ATRX encodes a novel member of the SNF2 family of proteins: mutations point to a common mechanism underlying the ATR-X syndrome
ATRX encodes a novel member of the SNF2 family of proteins: mutations point to a common mechanism underlying the ATR-X syndromeDavid J. Picketts, Douglas R. Higgs, Satvinder Bachoo, Derek J. Blake1, Oliver W. J. Quarrell2 and Richard J. Gibbons*
The Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK, 1Genetics Laboratory, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK and 2Centre for Human Genetics, 117, Manchester Road, Sheffield S10 5DN, UK
Received August 30, 1996;Revised and Accepted September 23, 1996
It was shown recently that mutations of the ATRX gene give rise to a severe, X-linked form of syndromal mental retardation associated with [alpha] thalassaemia (ATR-X syndrome). In this study, we have characterised the full-length cDNA and predicted structure of the ATRX protein. Comparative analysis shows that it is an entirely new member of the SNF2 subgroup of a superfamily of proteins with similar ATPase and helicase domains. ATRX probably acts as a regulator of gene expression. Definition of its genomic structure enabled us to identify four novel splicing defects by screening 52 affected individuals. Correlation between these and previously identified mutations with variations in the ATR-X phenotype provides insights into the pathophysiology of this disease and the normal role of the ATRX protein in vivo.
We have shown recently that mutations in a novel X-encoded protein (ATRX) give rise to a characteristic form of syndromal mental retardation (MR) associated with [alpha] thalassaemia (ATR-X syndrome) (1 ). As for many human genetic disorders, the disease gene was cloned without prior knowledge of the protein it encodes; hence the challenge to determine its normal role and elucidate the pathophysiology of its associated disease(s). Some information may be gained when such proteins show significant homology to other proteins or domains whose function is already known. In addition, naturally occurring mutations may be informative: their locations may indicate critical regions of the protein; their classes may infer the mechanisms by which qualitative or quantitative alterations of the protein cause disease; and their clinical effects may point to cellular or molecular targets of the disease gene.
Preliminary analysis of the ATRX protein demonstrated that it is a new member of a superfamily (SFII) of proven or putative helicases which share seven conserved, co-linear `helicase' motifs (2 ,3 ). On the basis of additional homology located between these motifs, ATRX was assigned to the SNF2 subgroup which includes a rapidly expanding group of proteins involved in a wide variety of cellular processes such as transcriptional regulation, recombination, replication and DNA repair (4 ,5 ). Further sequence comparisons, including the regions flanking the SNF2 helicase domain, have subdivided the family into structurally and functionally distinct groups. To some extent this has enabled the function of newly identified members of the group to be predicted (6 ). However, since the ATRX protein sequence was incomplete, it could not be assigned with confidence to any of the SNF2 subgroups and its functional role remains unknown.
To date, only 13 mutations of the ATRX gene have been described (1 ,7 -9 ) but, since the gene has only been partially characterised, the full repertoire of mutations and their phenotypic effects have not been established. At present, the best guide to the normal function of ATRX comes from an evaluation of the phenotypic effects of mutation. In contrast to other human genetic diseases resulting from mutations in SNF2-like genes, in the ATR-X syndrome there is no clinical evidence for UV sensitivity or the premature development of malignancy, implying that ATRX is not involved in DNA recombination and repair. However, the association of ATR-X syndrome with [alpha] thalassaemia suggests that the protein is involved in the regulation of gene expression (1 ).
To increase our understanding of the normal function and role of the ATRX protein, we have established the structure of its full-length cDNA and predicted protein to compare with other members of the SNF2 family. In addition, to enable the full range of ATRX mutations to be identified, we have determined the genomic structure and intron-exon boundaries of the ATRX gene. Ultimately this will allow us to correlate the full range of molecular defects underlying the ATR-X syndrome with the clinical and haematologic phenotype and infer the mechanism(s) by which ATRX mutations may perturb expression of target genes.
The ATRX gene encodes two closely migrating mRNA transcripts of ~10 kb but, at present, the corresponding cDNA contig (referred to as XH2) represents only 6.1 kb (Fig. 1 A). We recently have extended the sequence of the 3' end of the cDNA to the poly(A) tail (X83753). To complete the cDNA sequence, we identified clones from the uncharacterised 5' end of the mRNA transcript by screening a human fetal brain cDNA library with a PCR-amplified probe (72/73) corresponding to the extreme 5' end of the cDNA contig. This identified the clone 72/73-23 which extends the contig by 2 kb (Fig. 1 B).
Although translation of the ATRX protein previously was thought to initiate at nucleotide 2911 of the mRNA transcript (10 ), analysis of the full-length cDNA has shown that the open reading frame (ORF) includes an additional 742 amino acids; the first in-frame stop codon occurs at nucleotides 7810-7812. The alternatively spliced transcripts encode three different sized proteins. The inclusion of exons 6 and 7 in the longest transcript (F1) introduces in-frame stop codons and, in this case, the first methionine is encoded at 946 (M2) giving a predicted protein of 2288 amino acids (260 kDa). When exons 7, or 6 and 7, are spliced out, a methionine at nucleotide 563 (M1) represents the start of the ORF and this results in predicted proteins of 2375 amino acids (270 kDa) and 2337 amino acids (265 kDa) respectively.
As noted previously (10 ), the protein can be broadly divided into three regions (Fig. 1 A): an N-terminal hydrophilic segment (~1470 amino acids), a central region containing alternating hydrophilic and hydrophobic stretches (~770 amino acids) and a C-terminal domain (~135 amino acids). Almost 40% of the N-terminal region is comprised of three amino acids (serine, lysine and glutamic acid). In addition, this segment contains a nuclear localisation signal (NLS, amino acids 1025-1050) and a stretch of 21 glutamic acid residues (amino acids 1326-1347) which may be involved in protein-protein interactions. The central portion contains the helicase motifs and, as previously noted, this region is most similar to RAD54 (6 ,10 ). In the extreme C-terminal region, we identified a small motif (P, amino acids 2268-2284) which shows 40-50% identity to other SNF2-like proteins including brahma, BRG1, SNF2 and STH1. In addition, we noted that the entire carboxy-terminal region is rich (~20%) in glutamine residues, including a stretch of 9/11 glutamines (Q, codons 2298-2308), suggesting that this could also be a domain involved in protein-protein interactions.
Thus, although ATRX clearly belongs to the SNF2 subgroup, the extended sequence analysed here shows that it is an entirely novel member of this family. Previous suggestions that it is more closely related to RAD54 than other family members (6 ) are not substantiated by our analysis of newly acquired sequence from the flanking regions (see Discussion).
To obtain the genomic structure of the ATRX gene, we employed a combination of exon-exon amplification, vectorette PCR and genomic subcloning from a cosmid contig. Initially we used primers, originally designed for mutational analysis of exons by single strand conformation polymorphism (SSCP) (1 ), to compare the sizes of products obtained by PCR using cDNA or genomic DNA as a template. When genomic and cDNA products appeared identical in size they were assumed to represent individual exons. Amplified genomic products which differed in length when compared with their cDNA counterparts were sequenced to characterise the splice sites. When a pair of primers did not amplify genomic DNA, PCR fragments obtained using each exon primer with the vectorette primer were cloned and sequenced to identify intron-exon boundaries.
As a third approach, an X-only cosmid library [number 104 (L4/FSC X)] (11 ) was screened with a mixture of cDNA clones to generate a contig spanning the ATRX gene. Gaps in the contig were filled by screening a second cosmid library made from YAC 4551 using cDNA probes not contained in the original contig. This resource was used to subclone and sequence genomic fragments that contained intron-exon boundaries not identified by other methods. In this way, we have characterised 36 exons distributed throughout ~300 kb of genomic DNA encoding the entire cDNA sequence (Fig. 2 ). Exons range in size from 54 to ~3000 bp, and all splice sites conform to the consensus splice sequences (data are included with the submitted sequence, Accession nos U72900-U72935).
One vectorette clone (V117), generated from primer XNP117 located in exon 1, orientated towards the 5' end of the gene contained ~1.5 kb of 5'-flanking sequence of which the first ~400 nucleotides were sequenced and examined for promoter elements and CpG content. A plot of the proportion of CpG dinucleotides identified two CpG-rich regions (Fig. 3 ). The first encompasses the transcriptional start site (+1) and is associated with a nearby unmethylated SacII site; the second CpG island is located 176 nucleotides downstream of this. Since the second CpG island could have been associated with another transcriptional start site for ATRX, we used the oligonucleotide XNP139 for primer extension analysis but we only detected transcripts originating from the proximal CpG island (data not shown). The possibility that the distal CpG island is associated with a transcript on the other DNA strand has not been addressed. Sequence analysis upstream of the transcriptional start site (+1 to -377), containing the putative promoter, did not reveal a TATA box, but there were multiple CCAAT boxes and binding sites for the CTF family of transcription factors. No binding sites were identified for known erythroid-enriched transcription factors (12 ).
Establishing the complete intron-exon structure of the ATRX gene allowed us to undertake a more extensive mutational analysis than previously, including potential splicing and promoter defects. To screen for splicing defects we analysed ~8 kb (1-7986) of the cDNA from 52 affected individuals. Products of altered size were identified and sublocalised in four affected individuals.
In two ATR-X patients from pedigree 20 [case 11 (13 ), and his similarly affected cousin case 12], using primers XNP52 and XNP53 (1 ), which amplify exons 15 and 16, we detected the expected fragment (227 bp) and two additional, abnormal fragments of increased size (Fig. 4 A). Sequence analysis demonstrated a G4650A transition at the well conserved G adjacent to the invariant GT of the donor splice site [reviewed in (14 )]. This mutation decreases the efficiency of splicing at the normal site resulting in the use of other cryptic donor sites and the inclusion of the adjacent intronic sequence (intron 15) into the transcript. One such spliceform includes 53 nucleotides of intronic sequence causing the insertion of 17 amino acids and creating a stop codon where it is spliced to exon 16 (Fig. 4 A). These patients also produce some normally spliced product which incorporates the A -> G nucleotide change; however, the altered base (AAG -> AAA: Lys -> Lys) does not create an amino acid substitution and therefore these patients produce a reduced amount (~30%) of entirely normal mRNA. Interestingly, previously we have identified another change in the ATRX protein from these individuals which results in the substitution of serine for asparagine. We suggested that this may not represent the disease-causing mutation in this family due to the conservative nature of the change and its location within a non-conserved region of the protein (1 ). It now appears that N1743S represents a rare amino acid polymorphism.
In a further case [pedigree 15, case 6 (13 )] using primers Seq19 and XNP18 which amplify exons 28 and 29, we detected a normal band (173 bp) representing ~10% of the total product and at least two larger abnormal fragments (Fig. 4 B). Sequence analysis around the donor and acceptor splice sites of intron 28 were normal. Characterisation of three RT-PCR fragments revealed a normally spliced product and two abnormal spliceforms containing 124 bp of an Alu repeat at the 5' end. In addition, the larger spliceform included 200 bp of unique sequence derived from intron 28. Due to the nature of this abnormality lying within a large (>8 kb) intron, we have not yet identified the causative mutation.
We also identified a novel splicing defect in two apparently unrelated families (pedigrees 12 and N17). Amplification of cDNA from these patients [case 3 (13 ); N17 see Materials and Methods] with primers XNP100 and XNP103 resulted in a single fragment of decreased size (214 bp) compared with normal individuals (277 bp), localising the mutation between exons 8 and 9 (Fig. 4 C). Sequence analysis of this product identified an A869G transition in both patients: this creates a cryptic donor splice site (at nucleotide 865, exon 8) which is used exclusively and results in the loss of 63 nucleotides (865-927) of exon 8, thereby maintaining the translational reading frame.
To exclude the possibility that these two families might be related, we analysed several polymorphic markers. The two affected individuals have entirely different haplotypes around ATRX and in pedigree 12 the mutation appears to have arisen as a new event of grandpaternal origin (data not shown). Since this deletion of 63 bases leaves the remainder of the reading frame intact and in-phase it seemed possible that this might represent a rare polymorphism. However, we did not find this mutation in 13 normal X chromosomes or in any of the other 50 affected individuals we have studied. Although we cannot exclude this as a rare polymorphism, it seems probable that it is a disease-causing mutation which could have detrimental effects on protein folding due to the removal of 21 amino acids (61-81) including one cysteine residue.
Having established the complete structure of the ATRX gene, its cDNA and predicted protein, it is now possible to search for similarities to other well characterised members of the SNF2 family that might point to the normal role of this protein in vivo. Previous analysis of a partial cDNA identified two small segments (B and C in Fig. 1 A) in the central domain of ATRX which share 50-55% identity with the yeast RAD54 gene (10 ). ATRX was thus provisionally assigned to the RAD54 group of SNF2-like proteins, implying a role in DNA recombination or repair. The complete structure of the ATRX gene, established here, and the recent characterisation of the mouse and human homologues of RAD54 (15 ) have allowed us to re-evaluate the relationship between ATRX and the RAD54 group.
Alignment of the predicted ATRX protein with human, mouse and yeast RAD54 shows no homology beyond that described previously around elements B and C which are not exclusive to the RAD54 group (data not shown). Furthermore, we have found no significant similarity between ATRX and RAD54 in the N-terminal and C-terminal flanking regions which characterise the SNF2 subgroups (6 ). Therefore, although similarity between ATRX and RAD54 in the SNF2 central domain may point to a common ancestry, their divergent flanking regions imply that they are not closely related and probably subsume different functional roles in vivo.
We could not detect extensive similarity between ATRX and any of the SNF2 subgroups, implying that it is an entirely novel member of this family. However, several new findings support previous observations suggesting that ATRX is involved in the regulation of gene expression. The N-terminal region contains a nuclear localisation signal (NLS in Fig. 1 A), and provisional data using a polyclonal anti-ATRX antibody have confirmed that it is a nuclear protein (unpublished data). The C-terminal region is highly glutamine-rich, including a run of 9/11 glutamines, as previously observed in some well characterised transcription factors (16 -18 ). In addition, a 15 amino acid segment (P) in the C-terminal region is conserved (35-50% similarity) in several SNF2-like proteins that are involved in the regulation of gene expression (brahma, BRG1, SNF2 and STH1) but not in RAD54 which is involved in recombination and repair. It is interesting that ATRX mutations down-regulate [alpha]-globin expression, whereas the closely related [beta]-globin genes appear unaffected. One possible reason is that these genes are contained within different chromosomal environments and because of this are regulated differently via their interactions with chromatin (19 ). This is consistent with the emerging concept that all SNF2-like proteins may exert their effects by influencing protein-DNA interactions including well described effects on remodelling chromatin using the energy of ATP hydrolysis (20 -22 ).
Now that the ATRX gene has been completely characterised, it will be possible to establish the full range of disease-causing mutations. To date, 16 mutations have been documented (Table 1 ), including nine missense mutations; two nonsense mutations; five splicing defects, of which four cause frameshifts and protein truncation, and a small (2.0 kb) deletion. These mechanistically diverse mutations are quite evenly distributed throughout the gene and apparently not concentrated in structurally conserved regions. Thus, the common theme is that they all potentially reduce the amount of normal ATRX protein, leading to a loss of function. While this cannot yet be proven for the missense mutations, the principle is illustrated most clearly by the small deletion, in which ATRX mRNA is reduced to <1% of normal and may represent a null mutation (1 ). Similarly, in some of the splicing mutants, including some presented here, the levels of normal ATRX mRNA range from <1 to 30% (9 ). The fact that key features of the ATR-X syndrome are found in the presence of substantial amounts of normal ATRX mRNA suggests that the target pathways are sensitive to the dosage of this protein. One reason for such sensitivity could be that, like the related SNF2 protein (23 ,24 ), ATRX may be required in carefully controlled stoichiometric amounts for the formation of multiprotein complexes.
Patient N17, who has not been described previously, has severe mental retardation, characteristic facial dysmorphism, hiatus hernia and mild urogenital abnormalities. In contrast to other cases we have reported, no cells with HbH inclusions have been observed after incubation of fresh venous blood with 1% brilliant cresyl blue solution on several occasions. The clinical details of pedigrees 12, 15 and 20 and all other affected individuals discussed in the text have been reported previously (13 ).
To extend the published cDNA sequence, primers XNP72 and XNP73 were used to amplify a fragment which was used to screen a human fetal brain cDNA library prepared in the vector pcDNA II (28 ) by standard conditions (29 ). Primers XNP84 and XNP107 were used for 5' RACE analysis as described by Frohman et al. (30 ). Clone 72/73-23, and fragments F1, F2 and F3 cloned into pCR-Script (see text) were sequenced using a modified T7 polymerase (USB Biochemicals) and sequential truncations (31 ). Analysis of the alternate spliceforms was performed by RT-PCR as described by Brown et al. (32 ) using RNA isolated from Epstein-Barr virus-transformed lymphoblasts and the primers XNP125 and XNP103.
Primer extension with the primers XNP122 and XNP139 was performed on 10 [mu]g of total RNA isolated from the cell lines HT-29 (33 ), K562 (34 ), HeLa (35 ), HEL (36 ) and G401 (human renal Wilm's tumour; ECACC) as described by Hughes et al. (37 ), except that primers were annealed at 65oC. Primer XNP122 was also used to generate a sequencing ladder using the genomic clone pEco6kb, a 6 kb EcoRI genomic fragment including exon 1 and the 5'-flanking region of the ATRX gene.
PCR primers initially designed for SSCP analysis were used to amplify a normal genomic template as described by Gibbons et al. (1 ), with the addition of Taq PCR extender (Stratagene) following the manufacturer's recommendations. Amplified fragments were gel purified (Qiagen), subcloned into pCR-Script (Stratagene) and sequenced using M13 forward and reverse primers and a modified T7 polymerase (Sequenase).
Vectorette PCR was performed using DNA from YAC 4551 (38 ) after digestion with either RsaI, PvuII or HaeIII; or BglII, BclI and BamHI. Following the manufacturer's instructions, blunt vectorette linkers (Genosys) were ligated onto the digested genomic DNA and PCR amplified using a phosphorylated vectorette primer and a gene-specific primer. If the products were in the size range of 100-400 bp, prior to sequencing single-stranded template was produced by digestion with lambda exonuclease which degrades the strand containing the phosphorylated primer (39 ).
We would like to thank Drs H.M. Kingston, B.C.C. Davison, and A.O.M. Wilkie for referring patients for whom the clinical features have been previously described; Professor Sir D. Weatherall for his continued support and help throughout; Dr A. Monaco for YAC 4551; J. Sloane-Stanley for tissue culture work; and L. Rose for excellent secretarial skills. The families are gratefully acknowledged for their cooperation. This work was supported by grants from the MRC and the Wellcome Trust (Grant ref. no. 042152).
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*To whom correspondence should be addressed
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