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Cleft lip and cleft palate (CLP) are common disorders that occur either as part of a syndrome, where structures other than the lip and palate are affected, or in the absence of other anomalies. Van der Woude syndrome (VWS) and popliteal pterygium syndrome (PPS) are autosomal dominant disorders characterized by combinations of cleft lip, CLP, lip pits, skin-folds, syndactyly and oral adhesions which arise as the result of mutations in interferon regulatory factor 6 (IRF6). IRF6 belongs to a family of transcription factors that share a highly conserved N-terminal, DNA-binding domain and a less well-conserved protein-binding domain. To date, mutation analyses have suggested a broad genotype-phenotype correlation in which missense and nonsense mutations occurring throughout IRF6 may cause VWS; in contrast, PPS-causing mutations are highly associated with the DNA-binding domain, and appear to preferentially affect residues that are predicted to interact directly with the DNA. Nevertheless, this genotype-phenotype correlation is based on the analysis of structural models rather than on the investigation of the DNA-binding properties of IRF6. Moreover, the effects of mutations in the protein interaction domain have not been analysed. In the current investigation, we have determined the sequence to which IRF6 binds and used this sequence to analyse the effect of VWS- and PPS-associated mutations in the DNA-binding domain of IRF6. In addition, we have demonstrated that IRF6 functions as a co-operative transcriptional activator and that mutations in the protein interaction domain of IRF6 disrupt this activity.

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.

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.

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]