Tbx5 and Tbx4 transcription factors interact with a new chicken PDZ-LIM protein in limb and heart development.
The T-domain transcription factors, Tbx5 and Tbx4, play important roles in vertebrate limb and heart development. To identify interacting and potential Tbx-regulating proteins, we performed a yeast two-hybrid screen with the C-terminal domain of Tbx5 as bait. We identified a new PDZ-LIM protein composed of one N-terminal PDZ and three C-terminal LIM domains, which we named chicken LMP-4. Among the Tbx2, 3, 4, 5 subfamily, we observed exclusive interaction with Tbx5 and Tbx4 proteins. Tbx3 nor Tbx2 can substitute for LMP-4 binding. While chicken LMP-4 associates with Tbx5 or Tbx4, it uses distinct LIM domains to bind to the individual proteins. Subcellular co-localization of LMP-4 and Tbx proteins supports the protein interaction and reveals interference of LMP-4 with Tbx protein distribution, tethering the transcription factors to the cytoskeleton. The protein-protein interaction indicates regulation of Tbx function at the level of transcription factor nuclear localization. During chicken limb and heart development, Tbx5/LMP-4 and Tbx4/LMP-4 are tightly co-expressed in a temporal and spatial manner, suggesting that they operate in the same pathway. Surprisingly, chicken LMP-4 expression domains outside those of Tbx5 in the heart led to the discovery of Tbx4 expression in the outflow tract and the right ventricle of this organ. The Tbx4-expressing cells coincide with those of the recently discovered secondary anterior heart-forming field. The discrete posterior or anterior expression domains in the heart and the exclusive fore- or hindlimb expression of Tbx5 and Tbx4, respectively, suggest common pathways in the heart and limbs. The identification of a new Tbx5/4-specific binding factor further suggests a novel mechanism for Tbx transcription factor regulation in development and disease.
Department of Pediatrics, The Feinberg School of Medicine, Children's Memorial Institute for Education and Research, Northwestern University, Chicago, IL 60614, USA.
Ange
A
Krause
William
W
Zacharias
Troy
T
Camarata
Barbara
B
Linkhart
Evelyn
E
Law
Antje
A
Lischke
Erik
E
Miljan
Hans-Georg
HG
Simon
D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins; D019556:COS Cells; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D016000:Cluster Analysis; D003599:Cytoskeleton / Q000378:metabolism; D017931:DNA Primers; D004926:Escherichia coli; D005121:Extremities / Q000196:embryology*; D020869:Gene Expression Profiling; D005982:Glutathione Transferase / Q000378:metabolism; D006321:Heart / Q000196:embryology*; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D030161:Avian Proteins; D017931:DNA Primers; D020825:T-Box Domain Proteins; C103172:T-box transcription factor 5; C491196:TBX4 protein, Gallus gallus; D014157:Transcription Factors; D005982:Glutathione Transferase