HGSNAT has a TATA-less promoter with multiple starts of transcription.

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.

Impact of flanking chromosomal sequences on localization and silencing by the human non-coding RNA XIST.

BACKGROUND: X-chromosome inactivation is a striking example of epigenetic silencing in which expression of the long non-coding RNA XIST initiates the heterochromatinization and silencing of one of the pair of X chromosomes in mammalian females. To understand how the RNA can establish silencing across millions of basepairs of DNA we have modelled the process by inducing expression of XIST from nine different locations in human HT1080 cells. RESULTS: Localization of XIST, depletion of Cot-1 RNA, perinuclear localization, and ubiquitination of H2A occurs at all sites examined, while recruitment of H3K9me3 was not observed. Recruitment of the heterochromatic features SMCHD1, macroH2A, H3K27me3, and H4K20me1 occurs independently of each other in an integration site-dependent manner. Silencing of flanking reporter genes occurs at all sites, but the spread of silencing to flanking endogenous human genes is variable in extent of silencing as well as extent of spread, with silencing able to skip regions. The spread of H3K27me3 and loss of H3K27ac correlates with the pre-existing levels of the modifications, and overall the extent of silencing correlates with the ability to recruit additional heterochromatic features. CONCLUSIONS: The non-coding RNA XIST functions as a cis-acting silencer when expressed from nine different locations throughout the genome. A hierarchy among the features of heterochromatin reveals the importance of interaction with the local chromatin neighborhood for optimal spread of silencing, as well as the independent yet cooperative nature of the establishment of heterochromatin by the non-coding XIST RNA.

Identification of novel informative loci for DNA-based X-inactivation analysis.

The HUMARA assay, the most common method for evaluation of X-inactivation skewing in blood cells, has been reported to be usable in only about 80% of females, emphasizing the need for alternative methods for testing of HUMARA-uninformative individuals. We conducted an in silico search for potentially polymorphic tri-to-hexanucleotide repeats in the proximity of CpG islands located in 5' regions of X-chromosome genes to design five candidate assays (numbered I, II, III, IV, and V) combining methylation-specific restriction digest with PCR amplification in a manner similar to the HUMARA assay. The results obtained by these assays in 100 healthy females were compared to X-inactivation skewing measured by the AR-MSP method which is based on methylation-specific PCR amplification of the first exon of the AR gene. On the basis of statistical evidence, three of the novel assays (II, IV, and V), which were informative in 18%, 61%, and 55% of females in the cohort, respectively, may be used as alternatives or conjointly with the HUMARA assay to improve its reliability. The three new assays were combined with the HUMARA assay into a novel X-inactivation test leading to the increase of informative females in the cohort from 67% to 96%.

XIST-induced silencing of flanking genes is achieved by additive action of repeat a monomers in human somatic cells.

BACKGROUND: The establishment of facultative heterochromatin by X-chromosome inactivation requires the long non-coding RNA XIST/Xist. However, the molecular mechanism by which the RNA achieves chromosome-wide gene silencing remains unknown. Mouse Xist has been shown to have redundant domains for cis-localization, and requires a series of well-conserved tandem 'A' repeats for silencing. We previously described a human inducible XIST transgene that is capable of cis-localization and suppressing a downstream reporter gene in somatic cells, and have now leveraged these cells to dissect the sequences critical for XIST-dependent gene silencing in humans. RESULTS: We demonstrated that expression of the inducible full-length XIST cDNA was able to suppress expression of two nearby reporter genes as well as endogenous genes up to 3 MB from the integration site. An inducible construct containing the repeat A region of XIST alone could silence the flanking reporter genes but not the more distal endogenous genes. Reporter gene silencing could also be accomplished by a synthetic construct consisting of nine copies of a consensus repeat A sequence, consistent with previous studies in mice. Progressively shorter constructs showed a linear relationship between the repeat number and the silencing capacity of the RNA. Constructs containing only two repeat A units were still able to partially silence the reporter genes and could thus be used for site-directed mutagenesis to demonstrate that sequences within the two palindromic cores of the repeat are essential for silencing, and that it is likely the first palindrome sequence folds to form a hairpin, consistent with compensatory mutations observed in eutherian sequences. CONCLUSIONS: Silencing of adjacent reporter genes can be effected by as little as 94 bp of XIST, including two 'monomers' of the A repeat. This region includes a pair of essential palindromic sequences that are evolutionarily well-conserved and the first of these is likely to form an intra-repeat hairpin structure. Additional sequences are required for the spread of silencing to endogenous genes on the chromosome.

X-chromosome inactivation: molecular mechanisms from the human perspective.

X-chromosome inactivation is an epigenetic process whereby one X chromosome is silenced in mammalian female cells. Since it was first proposed by Lyon in 1961, mouse models have been valuable tools to uncover the molecular mechanisms underlying X inactivation. However, there are also inherent differences between mouse and human X inactivation, ranging from sequence content of the X inactivation center to the phenotypic outcomes of X-chromosome abnormalities. X-linked gene dosage in males, females, and individuals with X aneuploidies and X/autosome translocations has demonstrated that many human genes escape X inactivation, implicating cis-regulatory elements in the spread of silencing. We discuss the potential nature of these elements and also review the elements in the X inactivation center involved in the early events in X-chromosome inactivation.

Glucocerebrosidase gene has an alternative upstream promoter, which has features and expression characteristic of housekeeping genes.

Database searches have shown that a part of glucocerebrosidase (GBA) transcripts may originate at an alternative upstream promoter (P2) located 2.6 kb upstream of the known (P1) GBA promoter. The putative alternative transcripts contained one or two extra exons (exon -2 or exons -2, -1, respectively), but the first ATG codon and predicted amino-acid sequence are the same as in the transcript from P1. Luciferase assays confirmed promoter activity of both sites in HepG2 cells: the P1 construct exhibited the highest activity of luciferase (17.82±1.10 relative luciferase units), while the P2 construct reached 3.01±0.43 relative luciferase units. Serial 5' deletions of P2 led to changes in reporter activity, the most prominent decreases were observed in deletion constructs carrying bases -353 to -658, and -353 to -920 (numbered as in NM_001005750.1), respectively. This suggests that the P2 core promoter is contained within the region of -920bp to -1311bp. Three P2 transcription initiation sites were found by 5' RACE at positions 347, 380, and 413bp upstream of the +1 ATG. The expression stability of transcripts from P2, P1 was studied in 20 human tissues and was higher than that of GAPDH and ACTB, which are commonly used as reference housekeeping genes. The P2 contains an unmethylated CpG island, multiple Sp-1 consensus binding sites and, unlike P1, does not contain a TATA box, features all common to the majority of housekeeping gene promoters. We have examined DNA samples from a phenotypically diverse group of twenty Ashkenazi Jewish Gaucher patients homozygous for the common mild mutation N370S. Both P1 and P2, as well as exons -2 and -1, did not contain any sequence variations, with the exception of the known polymorphism rs10908459 found on one allele. The phenotypical differences in the patients were thus not explained by nucleotide variations in both promoters.

Disruption of OTC promoter-enhancer interaction in a patient with symptoms of ornithine carbamoyltransferase deficiency.

In a female patient with signs of ornithine carbamoyltransferase deficiency (OTCD), the only variation found was a heterozygous single nucleotide substitution c.-366A>G. Determination of transcription start sites of human OTC 95, 119 and 169 bp upstream of the initiation codon located the variation upstream of the 5'-untranslated region. We predicted the human promoter and enhancer elements from homology with rat and mouse, performed function analysis of both regulatory regions and assessed the impact of the promoter variation in functional studies using dual luciferase reporter assay. Our data indicate that: (i) Full transcriptional activity of human OTC promoter depends on an upstream enhancer, as do the rodent promoters. (ii) The promoter variation c.-366A>G does not affect the function of the promoter alone but it disrupts the interaction of the promoter with the enhancer. (iii) The promoter-enhancer interaction contributes to tissue specific expression of OTC in the liver. We conclude that mutations in the regulatory regions of OTC can lead to OTCD and should be included in genetic testing.

Getting to the center of X-chromosome inactivation: the role of transgenes.

X-chromosome inactivation is a fascinating epigenetic phenomenon that is initiated by expression of a noncoding (nc)RNA, XIST, and results in transcriptional silencing of 1 female X. The process requires a series of events that begins even before XIST expression, and culminates in an active and a silent X within the same nucleus. We will focus on the role that transgenic systems have served in the current understanding of the process of X-chromosome inactivation, both in the initial delineation of an active and inactive X, and in the function of the XIST RNA. X inactivation is strictly cis-limited; recent studies have revealed elements within the X-inactivation center, the region required for inactivation, that are critical for the initial regulation of Xist expression and chromosome pairing. It has been revealed that the X-inactivation center contains a remarkable compendium of cis-regulatory elements, ncRNAs, and trans-acting pairing regions. We review the functional componentry of the X-inactivation center and discuss experiments that helped to dissect the XIST/Xist RNA and its involvement in the establishment of facultative heterochromatin.

X chromosome inactivation: heterogeneity of heterochromatin.

The silent X chromosome in mammalian females is a classic example of facultative heterochromatin, the term highlighting the compacted and inactive nature of the chromosome. However, it is now clear that the heterochromatin of the inactive X is not homogeneous--as indeed, not all genes on the inactive X are silenced. We summarize known features and events of X inactivation in different mouse and human model systems, and highlight the heterogeneity of chromatin along the inactive X. Characterizing this heterogeneity is likely to provide insight into the cis-acting sequences involved in X chromosome inactivation.

A skewed view of X chromosome inactivation.

X chromosome inactivation involves a random choice to silence either X chromosome early in mammalian female development. Once silenced the inactive X is stably inherited through subsequent somatic cell divisions, and thus, females are generally mosaics, having a mixture of cells with one or the other parental X active. While in most females the number of cells with either X being active is roughly equal, skewing of X chromosome inactivation is observed in a percentage of women. In this issue of the JCI, Bolduc and colleagues address whether skewing of X chromosome inactivation in humans is influenced by an X-linked locus that can alter this initial random inactivation (see the related article beginning on page 333). Their data indicate that most of the skewing observed in humans results from secondary events rather than being due to an inherited tendency to inactivate a particular X chromosome.