Complexes between Forkhead and MADS-box transcription factors have previously been shown to be an important common combination involved in controlling the cyclical expression of cell cycle genes in S. cerevisiae. Here, we demonstrate that this combination of transcription factors is also functionally important in human cells, adding to the repertoire of transcription factor modules that function in metazoan systems. Specifically, we demonstrate that the human Forkhead transcription factor FOXK1 functionally interacts with the MADS-box protein SRF.
FOXK1 can form complexes with SRF in the absence of DNA through binding to the minimal core DNA-binding domain of SRF which includes the MADS-box (). Indeed, a mutant form of FOXK1 that cannot bind to DNA efficiently can still control SRF-dependent promoter activity in vivo
(). However, SRF is required for the efficient recruitment of FOXK1 to target promoters in vitro
and in vivo
( and ). This suggests a model whereby SRF acts as a platform to recruit FOXK1 (C). FOXK1 can then repress promoter activity. These observations are fully consistent with the known roles of SRF and other MADS-box proteins in acting as a platform for the assembly of many different types of transcriptional regulatory complexes, some of which like MRTFs make minimal DNA interactions (7
The paradigm for interactions between Forkhead and MADS-box transcription factors is the yeast Fkh2p-Mcm1p complex (2–5
). However, while there are important overall similarities between the Fkh2p-Mcm1p and human FOXK1-SRF complexes, their modes of interaction and regulation are not identical. Both Fkh2p and FOXK1 share a similar domain structure, with both possessing an N-terminally located FHA domain in addition to the Forkhead DNA-binding domain. FOXK1 is a transcriptional repressor protein, and Fkh2p can also repress transcription of its target genes during the early part of the cell cycle (3–5
). The mouse homologue of FOXK1, Foxk1/MNFβ represses transcription through the recruitment of the Sin3 corepressor (38
) and Fkh2p can also bind to Sin3 in vitro
(our unpublished data). In contrast, to date, no transcriptional activation capacity has been identified for mammalian FOXK1 proteins, and the region encompassing the FHA domain has repressive activity rather than the transactivation ability exhibited by the same region in Fkh2p (; 12,13,38). Moreover, to date, we have been unable to establish a role for the FOXK1-SRF complex in cell cycle control (our unpublished data), and instead, an alternative forkhead protein FOXM1 appears to perform the major role in controlling G2-M phase transcription in mammalian cells (18
). Our data indicate that FOXM1 does not function through binding and changing the activity of SRF (, data not shown).
There also seem to be important differences between human and mouse FOXK1 proteins. In mice, there are two isoforms of Foxk1/MNF, MNFα and the shorter splice form MNFβ. However, only the latter apparently shows DNA-binding and transcriptional regulatory capacity (15
). In contrast, full-length human FOXK1 (equivalent to MNFα) can bind to DNA and regulate transcription ( and ). Secondly, mutations designed to disrupt the phosphopeptide binding activity of the FHA domain of MNFβ partially diminished the repressive activity of MNFβ (15
) but were without effect in FOXK1 (data not shown). It is currently unclear why these proteins apparently function differently but these observations might reflect important evolutionary differences.
To establish the importance of the FOXK1-SRF interaction, we demonstrated that this complex functions on the SM α-actin
genes, and that FOXK1 has a repressive role in the complex. However, FOXK1 does not seem to be an obligate partner for SRF as we could not detect FOXK1 binding to the different target gene, SRF
(). Thus, FOXK1 is likely to be a substoichimetric partner for SRF, as suggested by our inability to co-immunoprecipitate endogenous FOXK1 and SRF. Biologically, FOXK1 is likely to restrict the expression of SM α-actin
expression in non-smooth muscle cell types such as the stem cell-like myogenic side population cells (33
). Recently, another forkhead transcription factor Foxo4 was shown to repress SM α-actin
expression in proliferating smooth muscle cells (39
). In common with FOXK1, Foxo4 repressed transcription in a DNA-binding independent manner and achieved this through interacting with and inhibiting the SRF-myocardin activator complex. Direct interactions between Foxo4 with SRF were not however shown. A different SRF partner protein Elk-1 was also shown to inhibit the expression of a number of smooth muscle-specific genes (40
). However, Elk-1 is ineffective against SM α-actin
. Thus, several different ways might have been devised to reduce the expression of different smooth muscle genes in different cell types through impacting on the activity of SRF.
In summary, we have identified a novel combination of functionally interacting human transcription factors, the Forkhead protein FOXK1 and MADS-box protein, SRF. Future studies will focus on how common this mode of SRF target gene regulation is in human cells.