biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsMALAY HALDARtranscription profiling by arrayMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-53796Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor Spic is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80+VCAM+ bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor Bach1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Further, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insight into iron homeostasis. Bone marrow derived macrophages were generated in vitro by culturing WT and Bach1 knock out bone marrow in the presence of GM-CSF and their global gene expression pattern were compared in the presence or absence of heme at the various indicated time points after treatment. GMP and MPP populations were sorted from fetal liver chimeras and pooled by donor genotype. RNA was isolated using an RNAqueous-Micro Kit (Ambion) and submitted for amplification, labeling and hybridization. Expression values were analyzed after RMA quantile normalization using ArrayStar software (DNASTAR).biostudies-arrayexpressSample Treatment - After 6 days in culture, cells were treated with either 40 µm heme (Sigma Aldrich) or Vehicle (0.15M NaCl containing 10% NH4OH).Hybridization - cRNA was hybridized to the GeneChip Mouse Gene 1.0 ST Array (Affymetrix).Growth Protocol - Bone marrow cells were harvested and cultured in Iscoves Modified Dulbecco's Medium (IMDM) supplemented with 10% final fetal calf serum, L-glutamine, non essential amino acids, 2-ME, penicillin/steptomycin antibiotics, and containing 20 nanograms/ml recombinant murine GM-CSF (peprotech).Nucleic Acid Extraction - 24 hours after treatment floating cells were removed by sucking out the media. Adherent cells were then collected by scraping and total RNA was extracted from these cells by using RNeasy mini kit (Qiagen) following the manufacturer's instructions.Scaning - Microarrays were scanned using an Affymetrix GCS3000 high resolution scanner.Labeling - Biotinylated cRNA was prepared using an Ovation PicoSL kit (NuGEN).MIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsMALAY HALDARKenneth MurphyMalay HaldarData Transformation - Expression values were analyzed after RMA quantile normalization using ArrayStar software (DNASTAR). ID_REF = VALUE = linear RMA signal intensityfalseHeme-induced inhibition of Bach1derepresses Spic.Splenic red pulp macrophages (RPM) degrade senescent erythrocytes and recycle heme-associated iron. The transcription factor Spic is selectively expressed by RPM and is required for their development, but the physiologic stimulus inducing Spic is unknown. Here, we report that Spic also regulated the development of F4/80+VCAM+ bone marrow macrophages (BMM) and that Spic expression in BMM and RPM development was induced by heme, a metabolite of erythrocyte degradation. Pathologic hemolysis induced loss of RPM and BMM due to excess heme but induced Spic in monocytes to generate new RPM and BMM. Spic expression in monocytes was constitutively inhibited by the transcriptional repressor Bach1. Heme induced proteasome-dependent BACH1 degradation and rapid Spic derepression. Further, cysteine-proline dipeptide motifs in BACH1 that mediate heme-dependent degradation were necessary for Spic induction by heme. These findings are the first example of metabolite-driven differentiation of a tissue-resident macrophage subset and provide new insight into iron homeostasis. Bone marrow derived macrophages were generated in vitro by culturing WT and Bach1 knock out bone marrow in the presence of GM-CSF and their global gene expression pattern were compared in the presence or absence of heme at the various indicated time points after treatment. GMP and MPP populations were sorted from fetal liver chimeras and pooled by donor genotype. RNA was isolated using an RNAqueous-Micro Kit (Ambion) and submitted for amplification, labeling and hybridization. Expression values were analyzed after RMA quantile normalization using ArrayStar software (DNASTAR).2014-03-14T00:00:00Z2023-09-04T08:35:23.139Z2022-03-14T22:58:15.782ZE-GEOD-53796GSE53796EFO_0002768