Molecular Structure of the Chicken Vitamin D-lnduced Calbindin-D_28KGene Reveals Eleven Exons, Six Ca²⁺-Binding Domains, and Numerous Promoter Regulatory Elements

Abstract

The seco-steroid hormone 1,25-dihydroxyvitamin D3 is known to induce the expression of a calcium binding protein termed calbindin-D28K in a variety of target tissues. In order to comprehend the mechanism of induction we have cloned and sequenced the chicken calbindin-D28K gene. The gene spans some 18.5 kilobases (kb) of chromosomal DNA from the putative Cap site to the polyadenylation site of the 2.8 kb mRNA. It is split into 11 coding exons by 10 intervening sequences. The promoter region of this gene is markedly G + C-rich (60-80%) extending from -225 to +400. Within this region we find 70 CpG dinucleotides, four G-C boxes, and numerous known promoter regulatory signals. These putative regulatory signals include a TATA box (ATAAATA) at -30 and a CAT box (CCAAT) at -326. Ten additional variant CAT boxes are found in the upstream promoter region (-218 to -770) of this gene. Furthermore we have identified a glucocorticoid-like responsive element at -410 (TCTACACACTGTTCC) and this element overlaps a metal responsive element (TGCACTC) and a variant CAT box (CCAAAT) and juxtaposes an enhancer-like core element (AAATGGT) on its 3'-side. In addition, the calbindin-D28K promoter is composed of a variety of simple repeated sequences, some of which are components of putative regulatory signals. All splice junctions were found to conform to the GT-AG rule. A consensus sequence of the 5'-splice junction reads AG/GTAAG-TTATA. A consensus sequence of the 3'-splice site consists of two elements: a pyrimidine track (mainly T) followed by ACAG/G-T. A two-dimensional model of calbindin-D28K was constructed which projects the existence of 6 alpha-helix-loop-alpha-helix regions characteristic of calcium binding domains. The 3'-end of the gene consists of a single large (2039 base pair) uninterrupted exon, an organizational feature common to other members of the calcium binding protein gene family which include calmodulin, parvalbumin, Spec I, myosin light chains, etc. Another feature common to the gene family is the presence of the repeated sequence ATTT or TTTA located in the 3'-untranslated exons. These simple repeat sequences could be involved in regulating mRNA degradation by serving as a ribonuclease recognition signal.

Structure of the rat osteocalcin gene and regulation of vitamin D-dependent expression.

“…There are only two nucleotide substitutions between the rat and human osteocalcin gene. Despite the sequence conservation of this element in the osteocalcin gene promoter, a homologous element is not found in the proximal promoter of the vitamin D-responsive genes alkaline phosphatase (22) and calbindin (24), suggesting a gene-specific and/or tissue-specific role for this promoter sequence. Sequences of other vitamin Dresponsive promoters and osteoblast synthesized proteins are not yet known.…”

“…1, a series of putative vitamin D-responsive elements are underlined. Although not restricted to vitamin D-responsive genes, sequences rich in AGAGG and GAGA have been found in promoter and structural regions of genes encoding three vitamin D-dependent proteins-alkaline phosphatase (22), osteonectin (23), and calbindin (24). The osteocalcin gene has several such sequences and the complementary nucleotide sequences (CCTCT) located in the promoter region; in introns 1, 3, and 4; in exon 2; and in the 3' flanking region (Fig.…”

Cloning and Expression of Secretagogin, a Novel Neuroendocrine- and Pancreatic Islet of Langerhans-specific Ca2+-binding Protein

“…In humans, calbindin D-28k and calretinin have been localized to chromosomes 8 and 16, respectively (10). The genomic structures of both proteins, which are highly evolutionarily conserved, have been elucidated and comprise 11 exons interrupted by 10 introns (11)(12)(13).…”

1,25-Dihydroxyvitamin D3 stimulates specifically the last steps of epidermal differentiation of cultured human keratinocytes

“…At the molecular level, 1,25(OH),-D3 has been shown to induce the transcription of calbindin genes in intestinal cells [33,34,40,621, of the osteocalcin gene in bone [14,29,37, 671 and to reduce the synthesis of collagen I in the latter tissue [25,301. Vitamin D, actually increases calcium and phosphorus uptake by intestine cells, increases calcium reabsorption by the kidney, and maintains normal bone growth.…”