally, Lawlor et al (58) compared gene expression profiles of α-TC1 cells with their primary mouse and human counterparts and showed a high level of discrepancy between them. One possibility for this discrepancy may be changes in gene expression in primary cells while cultured in vitro (17, 59).…”

Section: Discussionmentioning

confidence: 99%

Plasticity in the Glucagon Interactome Reveals Novel Proteins That Regulate Glucagon Secretion in α-TC1-6 Cells

Asadi

Dhanvantari

2019

Front. Endocrinol.

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Glucagon is stored within the secretory granules of pancreatic alpha cells until stimuli trigger its release. The alpha cell secretory responses to the stimuli vary widely, possibly due to differences in experimental models or microenvironmental conditions. We hypothesized that the response of the alpha cell to various stimuli could be due to plasticity in the network of proteins that interact with glucagon within alpha cell secretory granules. We used tagged glucagon with Fc to pull out glucagon from the enriched preparation of secretory granules in α-TC1-6 cells. Isolation of secretory granules was validated by immunoisolation with Fc-glucagon and immunoblotting for organelle-specific proteins. Isolated enriched secretory granules were then used for affinity purification with Fc-glucagon followed by liquid chromatography/tandem mass spectrometry to identify secretory granule proteins that interact with glucagon. Proteomic analyses revealed a network of proteins containing glucose regulated protein 78 KDa (GRP78) and histone H4. The interaction between glucagon and the ER stress protein GRP78 and histone H4 was confirmed through co-immunoprecipitation of secretory granule lysates, and colocalization immunofluorescence confocal microscopy. Composition of the protein networks was altered at different glucose levels (25 vs. 5.5 mM) and in response to the paracrine inhibitors of glucagon secretion, GABA and insulin. siRNA-mediated silencing of a subset of these proteins revealed their involvement in glucagon secretion in α-TC1-6 cells. Therefore, our results show a novel and dynamic glucagon interactome within α-TC1-6 cell secretory granules. We suggest that variations in the alpha cell secretory response to stimuli may be governed by plasticity in the glucagon “interactome.”

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Alpha TC1 and Beta-TC-6 genomic profiling uncovers both shared and distinct transcriptional regulatory features with their primary islet counterparts

Cited by 28 publications

References 110 publications

Stathmin-2 Mediates Glucagon Secretion From Pancreatic α-Cells

Stathmin-2 Mediates Glucagon Secretion From Pancreatic α-Cells

Combined analysis of dissimilar promoter accessibility and gene expression profiles identifies tissue-specific genes and actively repressed networks

Plasticity in the Glucagon Interactome Reveals Novel Proteins That Regulate Glucagon Secretion in α-TC1-6 Cells

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