MITF-M dimer binds the CDKN2A gene

Promoter analysis reveals multiple M- and E-box elements upstream of the transcription start site of the CDKN2A gene isoform p16 INK4A that are conserved between human and mouse. ChIP analysis in normal human melanocytes reveals that the promoter is bound in vivo by MITF to drive p16INK4A expression. MITF-dependent expression requires an intact binding site and the DNA-binding region of MITF, and drives upregulation of p16INK4A mRNA and protein levels (Loercher et al, 2005). Ectopic expression of MITF in mouse embryonic fibroblasts causes growth inhibition, morphological changes consistent with melanocyte differentiation and an accumulation of hypophosphorylated pRB (Loercher et al, 2005). Depletion of MITF with siRNA in normal melanocytes decreased the expression of p16INK4A, caused an accumulation of hypophosphorylated pRB and promoted reentry into the cell cycle as assessed by BrdU incorporation (Loercher et al, 2005). Expression of p16INK4A is required both for cell cycle arrest as well as for the morphological changes and S100 expression, both of which are consistent with differentiation into melanocytes in these cells (Loercher et al, 2005).

通过促进子分析，揭示了位于CDKN2A基因异构体p16INK4A转录起始位点上游的多个M-和E-box元件在人类和鼠类之间具有保守性。在正常人类黑色素细胞中进行的ChIP分析显示，该促进子通过MITF在体内结合，以驱动p16INK4A的表达。MITF依赖性表达需要完整的结合位点和MITF的DNA结合区域，并推动p16INK4A信使RNA和蛋白质水平的上调（Loercher等，2005）。在鼠胚胎成纤维细胞中MITF的异位表达引起生长抑制、与黑色素细胞分化一致的组织学变化以及去磷酸化pRB的积累（Loercher等，2005）。通过siRNA耗竭正常黑色素细胞中的MITF，降低了p16INK4A的表达，导致去磷酸化pRB的积累，并通过BrdU掺入评估促进了细胞周期的重新进入（Loercher等，2005）。p16INK4A的表达对于细胞周期停滞以及与这些细胞中黑色素细胞分化一致的组织学变化和S100表达都是必需的（Loercher等，2005）。