ChIP-seq was performed with H3K9ac antibody on MSC cells differentiated with adipogenic media for 7 days (MSCB7 or B7) treated with ethanol vehicle or 100 nM 1,25(OH)2D3 for 3 h prior to ChIP.
Vehicle - yellow
1,25(OH)2D3 - blue
Study Abstract
Terminal differentiation of multipotent stem cells is achieved through a coordinated cascade of activated transcription factors and epigenetic modifications that drive gene transcription responsible for unique cell fate. Within the mesenchymal lineage, factors such as RUNX2 and PPARγ are indispensable for osteogenesis and adipogenesis, respectively. We therefore investigated genomic binding of transcription factors and accompanying epigenetic modifications that occur during osteogenic and adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (MSC). As assessed by ChIP-seq and RNA-seq analyses, we found that genes vital for osteogenic identity were linked to RUNX2, C/EBPβ, RXR, and VDR binding sites, whereas adipocyte differentiation favored PPARγ, RXR, C/EBPβ, and C/EBPα binding sites. Epigenetic marks were clear predictors of active differentiation loci as well as enhancer activities and selective gene expression. These marrow-derived MSCs displayed an epigenetic pattern that suggested a default preference for the osteogenic pathway; however, these patterns were rapidly altered near the Adipoq, Cidec, Fabp4, Lipe, Plin1, Pparg and Cebpa genes during adipogenic differentiation. Surprisingly, we found that these cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice-versa) after commitment, as assessed by staining, gene expression, and ChIP-qPCR analysis. The osteogenic default pathway may be subverted during pathological conditions leading to skeletal fragility and increased marrow adipocity during aging, estrogen deficiency and skeletal unloading. Taken together, our data provide an increased mechanistic understanding of the epigenetic programs necessary for multipotent differentiation of MSCs that may prove beneficial in the development of therapeutic strategies.
Methods
Cell Culture and Differentiation
Marrow-derived MSCs were isolated from female C57bl/6 mice, cryopreserved, and expanded through no more than 10 passages (1,2). The MSCs were cultured in minimum Eagle medium alpha (MEMα) modification supplemented with 10% fetal bovine serum from Hyclone (Logan, UT), and 1% penicillin-streptomycin from Invitrogen. For differentiation, cells were grown to confluency and then transferred to either osteogenic differentiation media (10 mM βglycerophosphate and 50 μg/mL ascorbic acid) or adipogenic differentiation media (5 μg/mL insulin, 50 μM indomethacin, 0.1 μM dexamethasone) for the indicated periods, which was replenished every 2-3 days until assay.
Chromatin Immunoprecipitation (ChIP) followed by sequencing (ChIP-seq)
Chromatin immunoprecipitation was performed as described previously (3). Briefly, cells (both pre- and post-differentiation) were treated for 3 h with vehicle or 100 nM 1,25(OH)2D3 after differentiation as previously reported (4). Samples were subjected to immunoprecipitation using either a control IgG antibody or the indicated experimental antibody (5,6). All ChIP and ChIP-seq methodology including statistical information and data processing were performed as recently reported (6). ChIP-seq runs were all 50bp. Barcoded samples were run over 4 lanes total and the fastq data were concatenated prior to BOWTIE mapping (7). 12 barcoded samples were run in each lane, over 4 lanes total. Barcodes were decoded by Illumina HiSeq2000 software automatically. All samples were mapped from fastq files using BOWTIE [-m 1 -- best] to mm9 [UCSCmouse genome build 9]. Replicate lanes were analyzed separately for reproducibility and normalization of the peak calls. Peaks were called by using HOMER (http://homer.salk.edu/homer/ngs/index.html) and MACS (8,9). HOMER analysis was run using the default settings for peak finding. Histone peaks were called with a 2-fold enrichment over input instead of 4-fold (for transcription factors) given the nature of histone chip-seq. False Discovery Rate (FDR) cut off was 0.001 (0.1%) for all peaks.
Credits
Usage of these datasets should reference:
Meyer MB, Benkusky NA, Sen B, Rubin J, Pike JW. Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-Derived Mesenchymal Stem Cells. J Biol Chem. 2016 Jul 11 doi:10.1074/jbc.M1116.736538 PMID: 27402842
Raw data can be accessed through Gene Expression Omnibus record: GSE79813
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