Oct-1 Counteracts Autoinhibition of Runx2 DNA Binding To Form a Novel Runx2/Oct-1 Complex on the Promoter of the Mammary Gland-Specific Gene β-casein

Abstract

The transcription factor Runx2 is essential for the expression of a number of bone-specific genes and is primarily considered a master regulator of bone development. Runx2 is also expressed in mammary epithelial cells, but its role in the mammary gland has not been established. Here we show that Runx2 forms a novel complex with the ubiquitous transcription factor Oct-1 to regulate the expression of the mammary glandspecific gene ␤-casein. The Runx2/Oct-1 complex forms on a Runx/octamer element which is highly conserved in casein promoters. Chromatin immunoprecipitation, RNA interference, promoter mutagenesis, and transient expression analyses were used to demonstrate that the Runx2/Oct-1 complex contributes to the transcriptional regulation of the ␤-casein gene. Analysis of the complex revealed autoinhibitory domains for DNA binding in both the N-terminal and the C-terminal regions of Runx2. Oct-1 stimulates the recruitment of Runx2 to the ␤-casein promoter by interacting with the C-terminal region of Runx2, suggesting that Oct-1 stimulates Runx2 recruitment by relieving the autoinhibition of Runx2 DNA binding. These findings demonstrate that Runx2 collaborates with Oct-1 and contributes to the expression of a mammary gland-specific gene.The transcription factor Runx2 is a master regulator of bone development, and mutations in Runx2 are found in patients with the skeletal disorder cleidocranial dysplasia (15,28,32,33,40,43,45,58). Targeted deletion of the Runx2 gene in mice has demonstrated that Runx2 is a master regulator of osteoblast differentiation and is required for chondrocyte hypertrophy (28,32,45). Runx2 is a member of the Runt domain family of transcription factors (26,27,64,65). The Runt domain is a DNA-binding domain that specifically recognizes a consensus binding site (TGT/cGGT) found in the promoters of several cell type-specific genes (5,6,26,27,41,44,60). It has also been shown to regulate transcription in collaboration with several transcriptional regulators, including core-binding factor ␤, AP-1, Ets-1, androgen receptor, glucocorticoid receptor (GR), estrogen receptor, vitamin D 3 receptor, Smads, and STATs (12,

A role for Runx2 in normal mammary gland and breast cancer bone metastasis

“…Runx2 protein has since been observed in these latter cell lines and in HC11, MTSV1-7, and LCC-15MB cells, as determined by Western analysis and DNA-binding activity [Barnes et al, 2003;Inman and Shore, 2003;Selvamurugan et al, 2004;Inman et al, 2005]. Runx2 DNA-binding activity has also been detected in primary mammary epithelial cells from pregnant mice [Inman and Shore, 2003].…”

“…Work in my own laboratory has used this model cell line to investigate whether Runx2 contributes to the expression of genes known to be expressed in the mammary gland. Our studies have focused on two genes, the milk protein gene, b-casein, and OPN, a gene known to be normally expressed in both osteoblasts and mammary epithelial cells [Inman and Shore, 2003;Inman et al, 2005].…”

“…It is an established marker of mammary epithelial cell differentiation and a paradigm for hormonal regulation of gene transcription. The mouse b-casein gene promoter contains a Runx binding site which is essential for its lactogenic hormone stimulation [Saito and Oka, 1996;Inman et al, 2005]. A combination of RNAi, chromatin immunoprecipitation (ChIP) assays, and luciferase reporter assays demonstrated that Runx2 contributes to the activity of the b-casein promoter [Inman et al, 2005].…”

“…The mouse b-casein gene promoter contains a Runx binding site which is essential for its lactogenic hormone stimulation [Saito and Oka, 1996;Inman et al, 2005]. A combination of RNAi, chromatin immunoprecipitation (ChIP) assays, and luciferase reporter assays demonstrated that Runx2 contributes to the activity of the b-casein promoter [Inman et al, 2005]. In addition, it was noted that the Runx site in the b-casein promoter is adjacent to an octamer element and that this configuration is highly conserved in casein promoters from a wide range of mammals.…”

Runx2 in human breast carcinoma: its potential roles in cancer progression

“…(22,25) Runx2 was reported to be involved in the regulation of a mammary-gland-specific b-casein gene and osteopontin. (22,28,29) In regard to its potential roles at the sites of breast carcinoma metastasis to the bone, Runx2 was reported to regulate PTHrP expression of metastatic breast carcinoma cells in the microenvironment of bone metastasis and the cell cycle of carcinoma cells themselves.(30) Runx2 was also shown to modulate several factors, which can contribute to facilitating the process of metastasis including vascular endothelial growth factor (VEGF), (31) several matrix metalloproteinases (MMP) (24,32) and bone sialoprotein.(33) However, to the best of our knowledge, its roles in the early stage of breast cancer patients have not been studied at all. In addition, the correlation of Runx2 nuclear immunoreactivity in breast carcinoma cells and histopathological features of breast cancer were reported, (34) but the correlation between Runx2 expression and prognosis has still remained unknown.…”