BMP signaling: at the gate between activated melanocyte stem cells and differentiation [ATAC-seq]

Through recurrent bouts synchronous with the hair cycle, quiescent melanocyte stem cells (McSCs) become activated to generate proliferative progeny that differentiate into pigment-producing melanocytes. The signaling factors orchestrating these events remain incompletely understood. Here, we use single cell RNA-sequencing with comparative gene expression analysis to elucidate the transcriptional dynamics of McSCs through quiescence, activation, and melanocyte maturation. Unearthing signs of increased WNT and BMP signaling along this progression, we endeavored to understand how these pathways are integrated during differentiation. Employing conditional lineage-specific genetic ablation studies in mice, we find that loss of BMP signaling in the lineage leads to hair graying due to a block in melanocyte maturation. We show that interestingly, BMP signaling functions downstream of activated McSCs and maintains WNT effector, transcription factor LEF1. Employing pseudotime analysis, genetics, and promoter analyses, and chromatin landscaping, we show that following WNT-mediated activation of McSCs, BMP and WNT pathways collaborate to trigger the commitment of proliferative progeny by fueling LEF1 and MITF-dependent differentiation. Our findings shed light upon the signaling interplay and timing of cues that orchestrate melanocyte lineage progression in the hair follicle and underscore a key role for BMP signaling in driving complete differentiation. For ATAC-sequencing, cells were FACS purified using an 85 uM nozzle. qMcSCs (p60-80) were isolated as described in Fig. S2A; differentiating progeny were isolated form Dct-eGFP+ anagen skin at P9 (Dump-, Sca-1-, CD34-, eGFPhigh, CD117+ cells). Library preparation was performed as described (Buenrostro et al., 2013) with some modifications. After sorting, cells were washed with 1X PBS, pelleted, and resuspended in cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Lysis buffer was removed by centrifugation, and samples were incubated in the transposition reaction at 37C for 30 minutes (Illumina Nextera DNA Preparation Kit; TDE1 enzyme, 10uL). The reaction was terminated by addition of Tagmentaion Clean Up buffer (300 mM EDTA, 900 mM NaCl) and purified with the Qiagen MiniElue PCR purification kit. The DNA was then PCR amplified for 12-18 cycles, and the products were tested by D1000 Tape Station (Agilent) at 3 cycling conditions. Optimal samples were then pooled and bead purified before sequencing on NextSeq High Output 75 single read (40 x 40 bp paired end).