ChIP ChIP experiments have detected BRN binding at

ChIP-ChIP experiments have detected BRN2 binding at the NFIB locus in vivo using 501 Mel human melanoma cells (Kobi et al., 2010). This observation, coupled with the reciprocal expression of NFIB in response to gain or loss of BRN2 in 2D culture and 3D melanoma spheres, and the co-expression seen in vivo suggests that there is likely a direct regulation of NFIB by BRN2 in melanoma cells. MITF knockdown within these models is shown to increase both NFIB and EZH2 expression. While there has been no clear direct interaction between MITF and these genes, previous studies have shown that knockdown of MITF leads to an increase in BRN2 expression (Thurber et al., 2011), suggesting that MITF may regulate NFIB and EZH2 indirectly through interactions with BRN2. The increases seen in melanoma spheroid invasion in both BRN2 and NFIB stable melanoma cells coupled with the loss of enhanced migratory capacity of BRN2 over-expressing cells when NFIB is knocked down suggests that this factor is a pivotal component of driving this highly migratory/invasive melanoma cell phenotype within BRN2 expressing populations. Crucially, recent reports from studies using rodent models of Small Cell Lung Cancer (SCLC) have suggested a vital role for NFIB in triggering invasive behavior that drives metastatic spread of these tumours (Denny et al., 2016; Semenova et al., 2016). In these models NFIB expression was found to be necessary and sufficient to support metastatic spread, an observation further supported by a correlation between high NFIB expression and an advance metastatic tumour grade in neuro-endocrine tumours from human patients (Semenova et al., 2016). Our data presented here would suggest NFIB is capable of propagating the acquisition of a more invasive phenotype through broad changes in chromatin status, in large part by increasing expression and function of the histone methyl-transferase enzyme EZH2. Consistent with this idea, the pivotal role NFIB plays in the acquisition of a metastatic phenotype in SCLC cells was identified in studies initially aimed at characterizing genome wide alterations in chromatin accessibility during metastatic progression of SCLC. The notable enrichment of NFIB pyruvate dehydrogenase kinase in these hyper-accessible regions of the genome ultimately revealed the role for NFIB in driving metastasis and maintaining this permissive chromatin state (Denny et al., 2016). Expression of the chromatin modifying enzyme EZH2 has been shown to be directly controlled by NFIB in neuronal stem cells (Piper et al., 2014). Interestingly, EZH2 has previously been characterized as a potent driver of melanoma metastasis with high levels associated with a poor patient prognosis (Bachmann et al., 2006; Fan et al., 2011; Luo et al., 2013; Zingg et al., 2015). Previous in silico screens searching for high-affinity NFI-binding sites within various polycomb repressive complex gene sets identified two putative, highly conserved NFI-binding motifs in the basal promoter of EZH2 which was confirmed by in vitro and ChIP analyses (Piper et al., 2014). We were able to demonstrate that EZH2 is up-regulated following both BRN2 and NFIB stable overexpression in melanoma cells. EZH2 up-regulation is lost in A2058 and MM96L cells following NFIB siRNA knockdown, suggesting that NFIB expression is crucial to this regulation. Furthermore, mutating the NFI-binding sites in our EZH2-promoter driven luciferase construct effectively blocked the increases seen in EZH2-promoter driven expression. Interestingly, EZH2 expression has previous been linked with both increased growth and metastasis within tumours, whereas our model is predicated on a slowly proliferative cell phenotype. Unfortunately, NFIB cells failed to form tumours in xenograft models, which did not allow us to assess tumour growth. While melanoma cells often behave differently within the tumour microenvironment compared to cultured cells, it is possible that BRN2 and NFIB regulate additional genes that counteract the growth phenotype that might be anticipated with high EZH2 expression. Recent intravital imaging studies have found that EZH2 marks a heterogeneous, highly motile subpopulation of cells within melanoma tumours that is thought to promote early stages of metastasis (Manning et al., 2015). This study links these populations back to earlier intravital evidence showing that BRN2 expression is required for melanoma cell motility within tumours and dissemination into the blood stream during metastasis (Pinner et al., 2009). Our data provides strong evidence showing that these heterogeneous EZH2 expressing tumour populations are also BRN2 and NFIB positive and are likely mediated by BRN2 expression in an NFIB dependent manner. While our data demonstrates that NFIB directly promotes EZH2 expression downstream of BRN2, NFIB has been shown to repress EZH2 in primary cortical cells in vitro and during cortical development in-vivo (Piper et al., 2014) suggesting that cellular specificity and importantly cellular context may determine if NFIB functions as a repressor or activator at this locus. Computational predictions, coupled with multiplex experimental analysis suggest that in most contexts, the NFI family of transcription factors primarily act as transcriptional activators (Pjanic et al., 2011).