Inhibition of cyclin D1 gene transcription by Brg-1

The evolutionarily conserved SWI-SNF chromatin remodeling complex regulates cellular proliferation. A catalytic subunit, BRG-1, is frequently down regulated, silenced or mutated in malignant cells, however, the mechanism by which BRG-1 may function as a tumor suppressor or block breast cancer cellular proliferation is not understood. The cyclin D1 gene is a collaborative oncogene overexpressed in greater than 50% of human breast cancers. Herein, BRG-1 inhibited DNA synthesis and cyclin D1 expression in human MCF-7 breast cancer epithelial cells. The cyclin D1 promoter AP-1 and CRE sites were required for repression by BRG-1 in promoter assays. BRG-1 deficient cells abolished and siRNA to BRG-1 reduced, formation of the BRG-1 chromatin complex. The endogenous cyclin D1 promoter AP-1 site bound BRG-1. Estradiol treatment of MCF7 cells induced recruitment of BRG-1 to the endogenous hpS2 gene promoter. Estradiol, which induced cyclin D1 abundance, was associated with a reduction in recruitment of the co-repressors HP1α/HDAC1 to the endogenous cyclin D1 promoter AP-1/BRG-1 binding sites. These studies suggest the endogenous cyclin D1 promoter BRG-1 binding site functions as a molecular scaffold in the context of local chromatin upon which coactivators and corepressors are recruited to regulate cyclin D1.     

Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex

Growth factor-dependent accumulation of the cyclin D1 proto-oncogene is balanced by its rapid phosphorylation-dependent proteolysis. Degradation is triggered by threonine 286 phosphorylation, which promotes its ubiquitination by an unknown E3 ligase. We demonstrate that Thr286-phosphorylated cyclin D1 is recognized by a Skp1-Cul1-F box (SCF) ubiquitin ligase where FBX4 and alphaB crystallin govern substrate specificity. Overexpression of FBX4 and alphaB crystallin triggered cyclin D1 ubiquitination and increased cyclin D1 turnover. Impairment of SCF(FBX4-alphaB crystallin) function attenuated cyclin D1 ubiquitination, promoting cyclin D1 overexpression and accelerated cell-cycle progression. Purified SCF(FBX4-alphaB crystallin) catalyzed polyubiquitination of cyclin D1 in vitro. Consistent with a putative role for a cyclin D1 E3 ligase in tumorigenesis, FBX4 and alphaB crystallin expression was reduced in tumor-derived cell lines and a subset of primary human cancers that overexpress cyclin D1. We conclude that SCF(FBX4-alphaB crystallin) is an E3 ubiquitin ligase that promotes ubiquitin-dependent degradation of Thr286-phosphorylated cyclin D1.