The CCAAT-binding factor CBF/NF-Y regulates the human acetylcholinesterase promoter activity during calcium ionophore A23187-induced cell apoptosis

We previously reported that the expression of acetylcholinesterase during A23187-induced apoptosis of HeLa cells is regulated by Ca(2+) mobilization through the modulation of mRNA stability and acetylcholinesterase promoter activity. Transactivation of the human acetylcholinesterase promoter by A23187 was partially mediated by the distal CCAAT motif within the -1270 to -1248 fragment of the human acetylcholinesterase promoter, which was bound by the CCAAT binding factor (CBF/NF-Y). In the present study, we investigated the molecular mechanisms by which CBF/NF-Y regulates A23187-induced activation of the human acetylcholinesterase promoter. The results indicate that CBF/NF-Y binding to the distal CCAAT motif suppresses the promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that binding of CBF/NF-Y to the distal CCAAT motif decreased after A23187 treatment. Our results suggest that acetylcholinesterase promoter activation during A23187-induced HeLa cell apoptosis may result partly from the dissociation of CBF/NF-Y from the distal CCAAT motif in the acetylcholinesterase promoter, reversing this suppression.     

Activation of fibroblast procollagen alpha 1(I) transcription by mechanical strain is transforming growth factor-beta-dependent and involves increased binding of CCAAT-binding factor (CBF/NF-Y) at the proximal promoter.

During normal developmental tissue growth and in a number of diseases of the cardiopulmonary system, adventitial and interstitial fibroblasts are subjected to increased mechanical strain. This leads to fibroblast activation and enhanced collagen synthesis, but the underlying mechanisms involved remain poorly understood. In this study, we have begun to identify and characterize mechanical strain-responsive elements in the rat procollagen alpha 1(I) (COL1A1) gene and show that the activity of COL1A1 promoter constructs, transiently transfected into cardiac fibroblasts, was increased between 2- and 4-fold by continuous cyclic mechanical strain. This was accompanied by an approximately 3-fold increase in the levels of total active transforming growth factor-beta (TGF-beta) released into the medium. Inclusion of a pan-specific TGF-beta neutralizing antibody inhibited strain-induced COL1A1 promoter activation. Deletion analysis revealed the presence of two potential strain response regions within the proximal promoter, one of which contains an inverted CCAAT-box overlapping a GC-rich element. Both mechanical strain and exogenously added TGF-beta1 enhanced the binding activity of CCAAT-binding factor, CBF/NF-Y, at this site. Moreover, this element was sufficient to confer strain-responsiveness to an otherwise unresponsive SV40 promoter. In summary, this study demonstrates that strain-induced COL1A1 promoter activation in cardiac fibroblasts is TGF-beta-dependent and involves increased binding of CCAAT-binding factor at the proximal promoter. Furthermore, these findings suggest a novel and potentially important TGF-beta response element in the rat COL1A1 gene.     

Mapping of a human fibrillar collagen gene, pro alpha 1 (XI) (COL11A1), to the p21 region of chromosome 1

Type XI collagen is a minor and poorly characterized structural component of cartilage. Recently, cDNA and genomic clones coding for the pro alpha 1 chain of human Type XI collagen, formerly 1 alpha collagen, have been isolated and fully characterized. Here we have used one such probe to establish the chromosomal localization of the pro alpha 1 (XI) collagen gene (COL11A1) by hybridization to filter-bound DNA isolated from flow-sorted chromosomes and by in situ hybridization on metaphase chromosomes. This combination of approaches has enabled us to locate COL1A11 in the p21 region of chromosome 1. This represents the first mapping of a Type XI collagen gene and the first assignment of a collagen locus to chromosome 1. These studies also provide additional evidence for the nearly uniform dispersion of the human fibrillar collagen genes in the human genome.     

Fibrochondrogenesis results from mutations in the COL11A1 type XI collagen gene

Fibrochondrogenesis is a severe, autosomal-recessive, short-limbed skeletal dysplasia. In a single case of fibrochondrogenesis, whole-genome SNP genotyping identified unknown ancestral consanguinity by detecting three autozygous regions. Because of the predominantly skeletal nature of the phenotype, the 389 genes localized to the autozygous intervals were prioritized for mutation analysis by correlation of their expression with known cartilage-selective genes via the UCLA Gene Expression Tool, UGET. The gene encoding the α1 chain of type XI collagen (COL11A1) was the only cartilage-selective gene among the three candidate intervals. Sequence analysis of COL11A1 in two genetically independent fibrochondrogenesis cases demonstrated that each was a compound heterozygote for a loss-of-function mutation on one allele and a mutation predicting substitution for a conserved triple-helical glycine residue on the other. The parents who were carriers of missense mutations had myopia. Early-onset hearing loss was noted in both parents who carried a loss-of-function allele, suggesting COL11A1 as a locus for mild, dominantly inherited hearing loss. These findings identify COL11A1 as a locus for fibrochondrogenesis and indicate that there might be phenotypic manifestations among carriers.     

The human alpha 2(XI) collagen gene (COL11A2) maps to the centromeric border of the major histocompatibility complex on chromosome 6

Type XI collagen, a minor structural component of cartilage fibrils, is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI). Using a cloned fragment of the human alpha 2(XI) collagen gene (COL11A2) as a molecular probe for in situ hybridization and somatic cell hybrid mapping, we have localized the gene to the short arm of chromosome 6, region 21.3. By exploiting the rich source of probes provided by the major histocompatibility complex (MHC) genes, which also map to this chromosomal band, we have constructed macrorestriction maps of the region by pulsed-field gel electrophoresis and have localized the alpha 2(XI) collagen gene to the centromeric extreme of the MHC. Finally, we have demonstrated, by the isolation of overlapping cosmid clones, that the gene is 45 kb centromeric to the HLA-DPB2 locus and oriented with the 3' end toward the MHC. The COL11A2 locus thus demarcates the proximal boundary of the MHC. This finding may have implications for the understanding of certain MHC-linked diseases.     

The human alpha 2(XI) collagen (COL11A2) chain. Molecular cloning of cDNA and genomic DNA reveals characteristics of a fibrillar collagen with differences in genomic organization

We have isolated three overlapping cDNA clones encoding the pro alpha 2(XI) collagen chain from a human chondrocyte cDNA library. Together, the cDNAs code for 257 uninterrupted Gly-X-Y triplets (almost 80% of the triple helical domain) and about 200 amino acid residues of the carboxyl telopeptide and carboxyl propeptide. The identification of the clones as pro alpha 2(XI) cDNAs was based on the complete identity between the amino acid sequences of three tryptic peptides derived from human alpha 2(XI) collagen and the cDNA-derived sequence. We have also sequenced six exons within a human genomic alpha 2(XI) cosmid clone. This sequence shows that although type XI collagen belongs to the fibril-forming class of collagens, there are substantial differences in exon sizes at the 3' end of the gene when comparing the alpha 2(XI) gene with those of human types I, II, and III collagens. Finally, pro alpha 2(XI) cDNA has been used as a probe to determine the location of the gene by in situ hybridization of chromosome spreads. The results demonstrate that the gene is located close to the region p212 on chromosome 6. Northern blot analysis shows that the gene is expressed in cartilage but not in adult liver, skin, and tendon.