Review

The chromatin remodeller ATRX:

a repeat offender in human disease

David Clynes, Douglas R. Higgs, and Richard J. Gibbons

MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital,

Oxford OX3 9DS, UK

The regulation of chromatin structure is of paramount collaboration with its interaction partner death-associated

importance for a variety of fundamental nuclear process- 6 (DAXX), functions as a histone chaperone complex

es, including expression, DNA repair, replication, for the deposition of the histone variant H3.3 into peri-

and recombination. The ATP-dependent chromatin- centric, telomeric, and ribosomal repeat sequences [7–10].

remodelling factor ATRX (a thalassaemia/mental retar- ATR-X syndrome is characterised by a variety of clini-

dation X-linked) has emerged as a key player in each of cal features that include mental retardation, facial, skel-

these processes. Exciting recent developments suggest etal, and urogenital abnormalities, as well as mild a-

that ATRX plays a variety of key roles at tandem repeat thalassaemia (a blood disorder characterised by an im-

sequences within the genome, including the deposition balance of globin chain synthesis and anaemia) [11,12].

of a histone variant, prevention of replication fork stal- The latter is attributable to reduced expression of the a

ling, and the suppression of a homologous recombina- globin located on 16. ATRX was hence

tion-based pathway of maintenance. Here, we considered to be an X-chromosome-encoded trans-acting

provide a mechanistic overview of the role of ATRX in factor that facilitates the expression of a select repertoire

each of these processes, and propose how they may be of disparate genes. Subsequent studies in several model

connected to give rise to seemingly disparate human organisms have since uncovered defects in multiple im-

diseases. portant cellular processes upon perturbation of ATRX

function, including defective sister chromatid cohesion

ATRX: from thalassaemia to cancer and congression [13,14], telomere dysfunction [15], and

To understand the molecular mechanisms underlying many aberrant patterns of DNA methylation (at rDNA, sub-

human diseases we need to consider DNA in the context of telomeric, and heterochromatic repeats [16]). Exciting

chromatin. Chromatin consists of 147 bp of DNA wrapped recent developments have also implicated loss of ATRX

around a histone octamer that is subsequently assembled function in a specific subset of malignancies that depend

into compacted higher-order structures. The role of chroma- on a telomerase-independent pathway of telomere main-

tin extends far beyond that of a simple packaging tool; it is tenance called the ‘alternative lengthening of ’

post-translationally modified, facilitating the recruitment of (ALT) pathway [17–19]. Such a broad spectrum of pathol-

binding partners, and its structure is dynamically regulat- ogies led us to speculate as to whether the underlying

ed, allowing for the modulation of a wide variety of biological molecular mechanisms behind these pathologies are in-

processes (for extensive reviews see [1,2]). The identification terrelated. Here, we explore this possibility by outlining

of human diseases caused by mutations in genes that encode the recent advances in our understanding of ATRX cellu-

required for the remodelling or epigenetic modifi- lar function and what this implies about the role of

cation of chromatin has underscored the importance of chromatin in human disease.

chromatin structure in human disease. One such disorder,

ATRX: a repeat offender on heterochromatin

ATR-X syndrome, is caused by mutations in the ATRX gene.

A prominent clue to the physiological role of ATRX has

ATRX encodes a chromatin remodeller (ATRX), which is a

come from studying the distribution of ATRX in the nucle-

280-kDa protein that includes an unusual N-terminal plant

us. Indirect immunofluorescence studies have revealed

homeodomain (PHD) designated the ATRX-DNMT3-

that ATRX has a strong preference for binding within

DNMT3L (ADD) domain owing to its similarity to a protein

promyelocytic leukaemia (PML) bodies and also to repeti-

region found in the DNA methyl transferases (DNMTs) [3–

tive heterochromatic regions such as rDNA, telomeric, and

5]. Located at the C terminus are seven subdomains

pericentric DNA repeats [16,20,21].

that confer ATPase activity and identify ATRX as a snf2

Previous work indicates that localisation of ATRX to

family member of chromatin-associated proteins; many of

heterochromatin involves an interaction with the hetero-

which characteristically slide, remodel, or remove histones

chromatin protein HP1 and in neuronal cells methyl cpg

in in vitro assays [6]. It is now known that ATRX, in

binding protein 2 (MeCP2) is also implicated [22,23].

Corresponding author: Gibbons, R.J. ([email protected]).

Furthermore, targeting of ATRX to pericentric hetero-

Keywords: ATRX; G4-quadruplex DNA; DNA replication; alternative lengthening of

telomeres. chromatin is dependent on trimethylation of

at Lys9. Knockout of the methyltransferases Suv39H1

0968-0004/$ – see front matter

ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibs.2013.06.011 and Suv39H2, which are responsible for ‘writing’ this

Trends in Biochemical Sciences, September 2013, Vol. 38, No. 9 461

Review Trends in Biochemical Sciences September 2013, Vol. 38, No. 9

DAXX H3.3

HP1 ATRX

DAXX

Key: HP1 H3.3 Histone H3 K9me3 ATRX

Histone H3 K4me0

H3.3

TiBS

Figure 1. Recruitment of ATRX (a-thalassaemia/mental retardation X-linked) to chromatin. Binding of ATRX to histone H3 at heterochromatic sites occurs through interaction of

the ATRX ADD (ATRX-DNMT3-DNMT3L) domain with a histone H3 N-terminal tail that is trimethylated at Lys9 and unmodified at Lys4. Recruitment is enhanced by a third

interaction through heterochromatin protein (HP)1 that also recognises trimethylated H3 Lys9 [24,27] (left). Once recruited to its target sites ATRX, in combination with its

interaction partner death-associated protein 6 (DAXX), facilitates the deposition of the histone variant H3.3 [7–10], which may maintain DNA in the B-form [57].

modification, completely abrogates its recruitment [24]. the finding that it serves as a ‘hot spot’ for syndrome-

An appealing hypothesis to emerge from these two obser- associated mutations [28].

vations is that HP1 might recruit ATRX to pericentric

heterochromatin indirectly by serving as a protein scaf- ATRX: a histone chaperone

fold, facilitating the recruitment of ATRX through binding Once recruited to its target sites, how does ATRX ensure

of H3 K9me3 via its N-terminal chromodomains [25]. A appropriate gene expression and/or maintain genomic sta-

major caveat to this hypothesis, however, is that it pro- bility? It is now known that ATRX limits the deposition of

vides limited specificity given that HP1 is a widely dis- the histone variant macroH2A1 [29] (Box 1), whereas, in

tributed, constitutive component of heterochromatin with combination with its interaction partner (DAXX), it can

a large repertoire of binding partners [26]. Recent work by facilitate the incorporation of another histone variant,

Eustermann et al. and Iwase et al. elegantly addresses this H3.3, into pericentric, telomeric, and ribosomal repeat

question by showing that in addition to binding HP1, sequences [7–10]. DAXX functions as a highly specific

ATRX binds directly to the histone H3 tail. Importantly, histone chaperone that is able to discriminate H3.3 from

specificity for this interaction is governed through two

distinct binding pockets located in the N-terminal ADD Box 1. ATRX: sequestration of a histone variant

domain; one accommodating unmodified Lys4 and the

Recent research has identified an interaction between ATRX and

other restricted to di- or trimethylated Lys9. This allows

another histone variant, macroH2A1 [29]. MacroH2A1 is generally

for a combinatorial readout of K4me0 and K9me3 (or considered to associate with transcriptionally inert chromatin and is

K9me2) on the histone H3 N-terminal tail, with recruit- frequently found associated with heterochromatin [60]. Ratnamukar

et al. have shown that, in contrast to the role of ATRX/DAXX in

ment to K9me3 further enhanced by the previously char-

histone H3.3 deposition, ATRX acts as a negative regulator of

acterised interaction with HP1 [24,27] (Figure 1). ATRX

macroH2A chromatin association. Knockdown of ATRX results in an

recruitment to heterochromatin thereby adds credence to

increase in the accumulation of macroH2A1 at telomeres and at the

the existence of a multi-faceted ‘histone code’ that is ‘read’ a-globin cluster in a human erythroleukaemia cell line, suggesting

by a combination of multivalent effector–chromatin inter- that ATRX may additionally promote a-globin expression by

maintaining chromatin in an active configuration through seques-

actions. The importance of the ATRX ADD domain in the

tration of macroH2A1.

underlying aetiology of ATR-X syndrome is highlighted by

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Review Trends in Biochemical Sciences September 2013, Vol. 38, No. 9

the other major histone H3 variants H3.1 and H3.2, where- genes located several kilobases downstream of the TR DNA.

as ATRX is involved in targeting DAXX to repetitive Secondary DNA structures, such as G-quadruplexes, are

sequences and enhancing this deposition [7–9]. Under- known to form barriers to several nuclear processes, includ-

standing the function of H3.3 at ATRX target sequences ing DNA replication and transcription [35,37,38]. Consistent

is therefore likely to prove pivotal in understanding ATRX with this, recent studies have shown that loss of ATRX

function in relation to human disease. In contrast to the function is associated with replication defects, as identified

canonical histones, H3.3 is synthesised throughout the cell by an increase in replication fork stalling, a prolongation in S-

cycle and predominantly undergoes replication-indepen- phase, and a concomitant accumulation of p53 and phosphor-

dent deposition [30,31]. H3.3 was traditionally considered ylation of the histone variant H2AX at S139 (g-H2AX). g-

as a marker for highly dynamic, active chromatin and its H2AX and p53 accumulation particularly occurs at telo-

deposition in heterochromatic regions might therefore seem meres; a known site for G-quadruplex formation and ATRX

somewhat counterintuitive. Repetitive DNA, however, is binding [15,39,40]. Accumulating evidence is emerging that

predicted to be an inherently ‘unfriendly’ substrate for problems in replicating structured DNA can also cause per-

nucleosomes, leading to rapid turnover. ATRX/DAXX-de- turbations in local gene expression. The accurate propagation

pendent H3.3 deposition may be necessary to stabilise a B- of histone marks during chromosomal replication is thought

DNA conformation in tandem repetitive ATRX target sites. to rely on the tight coupling of replication with the recycling of

This raises the question of whether the pathologies observed parental histones. Work by Sarkies et al. has shown that

upon loss of ATRX function could be at least partially interruption of processive DNA replication at G-quadruplex

attributable to an increase in structured DNA at these sites. sequences in cells through loss of various G-quadruplex

results in this tight coupling being lost, leading

ATRX and a-thalassaemia to the biased incorporation of newly synthesised histones

ATR-X syndrome is characteristically associated with a- (associated with H4 N-terminal acetylation but not other

thalassaemia, attributable to the downregulation in expres- marks of activation or repression). Genes that were previ-

sion of the a-globin genes located close to the telomere of ously active became repressed and conversely genes that

chromosome 16. Chromatin immunoprecipitation (ChIP) were previously repressed became active [41,42].

data suggest that tandem repetitive DNA does indeed have An alternative explanation is that epigenetic silencing

a crucial role to play in this phenomenon by showing that, might arise from the creation of a double-strand break,

instead of binding directly to the a-globin genes, ATRX which is known to be generated upon collapse of a stalled

predominantly localises several kilobases upstream within replication fork (for comprehensive reviews see [43,44]).

a tandem repeat (TR) sequence, denoted cz. Strikingly, in Work by the Greenberg laboratory has recently demon-

ATR-X syndrome patients, the genes immediately proximal strated such a phenomenon by showing that the induction

to this repeat are the most downregulated, and those fur- of a double-strand break can lead to the silencing of a

thest away (10 kb) are the least perturbed. It has further reporter gene some 4 kb downstream from the break site

been noted that the size of the cz TR varies between through H2A ubiquitylation-dependent pausing of RNA

patients, and the degree of thalassaemia is proportional polymerase (RNAP)II [45] (Figure 2). Further work is

to the length of the cz TR, explaining the variable degrees of needed to dissect whether either of these events is indeed

thalassaemia observed between patients with identical occurring at the a-globin in ATR-X syndrome,

ATRX mutations [32]. Approximately 50% of ATRX target through careful profiling of DNA replication through the

sites, including telomeres, are predicted to adopt the G- associated cz TR and the detection of any associated

quadruplex conformation [32] and there is mounting evi- epigenetic changes arising from this site.

dence that these structures form in vivo [33–35]. Electro- Given that ATRX appears to have general roles in the

phoretic mobility shift assays have demonstrated that deposition of a histone variant and DNA replication, one

recombinant ATRX protein preferentially binds to G4-struc- might expect that the cellular consequences of losing

tured DNA over an unfolded telomere-derived sequence ATRX function would include a loss of genome stability

[32], strongly suggesting ATRX may directly bind these and chromosomal congression defects. In the appropriate

DNA structures in vivo. cz TR adopts a G-quadruplex cellular context this does indeed appear to be the case,

conformation in vitro and we propose that an explanation with loss of ATRX associated with extensive genomic

for the repeat length dependency is that the longer the TR rearrangements, DNA damage, and telomere fusions

sequence, the higher the probability that it will adopt a [15,19,39]. Furthermore, loss of ATRX function has been

noncanonical secondary structure such as a G-quadruplex. shown to result in both mitotic and meiotic defects, in-

Consistent with this hypothesis, previous studies have cluding defective spindle formation, and chromosome

shown that the length of a G-quadruplex-forming minisa- condensation and alignment [13,14,39]. A link between

telllite, CEB1, is related to its instability upon deletion of the defective replication and chromosome condensation has

known G-quadruplex resolving helicase, petite integration long been established (for review see [46]). As such, it is

frequency 1 (Pif1), and this is likely related to its ability to plausible that some of the other clinical manifestations of

form a G-quadruplex structure [36]. ATR-X syndrome, such as microcephaly, may at least be

partially attributable to the DNA replication defects/in-

ATRX and genome stability creased genomic instability observed upon loss of ATRX

Assuming such a hypothesis is correct, it raises the intriguing function. Indeed, the use of mouse models suggests this is

question as to how the presence of an aberrant DNA second- the case with increased replicative damage associated

ary structure may lead to the transcriptional silencing of with specific knock out of Atrx in myoblast, Sertoli, and

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Review Trends in Biochemical Sciences September 2013, Vol. 38, No. 9

G-quadruplex DNA potenally formed on lagging strand

DAXX DAXX ATRX dependent maintenance of ATRX Stalling of replicaon fork at G-quadruplex DNA upon ATRX DNA in B-form allowing for

processive replicaon loss of ATRX funcon ATP ADP

Replicaon fork collapse and resecon to form double strand break

HR mediated repair Transcriponal silencing

RNF8 H2A K119 monoubiquitylaon RNF168

PolII pausing and Iniaon of ALT at telomeres transcriponal silencing

TiBS

Figure 2. Loss of ATRX (a-thalassaemia/mental retardation X-linked) function may lead to DNA replication stalling-dependent transcriptional silencing and aberrant

telomeric recombination. The persistence of DNA secondary structures, particularly on the lagging strand, in the absence of functional ATRX may lead to replication fork

stalling (right hand side). Upon collapse of the fork a double-strand break is generated, which may result in the silencing of genes distal to the break site through RING

finger protein (RNF)8- and RNF168-mediated ubiquitylation of H2A and the subsequent stalling of RNA polymerase (RNAP)II [45]. Double-strand breaks are a known trigger

for homologous recombination-mediated repair, which at telomeric sites may in turn trigger the alternative lengthening of telomeres (ALT) pathway in malignant cells

lacking ATRX. DAXX, death-associated protein 6.

neuroprogenitor cells, leading to compromised muscle in mouse relative to human may alter the cellular effects

growth, testicular defects, and increased neuronal loss of ATRX loss.

respectively [39,47–49]. We note, however, that specific

knockout of Atrx in chondrocytes has minimal effects on ATRX and cancer

skeletal development, with the mouse model therefore The first indication that acquired somatic mutations in

failing to recapitulate the short stature observed in ATR- ATRX may be linked with malignancy came from the obser-

X patients [50]. This may infer that there is a potential vation that a cohort of patients presenting with myelodys-

cell-specific role for ATRX, while highlighting the pitfalls plastic syndrome (MDS) (a neoplastic disorder of the myeloid

of using mouse models to investigate human disease, lineage of haematopoiesis), concurrently acquire a-thalas-

because for example, the different repeat distributions saemia [a-thalassaemia–myelodysplasia (ATMDS)]. It is

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Review Trends in Biochemical Sciences September 2013, Vol. 38, No. 9

now clear that the majority of cases likely harbour mutations Box 2. Outstanding questions

in ATRX [51,52]. It remains unclear, however, as to whether

 Is it the presence of abnormal DNA structures persisting in the

ATRX mutations are driver or merely passenger changes in

absence of ATRX that triggers the replication defect observed in

ATMDS, although it is tempting to speculate that any poten-

null cells?

tial loss of genomic integrity associated with a loss of ATRX  Does replication fork stalling occur at the cz TR sequence upon

loss of ATRX function, and if so does this trigger epigenetic

function may be a contributing factor in driving a neoplasm.

silencing of the proximal alpha globin genes?

Interest in this question has been reignited by the

 Loss of ATRX alone appears to be insufficient to trigger ALT, what

recent finding that mutations in ATRX are additionally

other factor or factors are required?

associated with a specific subgroup of tumours that use a  Is the haematological malignancy associated with ATMDS

telomerase-independent mechanism of telomere mainte- dependent on the ALT pathway for telomere maintenance?

nance, termed the ALT pathway. To grow indefinitely,

cancer cells must circumvent the progressive attrition of

required to determine which additional factors are re-

telomeric DNA that inevitably occurs with each cell divi-

quired to trigger ALT in an ATRX-null genetic landscape.

sion, ultimately leading to cellular senescence or crisis. In

This may explain the conundrum that ATR-X cases do not

the majority of cancer cells this is potentiated through

appear to be cancer prone [11], again suggesting that

reactivation of telomerase, a specialised reverse transcrip-

additional defects are required for malignancy.

tase that synthesises the tandem TTAGGG hexanucleotide

repeat by using its own RNA subunit as a template [53,54].

Concluding remarks

However, 10–15% of cancers lack detectable telomerase

The biological insights gained from ATR-X syndrome illus-

and are instead reliant on a telomere maintenance path-

trates the benefit of studying rare genetic disorders and

way that depends on homologous recombination [55] be-

highlights the importance of chromatin remodellers in

tween telomeric sequences (for extensive reviews see

human disease. From studies that elucidated how and

[56,57]). Strikingly, a recent study has documented that

where ATRX is recruited in the genome, and that identified

ATRX is lost in 90% of in vitro immortalised ALT cell lines

the ATRX/DAXX complex as a novel histone chaperone for

[19]. Moreover, mutations in ATRX and/or DAXX have

H3.3, we have now begun to understand how ATRX may

been identified in many tumours exhibiting ALT, including

regulate gene expression and function as an important

pancreatic neuroendocrine tumours, neuroblastomas, pae-

tumour suppressor. We propose that these roles may be

diatric glioblastomas, oligodendrogliomas, and medullo-

related to a role for ATRX in regulating repeat structures

blastomas [17–19], and appear to be mutually exclusive

in the genome; however, we do not exclude that ATRX may

to mutations in the TERT promoter that potentiate telo-

also have repeat independent roles that could also contrib-

merase expression [55]. Furthermore, it has been noted

ute/explain the physiological effects of ATRX loss. The

that the loss of wild type ATRX expression in somatic cell

work described here raises many outstanding questions

hybrids segregates with activation of the ALT pathway

that will undoubtedly help clarify many of the hypotheses

[58]; strongly suggesting that ATRX acts as a suppressor of

we have outlined (Box 2). Nonetheless it is clear that

the ALT pathway.

insights gained from the study of one disorder can be

We propose that in the absence of functional ATRX,

applied to understand the role of ATRX in seemingly

increased stalling of replication forks [a known trigger of

disparate pathologies, highlighting the importance of com-

homologous recombination (HR)] at telomeric sites may

bining basic research with clinical genetics.

contribute to the initiation of aberrant telomeric recombi-

nation and activation of the ALT pathway. Telomeres are

Acknowledgements

known to be inherently difficult sites to replicate and re-

We would like to thank Ricarda Gaentzsch and Barbara Xella for critical

semble fragile sites [59]; furthermore, the stabilisation of G-

reading of the manuscript. This work was supported by the Medical

quadruplex structures using a stabilising ligand has been Research Council and the University of Oxford.

shown to perturb DNA replication at telomeres and trigger a

DNA damage response [38]. This implies that an increased References

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