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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 gland-specific gene beta-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 beta-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 beta-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.

Targeted deletion of the Runx2 gene in mice has demonstrated that Runx2 is a master regulator of osteoblast differentiation. Runx2 has therefore largely been regarded as a bone-specific transcription factor. Runx2-/- mice die shortly after birth and therefore the role of Runx2 in later developing tissues remains unclear. Here we show that the Runx2 protein is expressed in several mammary epithelial cell lines and in primary mammary epithelial cells. In addition, we have also found that it has a functionally important role in gene regulation. Osteopontin (OPN) is expressed in mammary epithelial cells during pregnancy and lactation and has been shown to have a role in mammary gland differentiation. Here we show that a Runx2 site in the OPN promoter is required for activation of the promoter in mammary epithelial cells. Moreover, dominant-negative Runx proteins can inhibit both activation of an OPN promoter reporter in transient transfections and expression of the endogenous OPN gene in mammary epithelial cells. Our data suggest, for the first time, that the osteoblast transcription factor Runx2 has a role in the normal regulation of gene expression in mammary epithelial cells.

The transcription factor RUNX1 (AML-1, PEBP2alphaB and CBFA2) is essential for definitive haematopoiesis, and chromosomal translocations involving the RUNX1 gene are frequently found in acute leukaemias. The gene encoding the histone acetyltransferase MOZ is also rearranged in some acute leukaemias, resulting in the expression of MOZ fusion proteins. MOZ has recently been shown to interact directly with RUNX1, indicating that MOZ fusion proteins act by deregulating RUNX1 function. Macrophage inflammatory protein-1alpha (MIP-1alpha) is a proinflammatory cytokine that also inhibits proliferation of haematopoietic stem cells. Amongst the conserved sequence elements in the human MIP-1alpha promoter are two consensus RUNX sites. We have investigated the role of these RUNX sites in the regulation of the MIP-1alpha promoter by PMA/PHA stimulation in Jurkat T-cells. RUNX1 can specifically bind to both RUNX sites in vitro and chromatin immunoprecipitation assays demonstrated that endogenous RUNX1 is constitutively bound to the endogenous MIP-1alpha promoter. Mutation of the RUNX sites demonstrated that the proximal RUNX site is essential for PMA/PHA-stimulated activation of the MIP-1alpha promoter. Activation of the promoter can also be inhibited by heterologous expression of the repressor protein AML-1/ETO. We further demonstrate that MOZ can activate the MIP-1alpha promoter and that this activation is largely dependent upon the proximal RUNX site. Moreover, we show that co-expression of MOZ and RUNX1 can synergistically activate the MIP-1alpha promoter. The regulation of MIP-1alpha expression by RUNX1/MOZ is discussed in the context of MIP-1alpha's role as an inhibitor of haematopoietic stem cell proliferation and its potential importance in leukaemias associated with RUNX1 or MOZ chromosomal rearrangements.

The POU domain transcription factor, Oct-2, is essential for the B cell-specific expression of CD36 in mouse B cells. In order to determine how Oct-2 mediates expression of CD36 in B cells, we cloned and analysed the mouse CD36 promoter. In contrast to the human CD36 promoter, the mouse promoter contains a consensus octamer element of the type ATGCTAAT. This octamer element can be bound by either Oct-1 or Oct-2 but requires the expression of Oct-2 to activate transcription in B cells. Mutation of the octamer element renders the CD36 promoter refractory to activation by Oct-2. Furthermore, we demonstrate that the CD36 octamer element does not support recruitment of the B cell-specific co-activator OBF-1 and that CD36 expression is unaffected in primary B cells derived from obf-1(-/-) mice. We conclude that Oct-2 activates CD36 gene expression in mouse B cells via the octamer element in the promoter. Our data also demonstrate that CD36 is the first example of an Oct-2-dependent gene whose expression in B cells is independent of OBF-1. These findings support the notion that Oct-2 regulates gene transcription by both OBF-1-dependent and -independent mechanisms.

Background: Hypoxia stimulates metastasis in cancer and is linked to poor patient prognosis. In tumours, oxygen levels vary and hypoxic regions exist within a generally well-oxygenated tumour. However, whilst the heterogeneous environment is known to contribute to metastatic progression, little is known about the mechanism by which heterogeneic hypoxia contributes to cancer progression. This is largely because existing experimental models do not recapitulate the heterogeneous nature of hypoxia. The primary effector of the hypoxic response is the transcription factor Hypoxia inducible factor 1-alpha (HIF1-alpha). HIF1-alpha is stabilised in response to low oxygen levels in the cellular environment and its expression is seen in hypoxic regions throughout the tumour.Methods: We have developed a model system in which HIF1-alpha can be induced within a sub-population of cancer cells, thus enabling us to mimic the effects of heterogeneic HIF1-alpha expression.Results: We show that induction of HIF1-alpha not only recapitulates elements of the hypoxic response in the induced cells but also results in significant changes in proliferation, gene expression and mammosphere formation within the HIF1-alpha negative population.Conclusions: These findings suggest that the HIF1-alpha expressing cells found within hypoxic regions are likely to contribute to the subsequent progression of a tumour by modifying the behaviour of cells in the non-hypoxic regions of the local micro-environment. [ABSTRACT FROM AUTHOR]

Background: The transcription factor Runx2 has an established role in cancers that metastasize to bone. In metastatic breast cancer cells Runx2 is overexpressed and contributes to the invasive capacity of the cells by regulating the expression of several invasion genes. CBF β is a transcriptional co-activator that is recruited to promoters by Runx transcription factors and there is considerable evidence that CBF β is essential for the function of Runx factors. However, overexpression of Runx1 can partially rescue the lethal phenotype in CBF β-deficient mice, indicating that increased levels of Runx factors can, in some situations, overcome the requirement for CBF β. Since Runx2 is overexpressed in metastatic breast cancer cells, and there are no reports of CBF β expression in breast cells, we sought to determine whether Runx2 function in these cells was dependent on CBF β. Such an interaction might represent a viable target for therapeutic intervention to inhibit bone metastasis. Results: We show that CBF β is expressed in the metastatic breast cancer cells, MDA-MB-231, and that it associates with Runx2. Matrigel invasion assays and RNA interference were used to demonstrate that CBF β contributes to the invasive capacity of these cells. Subsequent analysis of Runx2 target genes in MDA-MB-231 cells revealed that CBF β is essential for the expression of Osteopontin, Matrixmetalloproteinase-13, Matrixmetalloproteinase-9, and Osteocalcin but not for Galectin-3. Chromatin immunoprecipitation analysis showed that CBF β is recruited to both the Osteopontin and the Galectin-3 promoters. Conclusions: CBF β is expressed in metastatic breast cancer cells and is essential for cell invasion. CBF β is required for expression of several Runx2-target genes known to be involved in cell invasion. However, whilst CBF β is essential for invasion, not all Runx2-target genes require CBF β. We conclude that CBF β is required for a subset of Runx2-target genes that are sufficient to maintain the invasive phenotype of the cells. These findings suggest that the interaction between Runx2 and CBF β might represent a viable target for therapeutic intervention to inhibit bone metastasis. [ABSTRACT FROM AUTHOR]

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 gland-specific gene β-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 β-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 [3-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. [ABSTRACT FROM AUTHOR]

Bim is a proapoptotic protein of the Bcl-2 family that shares only the short BH3 domain with other members. It has three isoforms, apparently produced by alternative splicing. The demonstration that Bim is essential for certain apoptotic responses and to prevent overproduction of hematopoietic cells suggests that it may be a tumor suppressor. We have, therefore, investigated the organization of the mouse Bim gene, delineating its promoter and splicing, and positioned the gene on both mouse and human chromosomes. Bim has six exons, but the third is a facultative intron that is spliced out in the mRNAs for the smaller isoforms (BimL and BimS), but not that encoding the largest isoform (BimEL). The 0.8-kb region 5′ to exon 1, which contains a TATA-less promoter and binding sites for several transcription factors, can drive expression of a reporter gene. Mouse Bim localizes to the distal third of Chromosome (Chr) 2, near the F-G boundary, and its human counterpart to Chr 2q12 or q13. Deletions of these bands have been reported in ten tumors (eight hematopoietic), reinforcing the possibility that Bim is a tumor suppressor. These findings should help to clarify the regulation of Bim expression and to assess whether mutations involving Bim contribute to neoplastic and other diseases. [ABSTRACT FROM AUTHOR]

Protein phosphorylation represents one of the major mechanisms for transcription factor activation. Here we demonstrate a molecular mechanism by which phosphorylation by mitogen-activated protein (MAP) kinases leads to changes in transcription factor activity. MAP kinases stimulate DNA binding and transcriptional activation mediated by the mammalian ETS-domain transcription factor Elk-1. Phosphorylation of the C-terminal transcriptional activation domain induces a conformational change in Elk-1, which accompanies the stimulation of DNA binding. C-terminal phosphorylation is coupled to activation of DNA binding by the N-terminal DNA-binding domain via all additional intermediary domain. Activation of DNA binding is mediated by all allosteric mechanism involving the key phosphoacceptor residues. Together, these results provide a molecular model for how phosphorylation induces changes in Elk-1 activity. [ABSTRACT FROM AUTHOR]

The MADS-box family of transcription factors has been defined on the basis of primary sequence similarity amongst numerous proteins from a diverse range of eukaryotic organisms including yeasts, plants, insects, amphibians and mammals. The MADS-box is a conserved motif found within the DNAbinding domains of these proteins and the name refers to four of the originally identified members: MCM1, AG, DEFA and SRF. Several proteins within this family have significant biological roles. For example, the human serum-response factor (SRF) is involved in co-ordinating transcription of the protooncogene c-fos, whilst MCM1 is central to the transcriptional control of cell-type specific genes and the pheromone response in the yeast Saccharomyces cerevisiae. The RSRF/MEF2 proteins comprise a subfamily of this class of transcription factors which are key components in muscle-specific gene regulation. Moreover, in plants, MADS-box proteins such as AG, DEFA and GLO play fundamental roles during flower development. The MADS-box is a contiguous conserved sequence of 56 amino acids, of which 9 are identical in all family members described so far. Several members have been shown to form dimers and consequently two functional regions within the MADS-box have been defined. The N-terminal half is the major determinant of DNA-binding specificity whilst the C-terminal half is necessary for dimerisation. This organisation allows the potential formation of numerous proteins, with subtly different DNA-binding specificities, from a limited number of genes by heterodimerisation between different MADS-box proteins. The majority of MADS-box proteins bind similar sites based on the consensus sequence CC(A/T)6GG although each protein apparently possesses a distinct binding specificity. Moreover, several MADS-box proteins specifically recruit other transcription factors into multi-component regulatory complexes. Such interactions with tither proteins appears to be a common theme within this family and play a pivotal role in the regulation of target genes. [ABSTRACT FROM AUTHOR]

Investigates the phosphorylation of the ternary complex factor (TCF) proteins Elk-1 and SAP-1 and examines the signaling pathways regulating serum response element (SRE)-mediated gene expression. Increased DNA binding, ternary complex formation and transcriptional activation in phosphorylated Elk-1; Integration of MAP kinase signaling pathways in vivo.

Specificity protein 1 (Sp1) is an important transcription factor implicated in numerous cellular processes. However, whether Sp1 is involved in the regulation of RNA polymerase III (Pol III)- directed gene transcription in human cells remains unknown. Here, we first show that filamin A (FLNA) represses Sp1 expression as well as expression of TFIIB-related factor 1 (BRF1) and general transcription factor III C subunit 2 (GTF3C2) in HeLa, 293T, and SaOS2 cell lines stably expressing FLNA-silencing shRNAs. Both BRF1 promoter 4 (BRF1P4) and GTF3C2 promoter 2 (GTF3C2P2) contain putative Sp1-binding sites, suggesting that Sp1 affects Pol III gene transcription by regulating BRF1 and GTF3C2 expression. We demonstrate that Sp1 knockdown inhibits Pol III gene transcription, BRF1 and GTF3C2 expression, and the proliferation of 293T and HeLa cells, whereas Sp1 overexpression enhances these activities. We obtained a comparable result in a cell line in which both FLNA and Sp1 were depleted. These results indicate that Sp1 is involved in the regulation of Pol III gene transcription independently of FLNA expression. Reporter gene assays showed that alteration of Sp1 expression affects BRF1P4 and GTF3C2P2 activation, suggesting that Sp1 modulates Pol III-mediated gene transcription by controlling BRF1 and GTF3C2 gene expression. Further analysis revealed that Sp1 interacts with and thereby promotes the occupancies of TATA box-binding protein, TFIIAα, and p300 at both BRF1P4 and GTF3C2P2. These findings indicate that Sp1 controls Pol III-directed transcription and shed light on how Sp1 regulates cancer cell proliferation. [ABSTRACT FROM AUTHOR]

RNA polymerase II (pol II) is required for the transcription of all protein-coding genes and as such represents a major enzyme whose activity is tightly regulated. Transcriptional initiation therefore requires numerous general transcriptional factors and cofactors that associate with pol II at the core promoter to form a pre-initiation complex. Transcription factor IIA (TFIIA) is a general cofactor that binds TFIID and stabilizes the TFIID-DNA complex during transcription initiation. Previous studies showed that TFIIA can make contact with the DNA sequence upstream or downstream of the TATA box, and that the region bound by TFIIA could overlap with the elements recognized by another factor, TFIIB, at adenovirus major late core promoter. Whether core promoters contain a DNA motif recognized by TFIIA remains unknown. Here we have identified a core promoter element upstream of the TATA box that is recognized by TFIIA. A search of the human promoter database revealed that many natural promoters contain a TFIIA recognition element (IIARE). We show that the IIARE enhances TFIIA-promoter binding and enhances the activity of TATA-containing promoters, but represses or activates promoters that lack a TATA box. Chromatin immunoprecipitation assays revealed that the IIARE activates transcription by increasing the recruitment of pol II, TFIIA, TAF4, and P300 at TATA-dependent promoters. These findings extend our understanding of the role of TFIIA in transcription, and provide new insights into the regulatory mechanism of core promoter elements in gene transcription by pol II. [ABSTRACT FROM AUTHOR]