Cloning, characterization and analysis of the 5' regulatory region of zebrafish xpd gene.

The gene XPD/ERCC2 encodes an ATP-dependent 5'-3' helicase of 760 amino acids. There are few transcription factors known to be involved in the regulation of XPD, and these include the hypoxia-inducible factor-1α (HIF-1α), p53, hepatitis B virus x protein (HBx) and specificity protein 1 (Sp1). To identify functional elements of xpd gene in fish we employed a comparative genomic approach. In our in silico attempt to identify cis-regulatory transcription factor binding sites (TFBSs) conserved among the xpd promoter sequences from five available fish species, we verified that in some cases the spatial disposition of the putative TFBSs was not maintained, but they were present in all the sequences, allowing the prediction of conserved regulatory elements within the promoter sequences analysed. We also investigated the transcriptional regulation of the zebrafish xpd gene. Our in vitro transient transfection-reporter analysis identified a region in the zebrafish xpd gene responsible for xpd regulation, along with regulatory regions that inhibit the activity of this promoter in somatic cells. Moreover, we found two p53 binding sites in the xpd promoter and upon co-transfection assays we observed a repression due to p53. This provides the first identification and characterization of promoter regions that regulate the transcription of the zebrafish xpd gene, and offers insights into the regulation of this gene by p53. Together, these studies further support the utility of comparative genomics to uncover gene regulatory sequences based on evolutionary conservation and provide the basic information to explore and better understand the regulation and expression of XPD.

MiR-29a is an enhancer of mineral deposition in bone-derived systems.

MicroRNAs (miRNAs) provide a mechanism for fine-tuning of intricate cellular processes through post-transcriptional regulation. Emerging evidences indicate that miRNAs play key roles in regulation of osteogenesis. The miR-29 family was previously implicated in mammalian osteoblast differentiation by targeting extracellular matrix molecules and modulating Wnt signaling. Nevertheless, the function of miR-29 in bone formation and homeostasis is not completely understood. Here, we provide novel insights into the biological effect of miR-29a overexpression in a mineralogenic cell system (ABSa15). MiR-29a gain-of-function resulted in significant increase of extracellular matrix mineralization, probably due to accelerated differentiation. We also demonstrated for the first time that miR-29a induced ß-catenin protein levels, implying a stimulation of canonical Wnt signaling. Our data also suggests that SPARC is a conserved target of miR-29a, and may contribute to the phenotype observed in ABSa15 cells. Finally, we provide evidences for miR-29a conservation throughout evolution based on sequence homology, synteny analysis and expression patterns. Concluding, miR-29a is a key player in osteogenic differentiation, leading to increased mineralization in vitro, and this function seems to be conserved throughout vertebrate evolution by interaction with canonical Wnt signaling and conservation of targets.

Evolutionary conservation of TFIIH subunits: implications for the use of zebrafish as a model to study TFIIH function and regulation.

Transcriptional factor IIH (TFIIH) is involved in cell cycle regulation, nucleotide excision repair, and gene transcription. Mutations in three of its subunits, XPB, XPD, and TTDA, lead to human recessive genetic disorders such as trichothiodystrophy and xeroderma pigmentosum, the latter of which is sometimes associated with Cockayne's syndrome. In the present study, we investigate the sequence conservation of TFIIH subunits among several teleost fish species and compare their characteristics and putative regulation by transcription factors to those of human and zebrafish. We report the following findings: (i) comparisons among protein sequences revealed a high sequence identity for each TFIIH subunit analysed; (ii) among transcription factors identified as putative regulators, OCT1 and AP1 have the highest binding-site frequencies in the promoters of TFIIH genes, and (iii) TFIIH genes have alternatively spliced isoforms. Finally, we compared the protein primary structure in human and zebrafish of XPD and XPB - two important ATP-dependent helicases that catalyse the unwinding of the DNA duplex at promoters during transcription - highlighting the conservation of domain regions such as the helicase domains. Our study suggests that zebrafish, a widely used model for many human diseases, could also act as an important model to study the function of TFIIH complex in repair and transcription regulation in humans.

Molecular cloning and expression analysis of xpd from zebrafish (Danio rerio).

The XPD gene, located in human chromosome 19, encodes one of the two helicase components of transcriptional factor IIH (TFIIH), a ten-subunit, multifunctional complex that is essential for multiple processes, including basal transcription initiation and DNA damage repair [1, 2]. Alterations in XPD resulting in defective TFIIH function are associated with UV-sensitive disorders including Xeroderma pigmentosum, Cockayne syndrome, and Trichothiodystrophy (TTD) [3, 4]. TTD mice exhibit many symptoms of premature aging, including osteoporosis, kyphosis and osteosclerosis [5]. This fact has triggered our interest in analyzing XPD involvement in bone biology using zebrafish as model organism. Although orthologs of xpd are present in all species analyzed, no specific data on its gene structure, regulation or function exists at this time in any fish system. In this study we isolated the zebrafish cDNA encoding xpd, and examined its spatial-temporal expression during early development as well as its tissue distribution in adult zebrafish. Only one gene was identified in zebrafish and its sequence analysis showed a molecular structure with 23 coding exons similar to other species. The amino acid sequences were also found to be largely conserved among all species analyzed, suggesting function maintenance throughout evolution. Gene expression analysis in different zebrafish tissues by qPCR showed xpd expression in all tissues examined with the highest expression in branchial arches. Analysis of xpd expression in zebrafish embryos showed maternal inheritance and presence of xpd transcripts in all developmental stages analyzed suggesting its implication in early zebrafish larval development.