Characterization of the DNA-binding properties of the Mohawk homeobox transcription factor

Abstract

The homeobox transcription factor Mohawk (Mkx) is a potent transcriptional repressor expressed in the embryonic precursors of skeletal muscle, cartilage, and bone. MKX has recently been shown to be a critical regulator of musculoskeletal tissue differentiation and gene expression; however, the genetic pathways through which MKX functions and its DNA-binding properties are currently unknown. Using a modified bacterial one-hybrid site selection assay, we determined the core DNA-recognition motif of the mouse monomeric Mkx homeodomain to be A-C-A. Using cell-based assays, we have identified a minimal Mkx-responsive element (MRE) located within the Mkx promoter, which is composed of a highly conserved inverted repeat of the core Mkx recognition motif. Using the minimal MRE sequence, we have further identified conserved MREs within the locus of Sox6, a transcription factor that represses slow fiber gene expression during skeletal muscle differentiation. Real-time PCR and immunostaining of in vitro differentiated muscle satellite cells isolated from Mkx-null mice revealed an increase in the expression of Sox6 and down-regulation of slow fiber structural genes. Together, these data identify the unique DNA-recognition properties of MKX and reveal a novel role for Mkx in promoting slow fiber type specification during skeletal muscle differentiation.

FIGURE 1.

Characterizing the monomeric Mkx recognition motif. A fragment encoding the mouse Mkx homeodomain was used to screen a random decamer library using a modified bacterial one-hybrid site selection assay. A, 21 clones from the selection were analyzed for overrepresented sequence motifs. B and C, diagram of key determinants within the homeodomain that are important for specifying the recognition motif for mouse Mkx is shown. Mkx showed little specificity for binding positions 1 and 2 but a strong preference for A-C-A at positions 3–5.

FIGURE 2.

Mkx is capable of negative autoregulation. Fragments of the mouse Mkx promoter cloned upstream of a luciferase reporter gene were assayed for their responsiveness to Mkx regulation in NIH3T3 cells. An Mkx promoter-luciferase construct encompassing −3,576 bp to +127 bp (3.5kbMkx-luc) was strongly repressed by co-transfection with a plasmid-encoding Myc tag full-length Mkx (MT-Mkx). Deletion of the three Mkx repressor domains (MT-Mkx(ΔMRD1-3)) or Helix III of the homeodomain (MT-Mkx(ΔH3)) abrogated this effect. An activated form of Mkx (MT-Mkx-VP16) resulted in specific activation of the Mkx promoter reporter. A smaller Mkx promoter construct encompassing −113 bp to +127 bp (113bpMkx-luc) responded in an identical manner.

FIGURE 3.

Identifying the Mkx response element within the Mkx promoter. A, Mkx promoter sequence (−113 bp to +127) was cloned as three individual fragments (fragment A, −113 to −17; B, −41 to +56; and C, +32 to +127) upstream of SV40 in the luciferase pGL3Promoter. B, of the three smaller Mkx promoter fragments, fragment A was the most responsive to MT-Mkx-VP16 activation. This sequence was renamed the MRE.

FIGURE 4.

Identification of the MBS within the MRE. A and B, MT-Mkx (A) and MT-Mkx-VP16 (B) were able to bind the MBS sequence but not a mutated MBS sequence in an electrophoresis mobility shift assay. The Mkx-MBS complex could be supershifted with an anti-Myc antibody (MT-Ab). Deletion mutants of Mkx lacking Helix III of the homeodomain (MT-Mkx(ΔH3) and MT-Mkx(ΔH3)-VP16) were not sufficient to shift MBS. EMSA primers are provided in supplemental Table S1. C, cell-based luciferase transcription assays demonstrate that the MBS sequence is responsive to Mkx-VP16 activation. An artificial MBS (aMBS) that removes 5′-flanking sequence and randomizes the central 21 bp was equally responsive. Increasing the copy number of the MBS resulted in increased activation by Mkx-VP16. Mutation of either half-site demonstrated that both are required for Mkx-mediated regulation.

FIGURE 5.

Mkx is able to form a homodimer. The ability of Mkx to form a homodimer was assayed using a cell-based mammalian two-hybrid. Mkx is capable of homodimerization dependent upon amino acids within the homeodomain and CD-A. Data are presented for each Gal4-Mkx bait as -fold activation relative to the level of luciferase activity obtained by co-transfection of the Gal4DBD alone with appropriate prey.

FIGURE 6.

Mkx-binding sites within the Sox6 locus. Mkx (ATGTT-N₁₁-AACAT)- and Irx (ACAnnTGT)-binding sites were identified in the locus of Sox6. A, EMSAs demonstrate that MT-Mkx-VP16 can physically interact with the Sox6 MBS and IBS sequences in vitro. B, cell-based luciferase assays demonstrate that these sequences are responsive to MT-Mkx-VP16 activation and are dependent upon sequences within Helix III of the homeodomain (MT-Mkx(ΔH3)-VP16) and CD-A (MT-Mkx(ΔCDA)-VP16). An activated form of mouse Irx2 (HA-Irx2-VP16) could only regulate the Sox6 IBS.

FIGURE 7.

Sox6 and MHC expression in Mkx^−/− satellite cells. Primary cultures of adult muscle satellite cells were isolated from Mkx knock-out (Mkx^−/−) and wild type (Mkx^+/+) mice and cultured in vitro. Total RNA was isolated from proliferating cultures (day 0) or cultures that were differentiated under low mitogenic conditions for 1 or 3 days (day 1 and day 3). A, quantitative real-time PCR revealed that Sox6 transcription is up-regulated in Mkx^−/− satellite cells. Transcription of Myh7 was reduced in differentiated Mkx^−/− myotubes, whereas fast MHC isoforms were up-regulated (Myh1) or unaffected (Myh2). B, expression of fast MHC (My32) and slow MHC (NOQ7.5.4D) were detected in differentiated myoblasts isolated from Mkx^−/− and Mkx^+/+ mice.