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.
Krause
Ange
A
Department of Pediatrics, The Feinberg School of Medicine, Children's Memorial Institute for Education and Research, Northwestern University, Chicago, IL 60614, USA.
Zacharias
William
W
Camarata
Troy
T
Linkhart
Barbara
B
Law
Evelyn
E
Lischke
Antje
A
Miljan
Erik
E
Simon
Hans-Georg
HG
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