Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth.The Polycomb group (PcG) genes were first identified by their roles in ensuring appropriate temporal and spatial expression of Homeotic (Hox) genes, which act as transcriptional repressors required for correct body patterning during development. Members of the PcG family also play important regulatory roles in adult organisms, including stem cell maintenance, differentiation, and malignant transformation (for a review, see references 15, 23, and 42).PcG members are generally subdivided into two groups, Polycomb repressive complex 1 (PRC1) and PRC2, and are associated with distinct multiprotein complexes (13,30). It is believed that a coordinated action between PRC1 and PRC2 complexes introduces heritable histone modifications at certain promoters, followed by the recruitment of additional transcriptional repressors to these sites. The PRC2 complex, which includes Ezh2 and EED, possesses histone H3 lysine 9 and 27 methyltransferase activity, a "silencing" histone mark (8,10,38). PRC1 components subsequently bind to methylated H3K27, inhibit SWI/SNF remodeling, and recruit histone deacetylases to silence gene expression (11,47). Biochemical histone modification by components of the PRC1 complex has also been described: reconstituted PRC1-like complexes containing Bmi1 and RING1B contain an E3 ubiquitin ligase activity capable of ubiquitinating histone H2A (7, 46).Bmi-1 and Mel-18 are highly homologous constituents of the PRC1 complex. They share 65% amino acid identity and contain a RING finger domain in the N terminus, as well as proline-rich sequences and a helix-turn-helix domain in the C-terminal tail. While they are viable and have...