The Dominant Role of Sp1 in Regulating the Cystathionine β-Synthase –1a and –1b Promoters Facilitates Potential Tissue-specific Regulation by Kruppel-like Factors

Abstract

Cystathionine ␤-synthase (CBS) catalyzes the condensation of serine with homocysteine to form cystathionine and occupies a crucial regulatory position between the methionine cycle and transsulfuration. The human cystathionine ␤-synthase gene promoters ؊1a and ؊1b are expressed in a limited number of tissues and are coordinately regulated with proliferation through a redox-sensitive mechanism. Site-directed mutagenesis, DNase I footprinting and deletion analysis of 5276 bp of 5 proximal ؊1b flanking sequence revealed that this region does not confer tissue-specific expression and that 210 bp of proximal sequence is sufficient for maximal promoter activity. As little as 32 bp of the ؊1b proximal promoter region is capable of driving transcription in HepG2 cells, and this activity is entirely dependent upon the presence of a single overlapping Sp1/Egr1 binding site. Co-transfection studies in Drosophila SL2 cells indicated that both promoters are transactivated by Sp1 and Sp3 but only the ؊1b promoter is subject to a site-specific synergistic regulatory interaction between Sp1 and Sp3. Sp1-deficient fibroblasts expressing both Sp3 and NF-Y were negative for CBS activity. Transfection of these cells with a mammalian Sp1 expression construct induced high levels of CBS activity indicating that Sp1 has a critical and indispensable role in the regulation of cystathionine ␤-synthase. Sp1 binding to both CBS promoters is sensitive to proliferation status and is negatively regulated by Kruppel-like factors in co-transfection experiments suggesting a possible mechanism for the tissue specific regulation of cystathionine ␤-synthase.

Ischemia/Reperfusion Reduces Transcription Factor Sp1-mediated Cystathionine β-Synthase Expression in the Kidney

“…As shown in the present study, the critical and indispensable role of Sp1 in the regulation of rat CBS expression in the kidney is in agreement with its role in regulating human CBS expression in other tissues or cells reported in previous studies (20,22,43). Post-translational modifications, such as phosphorylation and glycosylation, have been indicated as important mechanisms in the regulation of Sp1 binding to the promoter regions of targeted genes (44,45).…”

Cystathionine Protects against Endoplasmic Reticulum Stress-induced Lipid Accumulation, Tissue Injury, and Apoptotic Cell Death

“…Media and Mammalian Cell Culture-CBS and CGL positive HepG2 hepatocellular carcinoma cells and human embryonic kidney 293AD cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured as described previously (6). CBS and CGL negative Chinese hamster fibroblast a23 cells were obtained from the University of Colorado Intellectual Developmental Disabilities Research Center central collection and were cultured as described previously (6).…”

“…CBS and CGL negative Chinese hamster fibroblast a23 cells were obtained from the University of Colorado Intellectual Developmental Disabilities Research Center central collection and were cultured as described previously (6).…”

“…Preliminary experiments confirmed that both of these cell lines express both CBS and CGL (data not shown). To investigate the possible protective effects of cystathionine in the absence of transsulfuration, we repeated these experiments with the CBS and CGL negative Chinese hamster fibroblast line a23 (6).…”

The Therapeutic Potential of Cystathionine β-Synthetase/Hydrogen Sulfide Inhibition in Cancer

“…2A), and the kidney (29,127). The cellular levels of CBS are regulated by transcriptional (29,32,73,132), epigenetic (96), and posttranslational (41,107,123) mechanisms. CBS expression is also under the control of hormonal regulation.…”