TY - JOUR

T1 - Functional characterization of the human biglycan 5'-flanking DNA and binding of the transcription factor c-Krox

AU - Heegaard, Anne-Marie

AU - Gehron Robey, P

AU - Vogel, W

AU - Just, W

AU - Widom, R L

AU - Schøller, J

AU - Fisher, L W

AU - Young, M F

PY - 1997

Y1 - 1997

N2 - The transcriptional regulation of human biglycan expression under normal and pathological conditions was studied. The 5'-flanking regions of the human and mouse genes were isolated and analyzed; the two promoter regions share 81% identity. Both promoters are without a TATA and CAT box and contain multiple Sp1 sites. Human dermal fibroblasts were transiently transfected with progressive deletional human biglycan 5'-flanking DNA-CAT constructs, and a significant variation in activity among the individual constructs was found. A small deletion in several cases caused a more than 2-fold increase or decrease in promoter activity, thereby mapping the target sites for repressors or activators. Human biglycan expression is reduced in females with Ullrich-Turner syndrome (45,X) and increased in individuals with supernumerary sex chromosomes, and it has been speculated that biglycan plays a role in the short stature phenotype of Turner syndrome. Analysis of the transcriptional regulation of biglycan in individuals with sex chromosome anomalies showed that a -262 to -218 region of the biglycan promoter was differentially regulated. This region was extensively analyzed by DNAse footprinting and electrophoretic mobility shift assays, and a putative binding site for the transcription factor c-Krox was discovered. The binding of c-Krox to a site located at approximately -248 to -230 in the human biglycan promoter was confirmed by using extracts from COS cells expressing recombinant human c-Krox. The expression of c-Krox in bone was then examined by reverse-transcribed polymerase chain reaction and Northern blotting analysis; an approximately 3.4 kb transcript was detected in primary osteoblastic cells, in MG-63 cells, and in human bone marrow stromal cells. This is the first detection of c-Krox in bone cells, and it suggests that c-Krox, like another member of the Krox family, Krox-20, might play a regulatory role in bone.

AB - The transcriptional regulation of human biglycan expression under normal and pathological conditions was studied. The 5'-flanking regions of the human and mouse genes were isolated and analyzed; the two promoter regions share 81% identity. Both promoters are without a TATA and CAT box and contain multiple Sp1 sites. Human dermal fibroblasts were transiently transfected with progressive deletional human biglycan 5'-flanking DNA-CAT constructs, and a significant variation in activity among the individual constructs was found. A small deletion in several cases caused a more than 2-fold increase or decrease in promoter activity, thereby mapping the target sites for repressors or activators. Human biglycan expression is reduced in females with Ullrich-Turner syndrome (45,X) and increased in individuals with supernumerary sex chromosomes, and it has been speculated that biglycan plays a role in the short stature phenotype of Turner syndrome. Analysis of the transcriptional regulation of biglycan in individuals with sex chromosome anomalies showed that a -262 to -218 region of the biglycan promoter was differentially regulated. This region was extensively analyzed by DNAse footprinting and electrophoretic mobility shift assays, and a putative binding site for the transcription factor c-Krox was discovered. The binding of c-Krox to a site located at approximately -248 to -230 in the human biglycan promoter was confirmed by using extracts from COS cells expressing recombinant human c-Krox. The expression of c-Krox in bone was then examined by reverse-transcribed polymerase chain reaction and Northern blotting analysis; an approximately 3.4 kb transcript was detected in primary osteoblastic cells, in MG-63 cells, and in human bone marrow stromal cells. This is the first detection of c-Krox in bone cells, and it suggests that c-Krox, like another member of the Krox family, Krox-20, might play a regulatory role in bone.

KW - Animals

KW - Base Sequence

KW - Biglycan

KW - Cell Line

KW - Cloning, Molecular

KW - DNA

KW - DNA Footprinting

KW - DNA-Binding Proteins

KW - Electrophoresis, Polyacrylamide Gel

KW - Extracellular Matrix Proteins

KW - Fibroblasts

KW - Humans

KW - Mice

KW - Molecular Sequence Data

KW - Osteoblasts

KW - Protein Binding

KW - Proteoglycans

KW - RNA, Messenger

KW - Sequence Homology, Nucleic Acid

KW - Sex Chromosome Aberrations

KW - Transcription Factors

KW - Transfection

KW - Zinc Fingers

U2 - 10.1359/jbmr.1997.12.12.2050

DO - 10.1359/jbmr.1997.12.12.2050

M3 - Journal article

C2 - 9421237

VL - 12

SP - 2050

EP - 2060

JO - Journal of Bone and Mineral Research

JF - Journal of Bone and Mineral Research

SN - 0884-0431

IS - 12

ER -