The osteocalcin gene encodes a 6-kDa polypeptide, which represents one of the most abundant noncollagenous bone proteins, and the present studies establish that osteocalcin mRNA is detected only in bone tissue. An osteocalcin gene was isolated from a rat genomic DNA library, and sequence analysis indicated that the mRNA is represented in a 953-nucleotide segment of DNA consisting of four exons and three introns. A modular organization of the 5' flanking sequences of the gene is reflected by the presence of at least three classes of regulatory elements, which include the following: (i) RNA polymerase H canonical sequences; (U) a series of consensus sequences for hormone receptor binding sites and cyclic nucleotide responsive elements consistent with physiologic expression ofthe osteocalcin gene; and (Uii) a 24-nucleotide sequence in the proximal promoter region with a CAAT motif as a central element. We have designated this highly conserved sequence as an "osteocalcin box" since only 2 nucleotide substitutions are found in the rat and human osteocalcin genes. We have demonstrated two factors regulating osteocalcin gene expression. First, a 200-fold increase occurs in normal fetal calvaria osteoblasts producing a mineralizing matrix, compared to confluent osteoblasts in a nonmineralizing matrix. Second, contained within the 600 nucleotides immediately upstream from the transcription start site are sequences that support a 10-fold stimulated transcription of the gene by 1,25-dihydroxyvitamin D.There has been much interest in the vitamin K-dependent protein of bone, osteocalcin (bone Gla protein), since its discovery over a decade ago (1). A distinguishing feature of this 5.7-kDa protein (46-50 amino acids, depending on the species), and of functional significance, are 3 residues of the calcium binding amino acid, y-carboxyglutamic acid (Gla). Gla residues are posttranslationally synthesized from selected glutamic acid residues by a vitamin K-and C02-requiring enzyme complex (2). They are located at positions 17, 21, and 24 in all species from swordfish to mammals (1). This highly conserved sequence region from residues 20-34 in the central portion of the molecule, which also includes a disulfide loop (Cys-23-Cys-29), accounts for a structural conformation of the protein in the presence of calcium that promotes a tight binding of the protein to hydroxyapatite (1). The appearance of osteocalcin in embryonic bone coincident with mineral deposition (1), its association with the hydroxyapatite component of the matrix (3), its chemoattractant property for cells capable of bone resorption (4), and its modulated synthesis by the calcitrophic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (5-7) suggest a role for the protein in bone turnover. Although many properties of the protein have been identified, the precise function of osteocalcin is still unknown.Osteocalcin is synthesized de novo by osteoblasts as a 10,000-kDa precursor (8). While the majority ofthe processed osteocalcin peptide (5.7 kDa) is deposited in b...

A variant human histone H2B cDNA (HHC289) has been cloned and characterized and shown to have a complex pattern of regulation with respect to the HeLa S3 cell cycle and HL60 cell differentiation. The H2B protein coding region of HHC289 is flanked at the 3' end by a 1798-nt nontranslated trailer that contains a region of hyphenated dyad symmetry and a poly(A) addition sequence, followed by a poly(A) tail. Nuclear run-on transcription analysis revealed a 2-fold increase in transcription of the HHC289 gene during S phase, in comparison to replication-dependent human histone genes which exhibit a 2-3-fold increase in transcription during S phase. Northern blot analysis indicated that the levels of the 2300-nt HHC289 mRNA species did not vary significantly during the HeLa S3 cell cycle, in comparison to replication-dependent H2B mRNAs which are elevated 15-fold during S phase. Northern blot analysis also revealed a reciprocal relationship during the onset of HL60 differentiation between the expression of the HHC289 H2B gene and the replication-dependent H2B genes. The levels of the 2300-nt HHC289 H2B species increased approximately 10-fold during HL60 cell differentiation whereas the levels of cell cycle dependent H2B mRNAs decreased to less than 1% of those in proliferating cells. These results suggest that complex transcriptional and posttranscriptional regulatory mechanisms control cellular levels of mRNAs from various human H2B histone genes during progression through the cell cycle and at the onset of differentiation.

Expression of cell cycle (core and H1 histone) and cell growth (c-myc and c-fos) regulated genes was examined in primary cultures of chick calvarial osteoblasts during a developmental sequence associated with the progressive maturation of the osteoblast in a bonelike mineralized extracellular matrix. We have identified a transition point early in the developmental sequence which occurs when proliferation ceases and expression of genes related to the differentiated phenotype of osteoblasts is initiated. During this transition period, cellular levels of RNA transcripts from core and H1 histone genes and the c-myc and c-fos protooncogenes decrease in a parallel and coordinate manner. The decline in histone gene transcription that accompanies the loss of accumulated histone mRNA indicates that the downregulation of histone gene expression is at least, in part, transcriptionally mediated. In contrast, persistence of c-myc and c-fos transcription following completion of the proliferation period, when the mRNAs are no longer present at detectable levels, suggests that the initial downregulation of protooncogene expression is controlled at the level of messenger RNA stability. Thus, two types of signaling mechanisms are operative in the down-regulation of cell proliferation genes during osteoblast differentiation--one that impinges on regulatory sequences that influence the interactions of transcription factors with cis-acting promoter elements and a second that modulates messenger RNA turnover. Of significance, downregulation of the cell cycle regulated histone genes is accompanied by a reciprocal increase in the expression of a structurally distinct subset of the histone genes that are not coupled with DNA replication during the period of expression of osteoblast phenotype markers.(ABSTRACT TRUNCATED AT 250 WORDS)

A variant human H2B histone gene (GL105), previously shown to encode a 2300 nt replication independent mRNA, has been cloned. We demonstrate this gene expresses alternative mRNAs regulated differentially during the HeLa S3 cell cycle. The H2B-Gl105 gene encodes both a 500 nt cell cycle dependent mRNA and a 2300 nt constitutively expressed mRNA. The 3' end of the cell cycle regulated mRNA terminates immediately following the region of hyphenated dyad symmetry typical of most histone mRNAs, whereas the constitutively expressed mRNA has a 1798 nt non-translated trailer that contains the same region of hyphenated dyad symmetry but is polyadenylated. The cap site for the H2B-GL105 mRNAs is located 42 nt upstream of the protein coding region. The H2B-GL105 histone gene was localized to chromosome region 1q21-1q23 by chromosomal in situ hybridization and by analysis of rodent-human somatic cell hybrids using an H2B-GL105 specific probe. The H2B-GL105 gene is paired with a functional H2A histone gene and this H2A/H2B gene pair is separated by a bidirectionally transcribed intergenic promoter region containing consensus TATA and CCAAT boxes and an OTF-1 element. These results demonstrate that cell cycle regulated and constitutively expressed histone mRNAs can be encoded by the same gene, and indicate that alternative 3' end processing may be an important mechanism for regulation of histone mRNA. Such control further increases the versatility by which cells can modulate the synthesis of replication-dependent as well as variant histone proteins during the cell cycle and at the onset of differentiation.