All-trans retinoic acid induces chromatin remodeling at the promoter of the mouse liver, bone, and kidney alkaline phosphatase gene in C3H10T 1/2 cells.

The alkaline phosphatase (ALP) gene is an important marker of osteoblast differentiation and bone formation. Although the molecular mechanisms of increased ALP expression in response to all-trans retinoic acid (ATRA) have been reported, the role of ATRA in chromatin structure changes remains unknown. Our results show that the expression of mouse liver, bone, and kidney ALP (mL/B/K-ALP) induced by ATRA in C3H10T 1/2 cells was related to the retinoic acid nuclear receptors, RARα and RARß, which are not involved in the MAPK pathway. DNase I hypersensitivity analysis revealed an inducible hypersensitive site in the mL/B/K-ALP promoter at ~520 bp upstream of the transcription start site. Chromatin immunoprecipitation experiments showed a cascade of transcription cofactor recruitment events during ATRA-induced upregulation of mL/B/K-ALP. Together, our results provide a link between ATRA-induced mL/B/K-ALP gene transcription and chromatin remodeling.

Remodeling of chromatin structure within the promoter is important for bmp-2-induced fgfr3 expression.

Fibroblast growth factor receptor 3 (FGFR3) plays an important role in cartilage development. Although upregulation of FGFR3 expression in response to bone morphogenetic protein-2 (BMP-2) has been reported, the molecular mechanisms remain unknown. In this study, we used in vivo approaches to characterize BMP-2-induced alterations in the chromatin organization of the FGFR3 core promoter. Chromatin immunoprecipitation analysis demonstrated that the binding of Brg1, a component of the SWI/SNF remodeling complex, may selectively remodel a chromatin region (encompassing nucleotide -90 to +35), uncovering the transcription start site and three Sp1-binding sites, as revealed by nuclease digestion hypersensitivity assays. We then showed an increase in the association of Sp1 with the proximal promoter, followed by the recruitment of p300, resulting in a change of the histone 'code', such as in phosphorylation and methylation. Collectively, our study results suggest a model for BMP-2-induced FGFR3 expression in which the core promoter architecture is specifically regulated.

SP1 acts as a key factor, contributes to upregulation of ADAM23 expression under serum deprivation.

ADAM23 modulates many cellular functions, alteration of expression causes a number of tumor types; however, the mechanisms controlling ADAM23 expression remain unknown. Here we have identified a SP1 binding site (-202/-190) that binds SP1 at the proximal promoter of human ADAM23 gene; furthermore, serum deprivation enhances open chromatin accessibility and help expose the SP1 binding site; finally, SP1 binding recruits RNA polymerase II, which in turn results in upregulation of endogenous ADAM23 expression. Therefore, the present study delineates the fundamental elements of a core promoter structure that will be helpful for future studies of the regulation of ADAM23 gene.

Nuclear factor Y is required for basal activation and chromatin accessibility of fibroblast growth factor receptor 2 promoter in osteoblast-like cells.

Fibroblast growth factor receptor 2 (FGFR2) plays an important regulatory role in bone development. However, the regulatory mechanisms controlling FGFR2 expression remain poorly understood. Here we have identified a role for the nuclear factor Y (NF-Y) in constitutive activation of FGFR2. A unique DNase I hypersensitive site was detected in the region encompassing nucleotides -270 to +230 after scanning a large range covering 33.3 kilobases around the transcription start site of FGFR2. Using a PCR-based chromatin accessibility assay, an open chromatin conformation was detected around the proximal 5' fragment of FGFR2 gene. Deletion constructs of the 5'-flanking region of FGFR2 were fused to a luciferase reporter gene. After transient transfection in C3H10T1/2, ME3T3-E1, and C2C12 as well as primary osteoblasts, a minimal region -86/+139 that is highly homologous to the human sequence and bears a CCAAT box was identified as the core promoter. Electrophoretic mobility shift assay supershift and chromatin immunoprecipitation demonstrated that the CCAAT box was the binding site for NF-Y. Deletion of NF-Y consensus sequence resulted in the total loss of NF-Y promoter activity. Overexpression of NF-Y protein and transfection of NF-Y small interfering RNAs in the cells substantially changed the promoter activity. Moreover, NF-Y small interfering RNAs greatly inhibited the endogenous FGFR2 transcription level and the chromatin accessibility and H3 acetylation across the promoter. Taken together, our results demonstrate that interaction of NF-Y at the CCAAT box is pivotal to FGFR2 gene transcription partly through the construction of a local open chromatin configuration across the promoter.

[Expression of BMP-2/7 heterodimers with potent ability to stimulate osteogenic differentiation in CHO cells].

Coding sequences of BMP-2 and BMP-7 were amplified using PCR and ligated with a DNA sequence encoding a flexible peptide (Gly4Ser)5. The fusion gene was inserted into plasmid pIRESneo3. The expression level of BMP2/7 heterodimers in the transfected CHO-K1 cells was 230.75+/-13.34 ng/mL. Culture medium of stably tansfected clone pool was collected as conditional medium to treat osteoblast MC3T3 cells. Staining of Alkalin phosphatase and Alizarin red demonstrated that the conditional medium significantly promoted osteogenic differentiation to a higher extent than BMP-2 homodimers expressed in either CHO-K1 cells or E. coli. Transcriptional levels of Osteogenic phenotype-related molecular markers such as OC, ALP, Runx2 and Osx were increased (P<0.05), and BMP/Smad signal activities were significantly enhanced by BMP-2/7 heterodimers, comparing with BMP-2 homodimers (P<0.05). The results demonstrate that BMP-2/7 heterodimers expressed in CHO-K1 cells have potent ability to stimulate osteogenic differentiation.