Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene

Abstract

Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines, and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites, and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2, and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon, and kidney cell lines; activities consistently followed the pattern P1> >P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene.

MicroRNAs Are Absorbed in Biologically Meaningful Amounts from Nutritionally Relevant Doses of Cow Milk and Affect Gene Expression in Peripheral Blood Mononuclear Cells, HEK-293 Kidney Cell Cultures, and Mouse Livers

“…For studies in cell cultures, human embryonic kidney (HEK)-293 cells (American Type Culture Collection) were cultured in MEM containing 10% exosome-depleted FBS, 0.1% sodium pyruvate, 100,000 U/L penicillin, and 100 mg/L streptomycin for 10 d. Bovine serum was depleted of exosomes by ultracentrifugation at 130,000 3 g for 4 h, leading to the removal of 97% and 81% of miR-29b and miR-200c, respectively. Cells were transfected with miRNA reporter genes as described previously (26). Forty-eight hours after transfection, exosome-depleted media were replaced with exosome-sufficient media at final concentrations of 600 fmol/L miR-29b or 1000 fmol/L miR-200c.…”

Resveratrol compounds inhibit human holocarboxylase synthetase and cause a lean phenotype in Drosophila melanogaster

“…We also do not know whether effects of resveratrol compounds are stereospecific; trans isomers or piceid and resveratrol are more abundant than cis isomers in grape materials [25]. Third, he expression of HLCS is regulated by three promoters [42,43], but the responsiveness of regulatory elements in these promoters to HLCS inhibitors is unknown. These uncertainties are currently being addressed in our laboratories.…”

Organic Acidemias: Clinical Presentation in Neonates

“…Biotin then activates its own expression by binding its 3 promoters, named P1, P2, and P3. 144,145 • Alters gene expression by promoting biotin binding to lysine (K) residues in histones H2A, H3, and H4. 146,147 H3 biotinylation on its K9 and K18 has been studied in detail.…”