title: HGSNAT has a TATA-less promoter with multiple starts of transcription.
abstract: Acetyl-CoA:α-glucosaminide N-acetyltransferase (N-acetyltransferase) is a lysosomal membrane enzyme that catalyzes a key step in the lysosomal degradation of heparan sulfate. Its deficiency causes Sanfilippo syndrome type IIIC (Mucopolysaccharidosis type IIIC, MPS IIIC). Here we characterize the promoter region of HGSNAT, the gene encoding N-acetyltransferase, which is located in the pericentromeric region of chromosome 8. We show that HGSNAT transcription is driven by a TATA-less promoter whose key elements are contained within the 1054bp region upstream of exon 1. About 400 bases of the region's 3'-prime end overlap with an unmethylated CpG island. Reduced reporter activities from promoter serial deletion constructs suggested strong regulatory elements at positions -101 to -20bp and -1073 to -716bp of the downstream initiation codon (DS-ATG). Targeted mutagenesis of the first Specificity protein 1-A (Sp1-A) of the six in silico-predicted Sp1 sites in the region flanking the major transcription start sites (TSSs, +50/-101) led to a 55% decrease of reporter activity, while inactivation of each of Sp1-B and Sp1-C resulted in its almost two-fold increase. The binding of Sp1 to the region was confirmed by chromatin immunoprecipitation (ChIP). Overall, this confirms that Sp1 is important for regulation of the HGSNAT promoter. Promoter fragments in antisense orientation (constructs pGL4 -20/-1305 and pGL4 +50/-1305) led to reporter activities of about 50% of the pGL4 -1305/-20 activity, implying divergent initiation of transcription at the promoter. We identified two main TSSs at positions +1 and -15 from DS-ATG using Rapid amplification of cDNA ends (5'RACE). Transcripts initiating at the TSSs thus contain only DS-ATG. Five patients from our MPS IIIC cohort (n=23) carried the rs4523300 promoter variant and one the rs149596192 promoter variant. Both variants lowered the expression of the reporter down to 68% and 59%, respectively. However, white blood cell (WBC) N-acetyltransferase activities in individuals carrying the variants did not significantly differ from homozygotes for the wild-type alleles, suggesting only a partial impact of transcriptional regulation on N-acetyltransferase activities in vivo.
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, Prague 120 08, Czech Republic. Electronic address: evas@email.cz.
authors.forename: Eva
authors.initials: E
authors.lastname: Richtrova
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, Prague 120 08, Czech Republic. Electronic address: lmraz@lf1.cuni.cz.
authors.forename: Lenka S
authors.initials: LS
authors.lastname: Mrazova
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, Prague 120 08, Czech Republic. Electronic address: dita.musalkova@gmail.com.
authors.forename: Dita
authors.initials: D
authors.lastname: Musalkova
authors.affiliation: Laboratory of Experimental Hepatology, Institute of Clinical and Experimental Medicine, Vídeňská 1958/9, Prague 140 21, Czech Republic. Electronic address: ondrej.luksan@seqme.eu.
authors.forename: Ondrej
authors.initials: O
authors.lastname: Luksan
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, Prague 120 08, Czech Republic. Electronic address: larisa.stolnaja@lf1.cuni.cz.
authors.forename: Larisa
authors.initials: L
authors.lastname: Stolnaya
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, Prague 120 08, Czech Republic. Electronic address: minksj@interchange.ubc.ca.
authors.forename: Jakub
authors.initials: J
authors.lastname: Minks
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, Prague 120 08, Czech Republic. Electronic address: jan.lukas@lf1.cuni.cz.
authors.forename: Jan
authors.initials: J
authors.lastname: Lukas
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, Prague 120 08, Czech Republic. Electronic address: lenka.dvorakova@lf.cuni.cz.
authors.forename: Lenka
authors.initials: L
authors.lastname: Dvorakova
authors.affiliation: Laboratory of Experimental Hepatology, Institute of Clinical and Experimental Medicine, Vídeňská 1958/9, Prague 140 21, Czech Republic. Electronic address: miji@ikem.cz.
authors.forename: Milan
authors.initials: M
authors.lastname: Jirsa
authors.affiliation: Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 2, Prague 120 08, Czech Republic. Electronic address: mhreb@lf1.cuni.cz.
authors.forename: Martin
authors.initials: M
authors.lastname: Hrebicek
mesh_terms: D000123:Acetyltransferases / Q000235:genetics*; D016022:Case-Control Studies; D056945:Hep G2 Cells; D006801:Humans; D009084:Mucopolysaccharidosis III / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D016329:Sp1 Transcription Factor / Q000378:metabolism; D016385:TATA Box; D024363:Transcription Initiation Site
chemical_list: D016329:Sp1 Transcription Factor; D000123:Acetyltransferases; C514442:HGSNAT protein, human
keywords: HGSNAT; Housekeeping gene; MPS IIIC; N-acetyltransferase; Promoter; Sp1