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ABSTRACT: Heart formation requires a highly balanced network of transcriptional activation of genes. The homeodomain transcription factor, Shox2, is essential for the formation of the sinoatrial valves and for the development of the pacemaking system. The elucidation of molecular mechanisms underlying the development of pacemaker tissue has gained clinical interest as defects in its patterning can be related to atrial arrhythmias. We have analyzed putative targets of Shox2 and identified the Bmp4 gene as a direct target. Shox2 interacts directly with the Bmp4 promoter in chromatin immunoprecipitation assays and activates transcription in luciferase-reporter assays. In addition, ectopic expression of Shox2 in Xenopus embryos stimulates transcription of the Bmp4 gene, and silencing of Shox2 in cardiomyocytes leads to a reduction in the expression of Bmp4. In Tbx5(del/+) mice, a model for Holt-Oram syndrome, and Shox2(-/-) mice, we show that the T-box transcription factor Tbx5 is a regulator of Shox2 expression in the inflow tract and that Bmp4 is regulated by Shox2 in this compartment of the embryonic heart. In addition, we could show that Tbx5 acts cooperatively with Nkx2.5 to regulate the expression of Shox2 and Bmp4. This work establishes a link between Tbx5, Shox2 and Bmp4 in the pacemaker region of the developing heart and thus contributes to the unraveling of the intricate interplay between the heart-specific transcriptional machinery and developmental signaling pathways.
Human Molecular Genetics 12/2010; 19(23):4625-33. · 7.64 Impact Factor
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Rüdiger J Blaschke,
Nathan D Hahurij,
Sanne Kuijper,
Steffen Just,
Lambertus J Wisse,
Kirsten Deissler,
Tina Maxelon,
Konstantinos Anastassiadis,
Jessica Spitzer,
Stefan E Hardt,
Hans Schöler,
Harma Feitsma,
Wolfgang Rottbauer,
Martin Blum,
Frits Meijlink,
Gudrun Rappold,
Adriana C Gittenberger-de Groot
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ABSTRACT: Identifying molecular pathways regulating the development of pacemaking and coordinated heartbeat is crucial for a comprehensive mechanistic understanding of arrhythmia-related diseases. Elucidation of these pathways has been complicated mainly by an insufficient definition of the developmental structures involved in these processes and the unavailability of animal models specifically targeting the relevant tissues. Here, we report on a highly restricted expression pattern of the homeodomain transcription factor Shox2 in the sinus venosus myocardium, including the sinoatrial nodal region and the venous valves.
To investigate its function in vivo, we have generated mouse lines carrying a targeted mutation of the Shox2 gene. Although heterozygous animals did not exhibit obvious defects, homozygosity of the targeted allele led to embryonic lethality at 11.5 to 13.5 dpc. Shox2-/- embryos exhibited severe hypoplasia of the sinus venosus myocardium in the posterior heart field, including the sinoatrial nodal region and venous valves. We furthermore demonstrate aberrant expression of connexin 40 and connexin 43 and the transcription factor Nkx2.5 in vivo specifically within the sinoatrial nodal region and show that Shox2 deficiency interferes with pacemaking function in zebrafish embryos.
From these results, we postulate a critical function of Shox2 in the recruitment of sinus venosus myocardium comprising the sinoatrial nodal region.
Circulation 05/2007; 115(14):1830-8. · 14.74 Impact Factor
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ABSTRACT: Mutations within the homeobox SHOX gene have been associated with short stature and the skeletal deformities found in Léri-Weill, Turner and Langer syndromes implying an involvement of SHOX in growth and bone formation. Despite its clinical significance, the precise role of SHOX and the mechanisms that modulate its functions remain unknown. We reported previously that SHOX is a nuclear protein that specifically binds DNA and acts as a transcriptional activator. We have shown that ectopic expression of SHOX leads to cell-cycle arrest and apoptosis in osteosarcoma and primary cells. To further characterize SHOX, we investigated whether the protein could be a target for phosphorylation. Here, we report that SHOX is phosphorylated exclusively on serine residues in vivo. Two-dimensional phospho-peptide mapping showed that SHOX is phosphorylated to various extents on multiple sites. Site-directed mutagenesis demonstrated that serine 106 is the major SHOX phosphorylation site. We show also that casein kinase II phosphorylates SHOX on serine 106 efficiently in vitro and specific casein kinase II inhibitors reduce SHOX phosphorylation strongly in vivo. Finally, we provide evidence that phosphorylation may play an important role in modulating SHOX biological activities, since a S106A SHOX mutant, defective in phosphorylation, does not activate transcription and fails to induce cell-cycle arrest and apoptosis.
Journal of Molecular Biology 02/2006; 355(3):590-603. · 4.00 Impact Factor
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ABSTRACT: Haploinsufficiency of the short stature homeobox gene SHOX has been found in patients with idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). In addition to complete gene deletions and nonsense mutations, several missense mutations have been identified in both patient groups, leading to amino acid substitutions in the SHOX protein. The majority of missense mutations were found to accumulate in the region encoding the highly conserved homeodomain of the paired-like type. In this report, we investigated nine different amino acid exchanges in the homeodomain of SHOX patients with ISS and LWD. We were able show that these mutations cause an alteration of the biological function of SHOX by loss of DNA binding, reduced dimerization ability, and/or impaired nuclear translocation. Additionally, one of the mutations (c.458G>T, p.R153L) is defective in transcriptional activation even though it is still able to bind to DNA, dimerize, and translocate to the nucleus. Thus, we demonstrate that single missense mutations in the homeodomain fundamentally impair SHOX key functions, thereby leading to the phenotype observed in patients with LWD and ISS.
Human Mutation 07/2005; 26(1):44-52. · 5.69 Impact Factor
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Antonio Marchini,
Tiina Marttila,
Anja Winter,
Sandra Caldeira,
Ilaria Malanchi, Rüdiger J Blaschke,
Beate Häcker,
Ercole Rao,
Marcel Karperien,
Jan M Wit,
Wiltrud Richter,
Massimo Tommasino,
Gudrun A Rappold
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ABSTRACT: Mutations in the homeobox gene SHOX cause growth retardation and the skeletal abnormalities associated with Léri-Weill, Langer, and Turner syndromes. Little is known about the mechanism underlying these SHOX-related inherited disorders of bone formation. Here we demonstrate that SHOX expression in osteogenic stable cell lines, primary oral fibroblasts, and primary chondrocytes leads to cell cycle arrest and apoptosis. These events are associated with alterations in the expression of several cellular genes, including pRB, p53, and the cyclin kinase inhibitors p21(Cip1) and p27(Kip1). A SHOX mutant, such as seen in Léri-Weill syndrome patients, does not display these activities of the wild type protein. We have also shown that endogenous SHOX is mainly expressed in hypertrophic/apoptotic chondrocytes of the growth plate, strongly suggesting that the protein plays a direct role in regulating the differentiation of these cells. This study provides the first insight into the biological function of SHOX as regulator of cellular proliferation and viability and relates these cellular events to the phenotypic consequences of SHOX deficiency.
Journal of Biological Chemistry 09/2004; 279(35):37103-14. · 4.77 Impact Factor
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ABSTRACT: We report the characterization of the nuclear localization signal (NLS) of the short stature homeobox gene SHOX. Mutations within the SHOX gene cause Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LD) as well as idiopathic short stature (ISS). Furthermore, haploinsufficiency of SHOX has also been implicated in Turner syndrome. SHOX has been shown to be a cell-type-specific transcriptional activator that localizes to the nucleus. The SHOX protein contains a central homeodomain that together with its transactivation domain regulates the transcription of its target sequences within the nucleus. The sequences for its nuclear localization have not been identified yet. Experimental characterization of SHOX-NLS by deletion mapping identified a non-classic type basic signal, AKCRK, in the recognition helix of the homeodomain. Fusion of this stretch of five amino acids to a cytoplasmic reporter protein resulted in its nuclear translocation. Functional analysis of a missense mutation R173C (C517T) affecting the identified SHOX-NLS in two families with LWS and LD showed that the mutated SHOX protein is unable to enter the nucleus. Conversely, we can demonstrate that insertion of the identified signal adjacent to the mutant site can restore its nuclear translocation. These results establish impairment of nuclear localization as a mechanistic basis for SHOX-related diseases.
Journal of Cell Science 07/2004; 117(Pt 14):3041-8. · 6.11 Impact Factor
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ABSTRACT: Deletion of the SHOX region on the human sex chromosomes has been shown to result in idiopathic short stature and proposed
to play a role in the short stature associated with Turner syndrome. We have identified a human paired-related homeobox gene,
SHOT, by virtue of its homology to the human SHOX and mouse OG-12 genes. Two different isoforms were isolated, SHOTa and SHOTb,
which have identical homeodomains and share a C-terminal 14-amino acid residue motif characteristic for craniofacially expressed
homeodomain proteins. Differences between SHOTa and b reside within the N termini and an alternatively spliced exon in the
C termini. In situ hybridization of the mouse equivalent, OG-12, on sections from staged mouse embryos detected highly restricted transcripts
in the developing sinus venosus (aorta), female genitalia, diencephalon, mes- and myelencephalon, nasal capsula, palate, eyelid,
and in the limbs. SHOT was mapped to human chromosome 3q25-q26 and OG-12 within a syntenic region on chromosome 3. Based on
the localization and expression pattern of its mouse homologue during embryonic development, SHOT represents a candidate for
the Cornelia de Lange syndrome.
Proceedings of the National Academy of Sciences 03/1998; 95(5):2406-2411. · 9.68 Impact Factor
