Identification of a new pebp2 alpha A2 isoform from zebrafish runx2 capable of inducing osteocalcin gene expression in vitro

Introduction: RUNX2 (also known as CBFA1/Osf2/AML3/PEBP2 alpha A) is a transcription factor essential for bone formation in mammals, as well as for osteoblast and chondrocyte differentiation, through regulation of expression of several bone- and cartilage-related genes. Since its discovery, Runx2 has been the subject of intense studies, mainly focused in unveiling regulatory targets of this transcription factor in high vertebrates. However, no single study has been published addressing the role of Runx2 in bone metabolism of low vertebrates. While analyzing the zebrafish (Danio rerio) runx2 gene, we identified the presence of two orthologs of RUNX2, which we named runx2a and runx2b and cloned a pebp2 alpha A-like transcript of the runx2b gene, which we named pebp2 alpha A2. Materials and Methods: Zebrafish runx2b gene and cDNA were isolated by RT-PCR and sequence data mining. The 3D structure of runx2b runt domain was modeled using mouse Runx1 runt as template. The regulatory effect of pebp2 alpha A2 on osteocalcin expression was analyzed by transient co-transfection experiments using a luciferase reporter gene. Phylogenetic analysis of available Runx sequences was performed with TREE-PUZZLE 5.2. and MrBayes. Results and Conclusions: We showed that the runx2b gene structure is highly conserved between mammals and fish. Zebrafish runx2b has two promoter regions separated by a large intron. Sequence analysis suggested that the runx2b gene encodes three distinct isoforms, by a combination of alternative splicing and differential promoter activation, as described for the human gene. We have cloned a pebp2 alpha A-like transcript of the runx2b gene, which we named pebp2 alpha A2, and showed its high degree of sequence similarity with the mammalian pebp2 alpha A. The cloned zebrafish osteocalcin promoter was found to contain three putative runx2-binding elements, and one of them, located at -221 from the ATG, was capable of mediating pebp2 alpha A2 transactivation. In addition, cross-species transactivation was also confirmed because the mouse Cbfa1 was able to induce the zebrafish osteocalcin promoter, whereas the zebrafish pebp2 alpha A2 activated the murine osteocalcin promoter. These results are consistent with the high degree of evolutionary conservation of these proteins. The 3D structure of the runx2b runt domain was modeled based on the runt domain of mouse Runx1. Results show a high degree of similarity in the 3D configuration of the DNA binding regions from both domains, with significant differences only observed in non-DNA binding regions or in DNA-binding regions known to accommodate considerable structure flexibility. Phylogenetic analysis was used to clarify the relationship between the isoforms of each of the two zebrafish Runx2 orthologs and other Runx proteins. Both zebrafish runx2 genes clustered with other Runx2 sequences. The duplication event seemed, however, to be so old that, whereas Runx2b clearly clusters with the other fish sequences, it is unclear whether Runx2a clusters with Runx2 from higher vertebrates or from other fish.info:eu-repo/semantics/publishedVersio

The Transcriptional Cofactor Nab2 Is Induced by TGF-β and Suppresses Fibroblast Activation: Physiological Roles and Impaired Expression in Scleroderma

By stimulating collagen synthesis and myofibroblasts differentiation, transforming growth factor-β (TGF- β) plays a pivotal role in tissue repair and fibrosis. The early growth response-1 (Egr-1) transcription factor mediates profibrotic TGF-β responses, and its expression is elevated in biopsies from patients with scleroderma. NGF1-A-binding protein 2 (Nab2) is a conserved transcriptional cofactor that directly binds to Egr-1 and positively or negatively modulates Egr-1 target gene transcription. Despite the recognized importance of Nab2 in governing the intensity of Egr-1-dependent responses, the regulation and function of Nab2 in the context of fibrotic TGF-β signaling is unknown. Here we show that TGF-β caused a time-dependent stimulation of Nab2 protein and mRNA in normal fibroblasts. Ectopic expression of Nab2 in these cells blocked Egr-1-dependent transcriptional responses, and abrogated TGF-β-induced stimulation of collagen synthesis and myofibroblasts differentiation. These inhibitory effects of Nab2 involved recruitment of the NuRD chromatin remodeling complex to the COL1A2 promoter and were accompanied by reduced histone H4 acetylation. Mice with targeted deletion of Nab2 displayed increased collagen accumulation in the dermis, and genetic or siRNA-mediated loss of Nab2 in fibroblasts was associated with constitutively elevated collagen synthesis and accentuation of Egr-1-dependent TGF-β responses in vitro. Expression of Nab2 was markedly up-regulated in skin biopsies from patients with scleroderma, and was localized primarily to epidermal keratinocytes. In contrast, little Nab2 could be detected in dermal fibroblasts. These results identify Nab2 as a novel endogenous negative regulator of Egr-1-dependent TGF-β signaling responsible for setting the intensity of fibrotic responses. Defective Nab2 expression or function in dermal fibroblasts might play a role in persistent fibrotic responses in scleroderma

Sclerostin: Current Knowledge and Future Perspectives

In recent years study of rare human bone disorders has led to the identification of important signaling pathways that regulate bone formation. Such diseases include the bone sclerosing dysplasias sclerosteosis and van Buchem disease, which are due to deficiency of sclerostin, a protein secreted by osteocytes that inhibits bone formation by osteoblasts. The restricted expression pattern of sclerostin in the skeleton and the exclusive bone phenotype of good quality of patients with sclerosteosis and van Buchem disease provide the basis for the design of therapeutics that stimulate bone formation. We review here current knowledge of the regulation of the expression and formation of sclerostin, its mechanism of action, and its potential as a bone-building treatment for patients with osteoporosis

HMMerThread: Detecting Remote, Functional Conserved Domains in Entire Genomes by Combining Relaxed Sequence-Database Searches with Fold Recognition

Conserved domains in proteins are one of the major sources of functional information for experimental design and genome-level annotation. Though search tools for conserved domain databases such as Hidden Markov Models (HMMs) are sensitive in detecting conserved domains in proteins when they share sufficient sequence similarity, they tend to miss more divergent family members, as they lack a reliable statistical framework for the detection of low sequence similarity. We have developed a greatly improved HMMerThread algorithm that can detect remotely conserved domains in highly divergent sequences. HMMerThread combines relaxed conserved domain searches with fold recognition to eliminate false positive, sequence-based identifications. With an accuracy of 90%, our software is able to automatically predict highly divergent members of conserved domain families with an associated 3-dimensional structure. We give additional confidence to our predictions by validation across species. We have run HMMerThread searches on eight proteomes including human and present a rich resource of remotely conserved domains, which adds significantly to the functional annotation of entire proteomes. We find ∼4500 cross-species validated, remotely conserved domain predictions in the human proteome alone. As an example, we find a DNA-binding domain in the C-terminal part of the A-kinase anchor protein 10 (AKAP10), a PKA adaptor that has been implicated in cardiac arrhythmias and premature cardiac death, which upon stress likely translocates from mitochondria to the nucleus/nucleolus. Based on our prediction, we propose that with this HLH-domain, AKAP10 is involved in the transcriptional control of stress response. Further remotely conserved domains we discuss are examples from areas such as sporulation, chromosome segregation and signalling during immune response. The HMMerThread algorithm is able to automatically detect the presence of remotely conserved domains in proteins based on weak sequence similarity. Our predictions open up new avenues for biological and medical studies. Genome-wide HMMerThread domains are available at http://vm1-hmmerthread.age.mpg.de

Novel mutants of NAB corepressors enhance activation by Egr transactivators.

The NGFI-A binding corepressors NAB1 and NAB2 interact with a conserved domain (R1 domain) within the Egr1/NGFI-A and Egr2/Krox20 transactivators, and repress the transcription of Egr target promoters. Using a novel adaptation of the yeast two-hybrid screen, we have identified several point mutations in NAB corepressors that interfere with their ability to bind to the Egr1 R1 domain. Surprisingly, NAB proteins bearing some of these mutations increased Egr1 activity dramatically. The mechanism underlying the unexpected behavior of these mutants was elucidated by the discovery that NAB conserved domain 1 (NCD1) not only binds to Egr proteins but also mediates multimerization of NAB molecules. The activating mutants exert a dominant negative effect on NAB repression by multimerizing with native NAB proteins and preventing binding of endogenous NAB proteins with Egr transactivators. To examine NAB repression of a native Egr target gene, we show that NAB2 represses Egr2/Krox20-mediated activation of the bFGF/FGF-2 promoter, and that repression is reversed by coexpression of dominant negative NAB2. Because of their specific ability to alleviate NAB repression of Egr target genes, the dominant negative NAB mutants will be useful in elucidating the mechanism and function of NAB corepressors