The mammalian Polycomblike protein PHF1 was previously shown to interact with the Polycomb group (PcG) protein Ezh2, a histone methyltransferase whose activity is pivotal in sustaining gene repression during development and in adulthood. As Ezh2 is active only when part of the Polycomb Repressive Complexes (PRC2-PRC4), we examined the functional role of its interaction with PHF1. Chromatin immunoprecipitation experiments revealed that PHF1 resides along with Ezh2 at Ezh2-regulated genes such as the HoxA loci and the non-Hox MYT1 and WNT1 genes. Knockdown of PHF1 or of Ezh2 led to up-regulated HoxA gene expression. Interestingly, depletion of PHF1 did correlate with reduced occupancy of Bmi-1, a PRC1 component. As expected, knockdown of Ezh2 led to reduced levels of its catalytic products H3K27me2/H3K27me3. However, reduced levels of PHF1 also led to decreased global levels of H3K27me3. Notably, the levels of H3K27me3 decreased while those of H3K27me2 increased at the up-regulated HoxA loci tested. Consistent with this, the addition of PHF1 specifically stimulated the ability of Ezh2 to catalyze H3K27me3 but not H3K27me1/H3K27me2 in vitro. We conclude that PHF1 modulates the activity of Ezh2 in favor of the repressive H3K27me3 mark. Thus, we propose that PHF1 is a determinant in PcG-mediated gene repression.

The MAGE-A, MAGE-B, and MAGE-C protein families comprise the class-I MAGE/cancer testes antigens, a group of highly homologous proteins whose expression is suppressed in all normal tissues except developing sperm. Aberrant expression of class I MAGE proteins occurs in melanomas and many other malignancies, and MAGE proteins have long been recognized as tumor-specific targets; however, their functions have largely been unknown. Here, we show that suppression of class I MAGE proteins induces apoptosis in the Hs-294T, A375, and S91 MAGE-positive melanoma cell lines and that members of all three families of MAGE class I proteins form complexes with KAP1, a scaffolding protein that is known as a corepressor of p53 expression and function. In addition to inducing apoptosis, MAGE suppression decreases KAP1 complexing with p53, increases immunoreactive and acetylated p53, and activates a p53 responsive reporter gene. Suppression of class I MAGE proteins also induces apoptosis in MAGE-A–positive, p53^wt/wt parental HCT 116 colon cancer cells but not in a MAGE-A–positive HCT 116 p53^−/− variant, indicating that MAGE suppression of apoptosis requires p53. Finally, treatment with MAGE-specific small interfering RNA suppresses S91 melanoma growth in vivo, in syngenic DBA2 mice. Thus, class I MAGE protein expression may suppress apoptosis by suppressing p53 and may actively contribute to the development of malignancies and by promoting tumor survival. Because the expression of class I MAGE proteins is limited in normal tissues, inhibition of MAGE antigen expression or function represents a novel and specific treatment for melanoma and diverse malignancies. [Cancer Res 2007;67(20):9954–62]

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The repair of DNA damage in highly compact, transcriptionally silent heterochromatin requires that repair and chromatin packaging machineries be tightly coupled and regulated. KAP1 is a heterochromatin protein and co-repressor that binds to HP1 during gene silencing but is also robustly phosphorylated by Ataxia telangiectasia mutated (ATM) at serine 824 in response to DNA damage. The interplay between HP1-KAP1 binding/ATM phosphorylation during DNA repair is not known. We show that HP1α and unmodified KAP1 are enriched in endogenous heterochromatic loci and at a silent transgene prior to damage. Following damage, γH2AX and pKAP1-s824 rapidly increase and persist at these loci. Cells that lack HP1 fail to form discreet pKAP1-s824 foci after damage but levels are higher and more persistent. KAP1 is phosphorylated at serine 473 in response to DNA damage and its levels are also modulated by HP1. Unlike pKAP1-s824, pKAP1-s473 does not accumulate at damage foci but is diffusely localized in the nucleus. While HP1 association tempers KAP1 phosphorylation, this interaction also slows the resolution of γH2AX foci. Thus, HP1-dependent regulation of KAP1 influences DNA repair in heterochromatin. Mol Cancer Res; 10(3); 401–14. ©2011 AACR.

Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial–mesenchymal transitions (EMT) occurring in wound-healing processes and the cancer stem cell–like compartment of tumors, including TGF-β dependence, we investigated the role of the Grainyhead gene, Grainyhead-like-2 (GRHL2) in oncogenic EMT. GRHL2 was downregulated specifically in the claudin-low subclass breast tumors and in basal-B subclass breast cancer cell lines. GRHL2 suppressed TGF-β–induced, Twist-induced or spontaneous EMT, enhanced anoikis sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by suppression of ZEB1 expression via direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription and it upregulated mir-200b/c as well as the TGF-β receptor antagonist, BMP2. Finally, ectopic expression of GRHL2 in MDA-MB-231 breast cancer cells triggered an MET and restored sensitivity to anoikis. Taken together, our findings define a major role for GRHL2 in the suppression of oncogenic EMT in breast cancer cells. Cancer Res; 72(9); 2440–53. ©2012 AACR.