ABSTRACT: MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains.Although it is well known that MafB is specifically expressed in macrophages, characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions macrophages, we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages. To investigate detail function of MafB in nonadherent macrophages, we performed microarray analysis.
Project description:MafB is a member of the large Maf family of transcription factors that share similar basic region/leucine zipper DNA binding motifs and N-terminal activation domains.Although it is well known that MafB is specifically expressed in macrophages, characterization of the null mutant phenotype in these tissues has not been previously reported. To investigate suspected MafB functions macrophages, we generated mafB/green fluorescent protein (GFP) knock-in null mutant mice. MafB deficiency was found to dramatically suppress F4/80 expression in nonadherent macrophages. To investigate detail function of MafB in nonadherent macrophages, we performed microarray analysis. Macrophages were derived from day 14.5 fetal livers of mafB- /- and WT mice. Suspensions of single fetal liver cells were prepared by mechanical disruption . A total of 106 cells in suspension were centrifuged at 1,200 rpm for 5 min, and the cell pellet was resuspended in Dulbeccoâs modified Eagleâs medium supplemented with 10% fetal calf serum (heat inactivated), streptomycin and penicillin (100 units/ml), and macrophage colony-stimulating factor (M-CSF) (10 ng/ml) and then seeded either onto a nonadhesive dishes coated with hydrophilic polymers (Hydrocell; Cell Seed, Tokyo). The culture medium was not changed throughout the experiment. M-CSF (final concentration, 10 ng/ml) was added every day from day 4 onwards. One, 2, 4, and 6 days after seeding, the cells were harvested and analyzed by flow cytometry. After 6 day culture, macrophages of Mafb-/- and WT were use microarray analysis .
Project description:Morphogenesis of the gonad requires cell-cell adhesion changes between diverse cell types. In the Drosophila gonad, the gene traffic jam regulates cell adhesion changes required for gonad formation and germ cell development (Li et al., 2003. Nature Cell Biol). To determine if the mammalian homologs of traffic jam in mammals, c-Maf and Mafb, also play a role in the transcription regulation of cell adhesion molecules in the mouse gonad, we performed a microarray analysis of FACS-purified Mafb-GFP-positive cells in E12.5 male control and c-Maf/Mafb mutant gonads. We used microarrays to determine genes affected by c-Maf mutation in E12.5 mouse gonad/mesonephros interstitial cells and macrophages E12.5 XY control (c-Maf+/-;Mafb-GFP+/-) and c-mutant (c-Maf-/-;Mafb-GFP+/-) gonad/interstitial interstitial cells and macrophages were obtained by FACS sorting of Mafb-GFP-positive cells. RNA was extracted for subsequent hybridization on Affymetrix microarrays.
Project description:Morphogenesis of the gonad requires cell-cell adhesion changes between diverse cell types. In the Drosophila gonad, the gene traffic jam regulates cell adhesion changes required for gonad formation and germ cell development (Li et al., 2003. Nature Cell Biol). To determine if the mammalian homologs of traffic jam in mammals, c-Maf and Mafb, also play a role in the transcription regulation of cell adhesion molecules in the mouse gonad, we performed a microarray analysis of FACS-purified Mafb-GFP-positive cells in E12.5 male control and c-Maf/Mafb mutant gonads. We used microarrays to determine genes affected by c-Maf mutation in E12.5 mouse gonad/mesonephros interstitial cells and macrophages
Project description:We utilized single-cell RNA-sequencing to identify candidate genetic targets of Mafb and c-Maf in the MGE-lineage using Nkx2.1-Cre generated neonatal wildtype (WT) and conditional Mafb/c-Maf double deletion mutant (cDKO) animals. We identified genes that likely contribute to Maf cDKO's phenotypes, which include preferential parvalbumin interneuron loss, overproduction of hippocampal interneurons and neurite outgrowth defects.
Project description:MafB and c-Maf deficient (Maf-DKO) or wt bone marrow cells were differentiated into macrophages by culture in DMEM/10%FCS supplemented with 20% M-CSF containing L-929 cell conditioned IMDM/0.5%FCS medium (LCM) with a half medium change on day 5 and then full medium changes every 4 days thereafter. RNA was extracted after 2 weeks in culture.
Project description:Analysis of the role of transcriptions factors MAF and MAFB on the phenotypic profles of human M-CSF-derived macrophages. Methods: Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from buffy coats from donors over a Lymphoprep gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell sorting using anti-CD14 microbeads (>95% CD14+ cells). Monocytes (0.5 x 106 cells/ml, >95% CD14+ cells) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) for 7 days in the presence of 10 ng/ml M-CSF to generate M-CSF-polarized macrophages (M-MØ). Macrophages were differentiated from peripheral blood monocytes from 3 healthy donors with M-CSF (M-MØ) to generate anti-inflammatory M-MØ. Macrophages were transfected with either Control siRNA or MAFB-specific siRNA or MAF-specific siRNA for 24h and global gene expression was analysed by RNA-Seq.
Project description:MafB is a member of the Maf family of bZip transcription factor and plays important roles in the developmental processes of various tissues, as well as in cell-type specific gene expression. MafB is expressed in differentiating keratinocytes in mice and is transcriptionally up-regulated upon human keratinocyte differentiation in vitro. In MafB-deficient mice, epidermal differentiation is partially impaired and the cornified layer is thinner. To gain insights into more detailed molecular mechanisms of MafB regulation of epidermal development, we performed microarray analysis of mRNAs isolated from dorsal skin epidermis of MafB-/- and wild-type mice at E18.5. Epidermis was separated from dorsal skin tissues of E18.5 mouse embryos (MafB-/- and WT) by Dispase (Life Technologies) treatment. Total RNA was isolated using Trizol reagent (Life Technologies), purified using an RNeasy mini kit (Qiagen), and subjected to microarray analysis.
Project description:Here we reveal a lncRNA-transcription factor network with the MAF/MAFB transcription factors as central players in directing the epidermal differentiation program.
Project description:MafA and MafB transcription factors have been shown to be key regulators of insulin and glucagon transcription. MafB is essential for alpha and beta cell differentiation, as MafB deficient mice produced fewer insulin+ and glucagon+ cells during development, with MafA expressed in remaining insulin+ cells. In contrast, beta cell development was reported to be normal in a total MafA knock out, although the animals developed beta cell dysfunction and diabetes as adults. However, we have found that MafB expression is elevated during development and retained in adult insulin+ cells after conditional removal of MafA in the pancreas. These studies will evaluate the broader significance of these insulin and glucagon regulators in alpha and beta cell development and function. Our efforts will focus on determining if the concerted actions of MafA and MafB factors are significant to beta cell formation, and we specifically plan to: Determine how alpha and beta cell differentiation is affected in MafA/MafB compound mutant mice during pancreas development. cDNA microarray studies (pancchip 6.0) with wild type, MafAKO, MafB-/-, and MafAKOMafB-/- mutant E18.5 pancreata will be performed to comprehensively identify genes controlled by MafA and MafB in developing alpha and beta cells.
Project description:MafB is a member of the Maf family of bZip transcription factor and plays important roles in the developmental processes of various tissues, as well as in cell-type specific gene expression. MafB is expressed in differentiating keratinocytes in mice and is transcriptionally up-regulated upon human keratinocyte differentiation in vitro. In MafB-deficient mice, epidermal differentiation is partially impaired and the cornified layer is thinner. To gain insights into more detailed molecular mechanisms of MafB regulation of epidermal development, we performed microarray analysis of mRNAs isolated from dorsal skin epidermis of MafB-/- and wild-type mice at E18.5.