Identifying mechanisms underlying early phases of melanocyte transformation and melanoma development is central to understanding the etiology of this devastating tumor, as well as for developing novel treatment approaches. Previous studies indicate the presence of mutant BRAF in melanocytic lesions, as well as its effect on pigment gene expression 
. The present study enhances our understanding of early events contributing to melanoma development. We demonstrate that loss of a transcriptionally active form of ATF2 in melanocytes inhibits melanoma development in an Nras/Ink4a model. Our quest to understand mechanisms underlying ATF2 activity in this process led us to identify an important role for ATF2 regulation of MITF, an important regulator of melanocyte biogenesis and a factor implicated in melanoma progression 
. Surprisingly, ATF2 negatively regulated MITF expression in mouse and human melanocytes, suggesting that ATF2 transcriptional activities limit MITF expression. We demonstrate that such negative regulation is elicited through downregulation of SOX10 by ATF2, in cooperation with JunB. A putative AP1 response element has been identified in SOX10 promoter sequences and ChIP analysis of this domain showed ATF2 and JunB binding. Overexpression of JunB efficiently suppressed SOX10 expression in an ATF2-dependent manner and inhibition of Jun transcriptional activities phenocopied the effect of shATF2, suggesting that negative regulation of SOX10 by ATF2 is direct, and is mediated in cooperation with JunB.
Importantly, ATF2-dependent negative regulation of Sox10 and consequently of MITF seen in melanocytes, but only in about 50% of the 18 melanoma cell lines studied here. Correspondingly, JunB, which is required for ATF2-dependent inhibition of Sox10 transcription, is no longer found on the promoter of SOX10 in melanoma cells (i.e. 501Mel) that exhibit positive regulation by ATF2. Rather, CREB and ATF2 are found on SOX10 and MITF promoters, pointing to a switch in ATF2 heterodimeric partners to enable positive regulation of these genes. Notably, melanoma cell lines that exhibit positive regulation of SOX10 and MITF by ATF2, also show high basal levels of MITF expression 
, suggesting that additional genetic or epigenetic changes distinguish these lines from melanocytes and the other melanoma lines in which ATF2 elicits negative regulation of MITF.
Notably, ATF2 control of MITF expression affected the ability of BRAF600E
-expressing melanocytes to exhibit transformed phenotype in culture, monitored by their ability to grow on soft agar. Inhibition of ATF2 abolished soft agar growth of BRAF600E
-expressing melanocytes, which was partially rescued upon KD of MITF. Interestingly, both the over expression or the KD of MITF resulted in inhibition of melanocytes ability to grow on soft agar, substantiating the notion that a fine balance of MITF expression must be maintained in order to ensure its contribution to cellular proliferation and transformation. We propose that excessively low or high MITF levels block melanocyte transformation, whereas intermediate levels allow transformation to occur. Overall, our observations demonstrate that ATF2 plays an important role in fine-tuning those levels and support the rheostat model proposed for MITF's role in melanoma development and progression 
. Of importance, ATF2 and MITF affect the ability of BRAF600E
-expressing melanocytes to grow on soft agar via distinct mechanisms. Whereas specific inhibition of ATF2 causes both accumulation of cells in G2 and induction of cell death, specific alteration of MITF protein levels—particularly depletion—significantly affects cell proliferation and inhibit growth on soft agar by non-lethally slowing cell cycle progression at G2/M. These observations are consistent with a report from Wellbrock and Marais 
, who showed that altered MITF expression inhibits melanocyte proliferation.
Importantly, inhibiting MITF expression in ATF2 KD melanocytes was sufficient to partially rescue melanocyte growth on soft agar. While supportive of our finding in the Nras::Ink4a
mouse melanoma model, where expression of transcriptionally inactive ATF2 inhibits melanoma formation, these observations provide the foundation for a model in which ATF2 inhibition causes increased MITF levels and concomitant inhibition of melanocyte growth, possible induction of cell death and delayed development. The latter is suggested by IHC analysis of mouse skin from ATF2md
mice, which shows notably reduced S100 staining indicative of delayed melanocyte development: ATF2
KO melanocytes appear to represent anagen stage IV, whereas WT represent anagen stage VI. This delay was seen at the 4- but not the 14-day time point, suggesting that an ATF2 effect might be limited to a specific subpopulation or phase of melanocyte development. The early (4 day) time point is within the time frame that allows induction of melanoma development by UV-irradiation of c-Met
mutant mice 
. It is therefore plausible that timely control of MITF expression by ATF2 determines melanocyte susceptibility to transformation.
Our analysis of genes whose expression is altered by ATF2 KD in melanocytes identified a cluster of pigmentation genes, many reportedly regulated by MITF 
. Therefore, changes in TYRP1, DCT and SILVER expression could be attributed to altered MITF expression. However, initial analysis points to a more complex mechanism since (i) the degree of changes in expression of these genes was often greater than that seen for MITF and (ii) expression of some pigmentation genes was found to be independent of MITF in some melanoma and melanocyte cultures. Hence, further studies are required to address mechanisms underlying ATF2 regulation of these pigmentation genes and the significance of such regulation to melanocyte transformation and melanoma development. While our present studies focused on the ATF2-MITF axis, it is expected that additional ATF2-regulated genes contribute to melanoma development 
. In agreement, our earlier studies using both human and mouse melanoma lines demonstrate that inhibition of ATF2 effectively inhibits tumorigenesis and blocks metastasis 
Important for ATF2 function is its subcellular localization. While findings presented here position ATF2 as an oncogene functioning in melanocyte transformation and melanoma development, earlier studies from our laboratory and others suggest that in keratinocytes and mammary glands, ATF2 elicits a tumor suppressor function 
. Of interest, assessing the localization of ATF2 in the melanoma cell lines studied here revealed that all express nuclear ATF2. Interestingly, in most cases the nuclear staining revealed a punctate staining, resembling the localization of ATF2 to DNA repair foci following DNA damage (Figure S9
). A possible link between the presence of ATF2 in repair foci in most melanoma cells points to the possible presence of activated DNA damage response which may be associated with genomic instability 
—aspects that will be explored in future studies. Significantly, the appearance of nuclear ATF2 is correlated with poor prognosis in melanoma, whereas melanomas that exhibit cytosolic ATF2 exhibit a better survival. Notably, cytosolic ATF2 is primarily seen in non-malignant skin tumors 
. Here we demonstrate that high nuclear ATF2/MITF ratios are associated with poor prognosis in primary melanomas, but not with metastatic melanomas. The latter finding attests for the important role ATF2 plays to control MITF expression in the early phase of melanocyte transformation and melanoma development.
Overall, using the mutant Nras/Ink4a melanoma model we provide genetic evidence for a central role for ATF2 in melanoma development. We demonstrate that in the absence of transcriptionally active ATF2, melanoma formation is largely inhibited. Furthermore, our data point to an unexpected role of ATF2 in fine-tuning of MITF transcription through regulation of its positive regulator SOX10. Mouse melanoma models and in vitro transformation studies indicate that this newly identified regulatory pathway is required for early phases of melanocyte transformation. Given that ATF2 affects activity of the oncogenes N-Ras (mouse model) and BRAF (melanocyte growth on soft agar); we expect that ATF2 play significant roles in melanomas that carry either of these mutations.