Modulation of Cardiac Growth and Development by HOP, an Unusual Homeodomain Protein

Chong Hyun Shin, Zhi-Ping Liu, Robert Passier, Chun-Li Zhang, Da-Zhi Wang, Thomas M. Harris, Hiroyuki Yamagishi, James A. Richardson, Geoffrey Childs, Eric N. Olson
2002 Cell  
quence referred to as the homeobox. Homeobox genes James A. Richardson, 2 Geoffrey Childs, 3 are found in all eukaryotic organisms, and over 160 have and Eric N. Olson 1,5 potentially been identified in the human genome. Ho-1 Department of Molecular Biology meodomain proteins can be categorized into different 2 Department of Pathology classes based on amino acid sequence homologies University of Texas Southwestern Medical Center within the homeodomain and their expression patterns. at Dallas A
more » ... ubset of homeobox genes located in clusters confers 6000 Harry Hines Boulevard positional information along the antero-posterior axis Dallas, Texas 75390 of the embryo. Numerous other homeobox genes are 3 Department of Molecular Genetics distributed throughout the genome and show unique Albert Einstein College of Medicine expression patterns. 1300 Morris Park Avenue The homeodomain is comprised of 60 amino acids Bronx, New York 10461 that adopt three ␣ helices, with a characteristic helix-4 Hubrecht Laboratory turn-helix motif (reviewed in Kornberg, 1993). Helix-3, Uppsalalaan 8 referred to as the recognition helix, lies in the major 3584CT Utrecht groove of the DNA binding site and makes direct contact The Netherlands with the DNA. Amino acid variations within the recognition helix dictate DNA binding site specificity of different homeodomain proteins. Additional specificity of DNA Summary binding is contributed by a flexible region, called the N-terminal arm, that extends from the first helix and We have discovered an unusual homeodomain prowraps around the DNA to make contacts with the minor tein, called HOP, which is comprised simply of a hogroove. meodomain. HOP is highly expressed in the develop- Target gene specificity of different homeodomain proing heart where its expression is dependent on the teins that bind the same DNA sequence is achieved cardiac-restricted homeodomain protein Nkx2.5. HOP through differential association with positive and negadoes not bind DNA and acts as an antagonist of serum tive cofactors. Such interactions can occur between horesponse factor (SRF), which regulates the opposing meodomain proteins and other transcriptional regulaprocesses of proliferation and myogenesis. Mice hotors that bind adjacent sequences in regulatory DNA or mozygous for a HOP null allele segregate into two by direct protein-protein interactions in the absence of phenotypic classes characterized by an excess or defi-DNA binding. A classic example of such combinatorial ciency of cardiac myocytes. We propose that HOP interactions is illustrated by the yeast homeodomain modulates SRF activity during heart development; its protein MAT␣2, which interacts with the MADS (MCM1, absence results in an imbalance between cardiomyo-Agamous, Deficiens, and serum response factor) box cyte proliferation and differentiation with consequent transcription factor MCM1, resulting in repression of abnormalities in cardiac morphogenesis. a-specific genes (Smith and Johnson, 1992). Similarly, homeodomains of the Paired-type associate with serum Introduction response factor (SRF), a MADS box factor that controls the expression of genes involved in cell proliferation and Organ formation during embryogenesis requires the myogenesis (Norman et al., 1988; Reecy et al., 1998). commitment of multipotent progenitor cells to specific Association of SRF with the phox/prx1 homeodomain lineages, the activation of tissue-specific genes, and the protein enhances the binding of SRF to DNA (Grueneprecise spatial organization of specialized cell types. A berg et al., 1992). The association of MADS box proteins tightly regulated balance between cell proliferation and like MCM1 and SRF with homeodomain proteins coudifferentiation is also required to generate and maintain ples cell identity with signal responsiveness, thereby the size and shape of the mature organ. These proproviding a mechanism for cell type-specific responses cesses are controlled by combinatorial interactions beto "generic" signals. tween cell-specific and broadly expressed transcription The cardiac-restricted homeodomain protein, Nkx2.5, factors that act through positive and negative mechaalso associates with SRF, resulting in cooperative actinisms to govern arrays of target genes in distinct tempovation of cardiac genes (Chen and Schwartz, 1996; Seprospatial patterns. ulveda et al., 2002). Nkx2.5, which is among the earliest Members of the homeodomain family of transcription markers of heart formation in vertebrate embryos (Lints factors play key roles in organogenesis and embryonic et al., 1993), is an ortholog of tinman, a homeobox gene patterning by interpreting positional information in the required for heart formation in Drosophila (reviewed in Harvey, 1996). Nkx2.5 is expressed throughout the developing and adult heart (Lints et al., 1993). Mice homozy-5 Correspondence: eolson@hamon.swmed.edu 6 These authors contributed equally to this work. gous for an Nkx2.5 null allele die during embryogenesis Cell 726 SRF or GST (as negative control) were incubated with 10 l of References 35 S-labeled HOP in 250 l GST binding buffer (PBS with 0.5% NP-40 and 5 mM EDTA) for 2 hr at 4ЊC. After washing with GST binding Benezra, R., Davis, R., Lockshon, D., Turner, L., and Weintraub, H. buffer, proteins associated with GST-agarose beads were analyzed (1990). The protein Id: a negative regulator of helix-loop-helix DNA with 4%-20% SDS-PAGE and visualized by autoradiography. One binding proteins. Cell 61, 49-59. l of in vitro translated 35 S-labeled HOP was loaded as input control. Chang, P.S., Li, L., McAnally, J., and Olson, E.N. (2001). Muscle specificity encoded by specific serum response factor-binding sites. J. Biol. Chem. 276, 17206-17212. Immunoprecipitation and Western Blot Analysis Expression vectors encoding Flag-tagged HOP and HA-tagged SRF Chen, C.Y., and Schwartz, R.J. (1996). Recruitment of the tinman were transfected into 293T cells. Cells were harvested 30 hr later homolog Nkx-2.5 by serum response factor activates cardiac ␣-actin and lysed in 1 ml of ice-cold PBS supplemented with complete gene transcription. Mol. Cell. Biol. 16, 6372-6384. protease inhibitors (Roche), 0.5% Triton X-100, 1 mM EDTA, and 40 Chen, J., and Fishman, M. (1996). Zebrafish tinman homolog demarunits of DNAase I (Roche Molecular Biochemicals). Immunoprecipicates the heart field and initiates myocardial differentiation. Develtations were carried out by incubating 500 l lysate with 20 l FLAGopment 122, 3809-3816. sepharose (Sigma) at 4ЊC for 3 hr. The beads were washed twice with Chen, F., Kook, H., Milewski, R., Gitler, A.D., Lu, M.M., Li, J., Nalysis buffer and boiled in SDS sample buffer. Immunoprecipitated zarian, R., Schnepp, R., Kuangyu, J., Biben, C., et al. (2002). Hop is proteins were analyzed by Western blotting using rat anti-HA antian unusual homeobox gene that modulates cardiac development. body. Anti-HOP antibody was generated by Cocalico Biologicals Cell 110, this issue, 713-723. (Reamstown, PA) using GST-HOP. Cleaver, O., Patterson, K., and Krieg, P. (1996). Overexpression of the tinman-related genes XNkx2.5 and XNkx2.3 in Xenopus embryos Gel Mobility Shift Assays results in myocardial hyperplasia. Development 122, 3549-3556. SRF (0.2 g cDNA) was transcribed and translated in vitro in the Croissant, J., Kim, J., Eichele, G., Goering, L., Lough, J., Prywes, presence of increasing amounts of HOP (0.2, 0.4, and 0.8 g cDNA) R., and Schwartz, R. (1996). Avian serum response factor expression in 25 l total volume with a TNT T7-coupled reticulocyte lysate restricted primarily to muscle cell lineages is required for ␣-actin system supplemented with [ 35 S]-methionine. Gel mobility shift gene transcription. Dev. Biol. 177, 250-264. assays were performed with 32 P-labeled oligonucleotides corresponding to the sequence of the c-fos SRE as described (Chang Guan, K.L., and Dixon, J.E. (1991). Eukaryotic proteins expressed et al., 2001). The amount of shifted probe was quantified with a in Escherichia coli: an improved thrombin cleavage and purification phosphoimager and normalized against the amount of translated procedure of fusion proteins with glutathione S-transferase. Anal. 35 S-labeled SRF protein in each binding reaction. Biochem. 192, 262-267. Tanaka, M., Chen, Z., Bartunkova, S., Yamasaki, N., and Izumo, S. (1999). The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. Development 126, 1269-1280. Treacy, M.N., He, X., and Rosenfeld, M.G. (1991). I-POU: a POUdomain protein that inhibits neuron-specific gene activation. Nature 350, 577-584. Vandromme, M., Gauthier-Rouviere, C., Carnac, G., Lamb, N., and Fernandez, A. (1992). Serum response factor p67 SRF is expressed and required during myogenic differentiation of both mouse C2 and rat L6 muscle cell lines.
doi:10.1016/s0092-8674(02)00933-9 pmid:12297046 fatcat:2qrlrmvfw5bajf4uyz6sjdxq5u