minicoopr was constructed by inserting a zebrafish mitfa minigene ( promoter + open reading frame + 3utr ) into the bglii site of pdesttol2pa2 . individual minicoopr clones were created by gateway multisite recombination using human , full - length open reading frames ( invitrogen ) . 25pg of each minicoopr - candidate clone and 25pg of tol2 transposase mrna were microinjected into one - cell embryos generated from an incross of tg(mitfa : braf ) ; p53(lf ) ; mitfa(lf ) zebrafish . sa-gal staining was performed as described , except that scales plucked from the dorsum of melanocyte - rescued zebrafish were stained instead of tissue sections . this assay was performed in an albino(b4 ) mutant background so melanin pigment would not obscure gal staining . experimental animals were injected with 20pg minicoopr - setdb1 + 10pg minicoopr - egfp and controls with 30pg minicoopr - egfp . total rna was extracted from four minicoopr - setdb1 melanomas and four minicoopr - egfp melanomas . total rna from each was used for synthesis of cdna , which was hybridized to a nimblegen 385k array ( catalog 071105_zv7_expr ) . zebrafish genes downregulated by setdb1 were selected by fold change ( egfp / setdb1 ) > 5 and filtered by a setdb1 specificity score , which was defined as a fold change of > 3 when comparing tg(mitfa : braf ) ; p53(lf ) melanomas to minicoopr - setdb1 melanomas . human melanoma tissue microarrays ( tmas ) were analyzed by immunohistochemistry for setdb1 using rabbit polyclonal ab ( sigma hpa018142 , 1:200 ) and a mouse monoclonal ab 4a3 ( sigma , wh0009869m7 , 1:400 ) with a purple substrate for the secondary antibody ( vector labs , vip substrate ) . minicoopr was constructed by inserting a zebrafish mitfa minigene ( promoter + open reading frame + 3utr ) into the bglii site of pdesttol2pa2 . individual minicoopr clones were created by gateway multisite recombination using human , full - length open reading frames ( invitrogen ) . 25pg of each minicoopr - candidate clone and 25pg of tol2 transposase mrna were microinjected into one - cell embryos generated from an incross of tg(mitfa : braf ) ; p53(lf ) ; mitfa(lf ) zebrafish . sa-gal staining was performed as described , except that scales plucked from the dorsum of melanocyte - rescued zebrafish were stained instead of tissue sections . this assay was performed in an albino(b4 ) mutant background so melanin pigment would not obscure gal staining . experimental animals were injected with 20pg minicoopr - setdb1 + 10pg minicoopr - egfp and controls with 30pg minicoopr - egfp . from zebrafish , total rna was extracted from four minicoopr - setdb1 melanomas and four minicoopr - egfp melanomas . total rna from each was used for synthesis of cdna , which was hybridized to a nimblegen 385k array ( catalog 071105_zv7_expr ) . zebrafish genes downregulated by setdb1 were selected by fold change ( egfp / setdb1 ) > 5 and filtered by a setdb1 specificity score , which was defined as a fold change of > 3 when comparing tg(mitfa : braf ) ; p53(lf ) melanomas to minicoopr - setdb1 melanomas . human melanoma tissue microarrays ( tmas ) were analyzed by immunohistochemistry for setdb1 using rabbit polyclonal ab ( sigma hpa018142 , 1:200 ) and a mouse monoclonal ab 4a3 ( sigma , wh0009869m7 , 1:400 ) with a purple substrate for the secondary antibody ( vector labs , vip substrate ) .
the most common mutation in melanoma , braf(v600e ) , activates the braf serine / threonine kinase and causes excessive mapk pathway activity1,2 . 
 braf(v600e)mutations are also present in benign melanocytic nevi3 , highlighting the importance of additional genetic alterations in the genesis of malignant tumors . 
 such changes include recurrent copy number variations that result in the amplification of oncogenes4,5 . for certain amplifications , 
 the large number of genes in the interval has precluded an understanding of cooperating oncogenic events . here , we have used a zebrafish melanoma model to test genes in a recurrently amplified region on chromosome 1 for the ability to cooperate with braf(v600e ) and accelerate melanoma . 
 setdb1 , an enzyme that methylates histone h3 on lysine 9 ( h3k9 ) , was found to significantly accelerate melanoma formation in the zebrafish . 
 chromatin immunoprecipitation coupled with massively parallel dna sequencing ( chip - seq ) and gene expression analyses revealed target genes , including hox genes , that are transcriptionally dysregulated in response to elevated setdb1 . 
 our studies establish setdb1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis .