Does Vitamin D play a role on Msx1 homeoprotein expression involving an endogenous antisense mRNA?

Msx1 homeobox gene, a member of Msx family, has been implicated in numerous organs. Its participation was established in different events, such as morphogenetic field determinism and epithelio-mesenchymal interactions. Most of Msx1 target organs are also known for their sensitivity to Vitamin D: such as bone, tooth germ, and hair follicle. Whereas, the expression of Msx2, another member of Msx family, has been shown to be controlled by Vitamin D, no information is available for Msx1. This study aims to analyze the potential relationships between Vitamin D and Msx1 through: (1) comparative analysis of Vitamin D receptor (VDR) and Msx1 protein expression, (2) investigation of Msx1 expression in VDR null mutant mice, and (3) study of Msx1 overexpression impact on osteocalcin VDR expression in immortalized MO6-G3 odontoblasts. Results show the existence of cross-talks between Vitamin D and Msx1 regulation pathways. In odontoblastic cells, Msx1 overexpression decrease VDR expression, whereas in rickets Msx1 sense transcript expression is decreased. These cross-talks may open a new window in the analysis of rickets mineralized tissues physiopathology. In Vitamin D null mutants, the study of the natural Msx1 antisense transcript which has been recently described should be informative.

Constitutive amplification of a zinc finger protein gene in cattle.

The human OZF gene encodes a protein consisting essentially of zinc finger motifs of the Krüppel type. Evolutionary studies revealed amplification of the bovine OZF gene in cattle. Domestic cattle includes two major subspecies: taurine (Bos primigenius taurus) and zebu (B. p. indicus). Amplified sequences were found in both subspecies and mapped to the same locus of chromosome X in band q11. No amplification was found in other bovids and in particular in Bison, the closest related genus to the genus Bos. One copy of the amplified locus was cloned and sequenced. It encodes a protein sharing 95% identity with the human OZF protein. The bovine OZF gene was detected in an additional locus on chromosome 18 in both Bos and Bison, in band q23-24, which is likely to represent the ancestral position of the OZF gene. This is the first evidence of gene amplification in a mammalian species during evolution.

Genomic organization and promoter identification of ZNF146, a gene encoding a protein consisting solely of zinc finger domains.

The ZNF146 gene (alias OZF) encodes a protein consisting solely of ten zinc finger motifs. It is amplified and overexpressed in pancreatic carcinomas. To better understand the mechanisms controlling its expression, we have isolated the human ZNF146 gene and performed an initial assessment of its promoter activity. ZNF146 encompasses 25 kb of sequence and consists of four non-coding exons located upstream of a single coding exon. The sequence of proximal 1.4 kb of ZNF146 promoter has a high GC content, is devoid of a TATA box and contains several potential transcriptional elements. This region directs high-level expression of a transfected reporter construct in human cell lines. Analysis of a series of 5'-deletion constructs indicated that the first 80 bp upstream of the potential start site of transcription carry minimal promoter activity whereas the first 550 bp are required for maximal promoter activity.

Partial nucleotide sequence and chromosomal localization of a bovine zinc finger gene ZNF164.

A clone carrying an open reading frame coding for a novel zinc finger protein of the Krüppel family was isolated from a bovine genomic library and designated ZNF 164 (zinc finger protein 164). Partial sequencing revealed that it contained at least 13 zinc finger motifs preceded by a lysine-rich region of 60 amino acids. The ZNF164 protein shared approximately 60% similarity with several zinc finger proteins but did not appear to be orthologous with a previously identified gene. Using fluorescence in situ hybridization, the ZNF164 gene was mapped to bovine chromosome band 17q24.

Msx1 homeogene antisense mRNA in mouse dental and bone cells.

Msx1 plays a key role in early dental and cranio-facial patterning. A systematic screening of Msx1 transcripts during late postnatal stages of development evidenced not only sense mRNA but also antisense mRNA in the skeleton. Natural antisenses are able to bind their corresponding sense RNAs and block protein expression. Specific reverse-transcription polymerase chain reaction (RT-PCR) Northern-blotting using riboprobes and primer extension analysis allowed to identify and sequence a mouse 2184-base Msx1 antisense transcript. The transcription start site was located in a region including a consensus TATA box. In situ hybridization evidenced an increase in antisense mRNA expression during dental and bone cell differentiation in prenatal (Theiler stages E15.5-18.5) and newborn mice. This upregulation was related to Msx1 protein downregulation in cells expressing Msx1 sense mRNA. In vitro, transient Msx1 sense and antisense mRNA overexpression was performed in MO6-G3 cells, which pertain to the odontoblast lineage (polarization and dentin sialoprotein and phosphoprotein synthesis). The balance between antisense and sense Msx1 mRNAs appeared to control Msx1 protein levels. These data suggest that a bidirectional transcription of Msx1 homeogene may control Msx1 protein levels, and therefore may be critical in cell communication and differentiation during dental and cranio-facial development and mineralization.

Amplification and over-expression of OZF, a gene encoding a zinc finger protein, in human pancreatic carcinomas.

The OZF gene encodes a protein consisting of 10 zinc finger motifs and is located on chromosome 19q3.1. We report here the amplification and over-expression of the OZF gene in pancreatic carcinomas. Increased gene copy number was detected in 3 of 12 tumour cell lines and 2 of 12 primary pancreatic carcinomas. Expression was detected in all cell lines, and the gene was over-expressed in cell lines with OZF gene amplification. Five of 8 tumours, including 2 primary tumours with OZF gene amplification, displayed high levels of OZF protein, whereas normal pancreas expressed low levels. Immuno-histochemical analysis showed that expression was restricted to tumour cells. Thus, high-level expression of OZF is frequent in pancreatic carcinomas and may contribute to the development or progression of this tumour.

Endogenous Msx1 antisense transcript: in vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals.

Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous Msx1 antisense RNA (Msx1-AS RNA) in mice, rats, and humans. In situ analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These in vivo data and overexpression of Msx1 sense and AS RNA in an odontoblastic cell line (MO6-G3) showed that the balance between the levels of the two Msx1 RNAs is related to the expression of Msx1 protein. To analyze the impact of this balance in the Msx-Dlx homeoprotein pathway, we analyzed the effect of Msx1, Msx2, and Dlx5 overexpression on proteins involved in skeletal differentiation. We showed that the Msx1-AS RNA is involved in crosstalk between the Msx-Dlx pathways because its expression was abolished by Dlx5. Msx1 was shown to down-regulate a master gene of skeletal cells differentiation, Cbfa1. All these data strongly suggest that the ratio between Msx1 sense and antisense RNAs is a very important factor in the control of skeletal terminal differentiation. Finally, the initiation site for Msx1-AS RNA transcription was located by primer extension in both mouse and human in an identical region, including a consensus TATA box, suggesting an evolutionary conservation of the AS RNA-mediated regulation of Msx1 gene expression.