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Differential roles of copper-binding transcription factors and the subfamilies of the SWI/SNF complex in the transcriptional regulation of the skeletal muscle lineage [CUT&RUN]

ABSTRACT: Copper (Cu)-binding transcription factors are emerging as important regulators of gene expression during skeletal muscle differentiation. Our group has shown that the classic Metal Transcription Factor 1, MTF1, works cooperatively with the myogenic factor MyoD to promote skeletal muscle differentiation and ensure viability of murine primary myoblasts. Preliminary evidence showed that MTF1 interacts with members of the mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) family of ATP-dependent chromatin remodeling enzymes to promote differential gene expression in proliferating and differentiating myoblasts. mSWI/SNF complexes are assembled into three major subfamilies that are distinguished by the presence of mutually exclusive subunits: cBAF (canonical BRG1 or BRM-Associated Factor), PBAF (Polybromo containing BAF), or ncBAF (non-canonical BAF). The mechanisms by which each subfamily contributes to the establishment or function of specific cell lineages are poorly understood. We determined the contributions of the BAF, ncBAF, and PBAF complexes to myoblast proliferation, differentiation, and metal homeostasis via knock-down (KD) of a distinguishing subunit of each complex (Baf250A, Brd9, and Baf180, respectively). Biochemical, molecular and RNA-seq analyses showed that Baf250A KD reduced myoblast proliferation rate and differentiation. Brd9 KD resulted in a minor proliferation defect and a delay in myogenesis. KD of Baf180 and other PBAF-specific subunits did not impact proliferation or differentiation but affected the maintenance of metal homeostasis in myoblasts. The data support a model for differential roles and interactions between MTF1 and a novel category of Cu-binding transcription factors and the SWI/SNF enzymes in the development and homeostasis of the skeletal muscle lineage.

ORGANISM(S): Mus musculus

PROVIDER: GSE253376 | GEO | 2024/10/26

REPOSITORIES: GEO

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Project description:Several versions of SWI/SNF complexes, BAF and PBAF, have been described based on unique subunit composition. We find that T cell development in the thymus and lymphoid periphery is largely normal in the absence of the PBAF-specific component BAF180. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus. These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription. mouse primary T helper lymphocytes, naïve and Th2 cells, resting and stimulated, comparison of wild-type and BAF180/Pbrm1 deficient cells RNA from T helper cells was compared in the presence (WT) and absence (KO) of the Pbrm1/BAF180 gene. Comparison was made in 2 T helper subsets (Naive and Th2), each under 2 condtions (resting and stimulated), in triplicate (A,B,C). Resting naïve CD4+ T cells (N) were purified to 95% purity from the spleens and lymph nodes of 4-6 week old Balb/c mice (WT, or BAF180 cKO) using CD4+CD62L+ T cell Purification Kit II per manufacturer’s instructions (Miltenyi). Stimulated Naive cells (S) were prepared from resting naive cells by stimulation for 24 hours on anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml. Resting Th2 cells (2U) were prepared from resting naive cells by plating onto anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml in the presence of 10ng/ml IL-4, 10ug/ml anti-IFN-gamma. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture. Stimulated Th2 cells (2S) were prepared from resting Th2 cells, stimulated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 1.5 hours. RNA was harvested from 3 replicates of the primary Th cells under each condition. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) Biotin labeled cRNA was hybridized to Illumina’s Sentrix MouseRef-8,v2 Expression BeadChips.

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Differential roles of copper-binding transcription factors and the subfamilies of the SWI/SNF complex in the transcriptional regulation of the skeletal muscle lineage [CUT&RUN]

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