The OC box of the rat osteocalcin promoter (nt -99 to -76) is the principal proximal regulatory element contributing to both tissue-specific and developmental control of osteocalcin gene expression. The central motif of the OC box includes a perfect consensus DNA binding site for certain homeodomain proteins. Homeodomain proteins are transcription factors that direct proper development by regulating specific temporal and spatial patterns of gene expression. We therefore addressed the role of the homeodomain binding motif in the activity of the OC promoter. In this study, by the combined application of mutagenesis and site-specific protein recognition analysis, we examined interactions of ROS 17/2.8 osteosarcoma cell nuclear proteins and purified Msx-1 homeodomain protein with the OC box. We detected a series ofrelated specific protein-DNA interactions, a subset of which were inhibited by antibodies directed against the Msx-1 homeodomain but which also recognize the Msx-2 homeodomain. Our results show that the sequence requirements for binding the Msx-1 or Msx-2 homeodomain closely parallel those necessary for osteocalcin gene promoter activity in vivo. This functional relationship was demonstrated by transient expression in ROS 17/2.8 osteosarcoma cells of a series of osteocalcin promoter (nt -1097 to +24)-reporter gene constructs containing mutations within and flanking the homeodomain binding site of the OC box. Northern blot analysis of several bone-related cell types showed that all of the cells expressed msx-1, whereas msx-2 expression was restricted to cells transcribing osteocalcin. Taken together, our results suggest a role for Msx-1 and -2 or related homeodomain proteins in transcription of the osteocalcin gene.Osteocalcin is one of the major noncollagenous proteins associated with mineralized bone extracellular matrix (1). Expression of the osteocalcin gene is dependent on cell type and stage-specific phenotypic development in osteoblasts (2-4). The osteocalcin gene is transcriptionally activated in postproliferative cells and expression is enhanced by 1,25-dihydroxyvitamin D3 (vitamin D) at the onset of extracellular matrix mineralization both in vivo and in vitro (refs. 5-9 and reviewed in ref. 10). Transcription in bone cells has been defined by the activity of a modular promoter containing multiple independent sites that exhibit recognition for transcription factors that transduce and integrate a broad spectrum of physiological regulatory signals (8,9,(11)(12)(13)(14)(15).Deletion analysis has demonstrated that the principal basal regulatory domain of the proximal osteocalcin promoter is localized between nt -108 and +24 (11,16). We have previously shown that critical transcriptional control mechanisms are associated with recognition by cognate transcription factors of a 24-nt element designated the OC box (nt -99 to -76). In vivo competition experiments demonstrate that expression of an osteocalcin promoter-reporter gene construct is blocked when cotransfected into osteoblastic cells with...

Two homeotic genes, Dlx and Msx, appear to regulate development of mineralized tissues, including bone, cartilage, and tooth. Expression of Msx-1 and Msx-2 has been studied during development of the osteoblast phenotype, but the role of Dlx in this context and in the regulation of bone-expressed genes is unknown. We used targeted differential display to isolate homeotic genes of the Dlx family that are expressed at defined stages of osteoblast differentiation. These studies were carried out with fetal rat calvarial cells that produce bone-like tissue in vitro. We observed a mineralization stage-specific mRNA and cloned the corresponding cDNA, which represents the rat homolog of Dlx-5. Northern blot analysis and competitive RT-PCR demonstrated that Dlx-5 and the bone-specific osteocalcin genes exhibit similar up-regulated expression during the mineralization period of osteoblast differentiation. This expression pattern differs from that of Msx-2, which is found predominantly in proliferating osteoblasts. Several approaches were pursued to determine functional consequences of Dlx-5 expression on osteocalcin transcription. Constitutive expression of Dlx-5 in ROS 17/2.8 cells decreased osteocalcin promoter activity in transient assays, and conditional expression of Dlx-5 in stable cell lines reduced endogenous mRNA levels. Consistent with this finding, antisense inhibition of Dlx-5 increased osteocalcin gene transcription. Osteocalcin promoter deletion analysis and binding of the in vitro translation product of Dlx-5 demonstrated that repressor activity was targeted to a single homeodomain-binding site, located in OC-Box I (-99 to -76). These findings demonstrate that Dlx-5 represses osteocalcin gene transcription. However, the coupling of increased Dlx-5 expression with progression of osteoblast differentiation suggests an important role in promoting expression of the mature bone cell phenotype.

Bone-specific expression of the osteocalcin gene is transcriptionally controlled. Deletion analysis of osteocalcin promoter sequences by transient transfection of osseous (ROS 17/2.8) and nonosseous (R2 fibroblast) cells revealed that the most proximal 108 nucleotides are sufficient to confer tissue-specific expression. By gel mobility shift assays with wild-type and mutated oligonucleotides and nuclear extracts from several different cell lines we identified a novel transcription factor complex which exhibits sequence-specific interactions with the primary transcriptional element, the OC box (nt -99 to -76). This OC box binding protein (OCBP) is present only in osteoblast-like cells. Methylation interference demonstrated association of the factor with OC box sequences overlapping the Msx homeodomain consensus binding site. By assaying several mutations of the OC box, both in gel shift and transient transfection studies using ROS 17/2.8, we show the following. First, binding of OCBP correlates with osteocalcin promoter activity in ROS 17/2.8 cells. Increased binding leads to a 2-3-fold increase in transcription, while decreased binding results in transcription 30-40% of control. Second, homeodomain protein binding suppresses transcription. However, Msx expression is critical for full development of the bone phenotype as determined by antisense studies. Last, we show that one of the mutations of the OC box permits expression of osteocalcin in non-osseous cell lines. In summary, we demonstrate association of at least two classes of tissue-restricted transcription factors with the OC box element, the OCBP and Msx proteins, supporting the concept that these sequences contribute to defining tissue specificity.

To explore the structural basis of alternative splicing, we have analyzed the splicing of pre-mRNAs containing an optional exon, E4, from the preprotachykinin gene. This gene encodes substance P and related tachykinin peptides by alternative splicing of a common pre-mRNA. We have shown that alternative splicing of preprotachykinin pre-mRNA occurs by preferential skipping of optional E4. The competing mechanism that incorporates E4 into the final spliced RNA is constrained by an initial block to splicing of the immediate upstream intervening sequence (IVS), IVS3. This block is relieved by sequential splicing, in which the immediate downstream IVS4 is removed first. The structural change resulting from the first splicing event is directly responsible for activation of IVS3 splicing. This structural rearrangement replaces IVS4 sequences with E5 and its adjacent IVS5 sequences. To determine how this structural change promoted IVS3 splicing, we asked what structural change(s) would restore activity of IVS3 splicing-defective mutants. The most significant effect was observed by a 2-nucleotide substitution that convened the 5' splice site of E4 to an exact consensus match, GUAAGU. Exon 5 sequences alone were found not to promote splicing when present in one or multiple copies. However, when a 15-nucleotide segment of IVS5 containing GUAAGU was inserted into a splicing-defective mutant just downstream of the hybrid exon segment E4E5, splicing activity was recovered. Curiously, the 72-nucleotide L2 exon of adenovirus, without its associated 5' splice site, activates splicing when juxtaposed to E4. Models for the activation of splicing by an RNA structural change are discussed.

Distorted sex ratios occur in hematologic disorders. For example, chronic lymphocytic leukemia (CLL) displays disproportionate sex ratios with a large male excess. However, the underlying genetics for these disparities are poorly understood, and gender differences for specific cytogenetic abnormalities have not been carefully investigated. We sought to provide an initial characterization of gender representation in genetic abnormalities in CLL by using fluorescence in situ hybridization (FISH). We confirm the well known skewed male-tofemale (M/F sex ratio) of ~1.5 in our CLL study population, but also determine the genotypic M/F sex ratio values corresponding to specific FISH DNA probes. Genetic changes in CLL detectable by four FISH probes were statistically compared with respect to gender. Initial FISH evaluations of 4698 CLL patients were retrospectively examined and new findings of the genotypic M/F sex ratios for these probes are reported. This study represents the largest CLL survey conducted in the United States using FISH probes. The CLL database demonstrated that FISH abnormalities (trisomy 12, 13q14.3 deletion and 17p13.1 deletion) probes had skewed M/F ratios of ~1.5. Also, by statistical analysis it was shown that ATM gene loss (11q22.3q23.1 deletion) solely or with other abnormalities was considerably higher in males with an M/F ratio of 2.5 and significantly different from M/F ratios of 1.0 or 1.5. We hypothesize that interactions involving these autosomal abnormalities (trisomy 12, and deletions of 11q22.3, 13q14.3, and 17p13.1), and the sex chromosomes may provide the genetic basis for the altered phenotypic M/F ratio in CLL.