Understanding tumor heterogeneity is an important challenge in current cancer research. Transcription and epigenetic profiling of cultured melanoma cells have defined at least two distinct cell phenotypes characterized by distinctive gene expression signatures associated with high or low/absent expression of microphthalmia-associated transcription factor (MITF). Nevertheless, heterogeneity of cell populations and gene expression in primary human tumors is much less well characterized. We performed single-cell gene expression analyses on 472 cells isolated from needle biopsies of 5 primary human melanomas, 4 superficial spreading, and one acral melanoma. The expression of -high and-low signature genes was assessed and compared to investigate intra- and intertumoral heterogeneity and correlated gene expression profiles. Single-cell gene expression analyses revealed varying degrees of intra- and intertumor heterogeneity conferred by the variable expression of distinct sets of genes in different tumors. Expression of partially correlated with that of its known target genes, while expression correlated best with and Nevertheless, cells simultaneously expressing -high and-low signature genes were observed both by single-cell analyses and RNAscope. Single-cell analyses can be performed on limiting numbers of cells from primary human melanomas revealing their heterogeneity. Although tumors comprised variable proportions of cells with the -high and-low gene expression signatures characteristic of melanoma cultures, primary tumors also comprised cells expressing markers of both signatures defining a novel cell state in tumors .

MIcrophthalmia-associated Transcription Factor (MITF) regulates melanocyte and melanoma physiology. We show that MITF associates the NURF chromatin-remodelling factor in melanoma cells. ShRNA-mediated silencing of the NURF subunit BPTF revealed its essential role in several melanoma cell lines and in untransformed melanocytes in vitro. Comparative RNA-seq shows that MITF and BPTF co-regulate overlapping gene expression programs in cell lines in vitro. Somatic and specific inactivation of Bptf in developing murine melanoblasts in vivo shows that Bptf regulates their proliferation, migration and morphology. Once born, Bptf-mutant mice display premature greying where the second post-natal coat is white. This second coat is normally pigmented by differentiated melanocytes derived from the adult melanocyte stem cell (MSC) population that is stimulated to proliferate and differentiate at anagen. An MSC population is established and maintained throughout the life of the Bptf-mutant mice, but these MSCs are abnormal and at anagen, give rise to reduced numbers of transient amplifying cells (TACs) that do not express melanocyte markers and fail to differentiate into mature melanin producing melanocytes. MSCs display a transcriptionally repressed chromatin state and Bptf is essential for reactivation of the melanocyte gene expression program at anagen, the subsequent normal proliferation of TACs and their differentiation into mature melanocytes.