ATAC-seq Reveals that Heme Regulates the Transcriptional Activity of Transcription Factors Driving CD8⁺ T Cell Exhaustion
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE312102 | GEO | 2026/01/25
REPOSITORIES: GEO
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Project description:Regulatory heme is known to control cellular behavior by regulating the activities of hemoproteins. We hypothesized that increased levels of regulatory heme may drive T cell exhaustion. To test this, we first confirmed that treatment with hemin, a heme analog, effectively increased intracellular regulatory heme levels in T cells. To evaluate the transcriptional changes in CD8+ T cells treated with hemin, we conducted RNA sequencing on CD8+ T cells with and without exogenous hemin treatment. The RNA-seq data revealed that hemin treatment strongly modulated gene expression patterns associated with terminally exhausted T cells. Pathway enrichment analysis further revealed that hemin suppressed pathways involved in proliferation and cytokine production while altering metabolic processes, such as cholesterol efflux and fatty acid biosynthesis, which are known to regulate T cell function. These results suggest that increased levels of regulatory heme drive T cell exhaustion differentiation.
2026-01-25 | GSE312103 | GEO
Project description:Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin and finally elution and identification by mass spectrometry. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in a SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization and vesicular trafficking, many of which have been associated with heme through complementary studies published recently. Taken together, these results provide support for the emerging role for heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.
2023-05-10 | PXD037165 | Pride
Project description:Macrophages are central in regulating iron homeostasis. Transcription repressor Bach2 regulates by heme. Here we investigated the relationship between heme-regulated Bach2 and macrophage in spleen. We found that gene expression were not many change between WT and Bach2 knock out mice in red-pulp macrophage.Our results suggest that the function of the red-pulp macrophage is not dependent on according to expression of Bach2.
2019-03-31 | GSE79806 | GEO
Project description:Macrophages are central in regulating iron homeostasis. Transcription repressor Bach2 regulates by heme. Here we investigated the relationship between heme-regulated Bach2 and macrophage in bone marrow. We identified RFP-positive and negative macrophage were in bone marrow. We found that RFP-positive macrophage related with iron-heme homeostasis maintenance and RPF-negative population related with immune response. In RFP positive macrophage, we also found the lysosomal heme transporter hrg-1 was Bach2 direct target gene. Our results suggest that the function of the bone marrow macrophage alters according to expression of Bach2.
2018-09-26 | GSE79697 | GEO
Project description:Control of intracellular heme levels by extracellular scavenger proteins and intracellular heme oxygenases are essential functions during disease states with enhanced extracellular heme release. During severe hemolysis or rhabdomyolysis uncontrolled heme exposure can cause acute kidney injury and endothelial damage. The cytotoxic activity of heme has been primarily attributed to its pro-oxidative potential. However, the mechanisms of heme toxicity have never been systematically explored. Besides its redox reactivity, heme could also adversely alter cellular functions through its broad binding affinity to multiple non-hemoproteins. Such interactions may impair protein functions and support heme toxicity. In this study we mapped the gene expression profile of Hb triggered acute kidney injury in old blood transfused guinea pigs by serial analysis of gene expression (SAGE). Additionally, the toxic heme response of mouse embryo fibroblasts was systematically characterized on the gene and protein expression levels by gene array experiments and quantitative mass-spectrometry of stable isotope labeled cells. In all these studies, in addition to oxidative stress signals, the most significant signals were reproducibly found for biologic networks related to altered protein degradation, which ultimately triggers the response to unfolded proteins and apoptosis. These screening data could be mechanistically explained by heme-proteasome interactions and a proteasome inhibitor activity of heme. Proteasome inhibition drastically reduced the threshold of cellular toxicity during heme exposure. We therefore propose a novel model of heme toxicity whereby proteasome inhibition by the porphyrin fuels a vicious cycle of oxidative protein modification, accumulation of damaged proteins, cell damage and apoptosis. A two color common reference design was chosen with 2-4 independent biological replicates of each condition. Each experimental sample (Cy5 labeled) was hybridized against a non-treated reference sample (Cy3 labeled). To compensate for dye bias control arrays with competitively hybridized Cy3- and Cy5-labeled non-treated reference samples were used. The latter allowed for a very robust statistical analysis with pair-wise comparison of treatment array replicates versus the corresponding control array replicates.
2016-08-21 | E-GEOD-50145 | biostudies-arrayexpress
Project description:Control of intracellular heme levels by extracellular scavenger proteins and intracellular heme oxygenases are essential functions during disease states with enhanced extracellular heme release. During severe hemolysis or rhabdomyolysis uncontrolled heme exposure can cause acute kidney injury and endothelial damage. The cytotoxic activity of heme has been primarily attributed to its pro-oxidative potential. However, the mechanisms of heme toxicity have never been systematically explored. Besides its redox reactivity, heme could also adversely alter cellular functions through its broad binding affinity to multiple non-hemoproteins. Such interactions may impair protein functions and support heme toxicity. In this study we mapped the gene expression profile of Hb triggered acute kidney injury in old blood transfused guinea pigs by serial analysis of gene expression (SAGE). Additionally, the toxic heme response of mouse embryo fibroblasts was systematically characterized on the gene and protein expression levels by gene array experiments and quantitative mass-spectrometry of stable isotope labeled cells. In all these studies, in addition to oxidative stress signals, the most significant signals were reproducibly found for biologic networks related to altered protein degradation, which ultimately triggers the response to unfolded proteins and apoptosis. These screening data could be mechanistically explained by heme-proteasome interactions and a proteasome inhibitor activity of heme. Proteasome inhibition drastically reduced the threshold of cellular toxicity during heme exposure. We therefore propose a novel model of heme toxicity whereby proteasome inhibition by the porphyrin fuels a vicious cycle of oxidative protein modification, accumulation of damaged proteins, cell damage and apoptosis. A two color common reference design was chosen with 2-8 independent biological replicates of each condition. Each experimental sample (Cy5 labeled) was hybridized against a non-treated reference sample (Cy3 labeled). To compensate for dye bias control arrays with competitively hybridized Cy3- and Cy5-labeled non-treated reference samples were used. The latter allowed for a very robust statistical analysis with pair-wise comparison of treatment array replicates versus the corresponding control array replicates.
2016-08-21 | E-GEOD-50146 | biostudies-arrayexpress
Project description:Bach2 is vital for the formation of progenitor exhausted T cells and for maintaining their stem-like properties. In B cells, Bach2 undergoes conformational changes that reduce its transcriptional repression activity due to decreased DNA-binding affinity and increased degradation, subsequently triggering Blimp1 expression. Therefore, we aimed to use scRNA-seq to determine whether mutation of the heme-binding site in Bach2 (Bach2MUT) could more efficiently preserve stem-like properties in tumor-infiltrating lymphocytes (TILs) compared to wild-type Bach2 (Bach2WT).
2026-01-25 | GSE312100 | GEO
Project description:The response regulator HrrA belonging to the HrrSA two-component system (previously named CgtSR11) is known to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Sequence analysis indicated an involvement of the HrrSA system in heme-dependent gene expression. Growth experiments revealed that the non-pathogenic soil bacterium C. glutamicum is able to use hemin or hemoglobin as sole iron source. In DNA microarray analyses a putative operon encoding the hemin-binding protein HtaA and the putative hemin ABC transporter HmuTUV showed a strong upregulation in heme-grown cells. Deletion of the hmu operon clearly affects heme utilization, but does not completely abolish growth on heme or hemoglobin. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of a hrrA deletion mutant and C. glutamicum wild type in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase (hmuO), an enzyme involved in the utilization of heme as iron source. Besides hmuO, HrrA was shown to activate the expression of heme-containing components of the respiratory chain, namely ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa3 oxidase and three subunits of the cytochrome bc1 complex. Furthermore, HrrA represses almost all genes involved in heme biosynthesis, including glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Thus, our data clearly emphasize a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum.
2010-12-17 | GSE26122 | GEO
Project description:The response regulator HrrA belonging to the HrrSA two-component system (previously named CgtSR11) is known to be repressed by the global iron-dependent regulator DtxR in Corynebacterium glutamicum. Sequence analysis indicated an involvement of the HrrSA system in heme-dependent gene expression. Growth experiments revealed that the non-pathogenic soil bacterium C. glutamicum is able to use hemin or hemoglobin as sole iron source. In DNA microarray analyses a putative operon encoding the hemin-binding protein HtaA and the putative hemin ABC transporter HmuTUV showed a strong upregulation in heme-grown cells. Deletion of the hmu operon clearly affects heme utilization, but does not completely abolish growth on heme or hemoglobin. As a central part of this study, we investigated the regulon of the response regulator HrrA via comparative transcriptome analysis of a hrrA deletion mutant and C. glutamicum wild type in combination with DNA-protein interaction studies with purified HrrA protein. Our data provide evidence for a heme-dependent transcriptional activation of heme oxygenase (hmuO), an enzyme involved in the utilization of heme as iron source. Besides hmuO, HrrA was shown to activate the expression of heme-containing components of the respiratory chain, namely ctaD and the ctaE-qcrCAB operon encoding subunits I and III of cytochrome aa3 oxidase and three subunits of the cytochrome bc1 complex. Furthermore, HrrA represses almost all genes involved in heme biosynthesis, including glutamyl-tRNA reductase (hemA), uroporphyrinogen decarboxylase (hemE), and ferrochelatase (hemH). Thus, our data clearly emphasize a central role of the HrrSA system in the control of heme homeostasis in C. glutamicum. Three biological replicates of each experiment were performed. Experiment 1: Transcriptome comparison of wild type grown und FeSO4 or heme as iron source; Exp. 2: WT vs. hrrA deletion mutant grown on FeSO4; Exp. 3: WT vs. hrrA mutant grown on heme. For analysis via DNA microarraysose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either 2.5 uM FeSO4 or 2.5 uM heme as iron source.
2010-12-17 | E-GEOD-26122 | biostudies-arrayexpress
Project description:Hemes are essential but potentially cytotoxic cofactors that participate in critical and diverse biological processes. Although the pathway and intermediates for heme biosynthesis have been well defined, the intracellular networks which mediate heme trafficking remain unknown. Caenorhabditis elegans and related helminths are natural heme auxotrophs requiring environmental heme for growth and development. We exploited this auxotrophy to identify HRG-1 and HRG-4 in C. elegans and show that they are essential for heme homeostasis and normal vertebrate development. We demonstrate that heme deficiency upregulates expression of hrg-4 and its evolutionarily conserved paralog hrg-1. Depletion of either HRG-1 or HRG-4 in worms results in disruption of organismal heme sensing and abnormal response to heme analogs. HRG-1 and HRG-4 are novel transmembrane proteins that bind heme and have evolutionarily conserved functions. Transient knockdown of hrg-1 in zebrafish leads to hydrocephalus, yolk tube malformations, and, most strikingly, profound defects in erythropoiesis - phenotypes that are fully rescued by worm HRG-1. These findings reveal unanticipated and conserved pathways for cellular heme trafficking in animals that defines the paradigm for eukaryotic heme transport. Uncovering the mechanisms of heme transport in C. elegans will provide novel insights into human disorders of heme metabolism and generate unique anthelmintics to combat worm infestations. Experiment Overall Design: As a first-step toward understanding heme homeostasis at the molecular level, we performed genome-wide microarrays to identify genes that are transcriptionally regulated by heme. For microarray analysis, synchronized F2 larvae were re-inoculated in mCeHR-2 medium supplemented with 4, 20 or 500 uM hemin and harvested at the late L4 stage for mRNA prep and probe hybridization to Affymetrix C. elegans Whole Genome Expression Arrays. Total RNA from three biological replicates were used at each hemin concentration. Data from worms grown in mCeHR-2 medium with 4 and 500 uM hemin were compared to data from worms grown in 20 uM hemin. Microarray data were verified with Microarray Suite 5.0 (Affymetrix) and Robust Multichip Average Method (RMA, R package). Results from MAS 5.0 and RMA analyses provided with 375 genes that showed 1.6 fold change in 4 and 500 uM hemin when compared to data from 20 uM hemin samples. Statistical analyses identified changes in 375 genes from worms grown in either 4 or 500 µM heme (see Methods). The microarray results were validated by qRT-PCR (Supplementary Fig. 1) and the 375 heme-responsive genes were classified into eight categories based on their relative changes in gene expression (Table I). The data from the microarray study show that �1.9 % of genes in the worm genome are transcriptionally responsive to heme. Notably, of the 375 genes, 164 had some sequence identity in human genome databases at the amino acid level, and >90 % of the genes had no ascribed function in the C.
2008-10-18 | E-GEOD-8696 | biostudies-arrayexpress