Special Issue: Human Genetics

Review

Functional Insights into

Chromatin Remodelling from

Studies on CHARGE Syndrome

1, 2

M. Albert Basson, * and Conny van Ravenswaaij-Arts

CHARGE syndrome is a rare genetic syndrome characterised by a unique com- Trends

bination of multiple organ anomalies. Dominant loss-of-function mutations in the

The expressivity and penetrance of

gene encoding chromodomain helicase DNA binding protein 7 (CHD7), which is CHARGE phenotypes show little cor-

relation with the CHD7 genotype.

an ATP-dependent chromatin remodeller, have been identified as the cause of

CHARGE syndrome. Here, we review recent work aimed at understanding the The hypothesis that mutations or poly-

morphisms in other genes modify dis-

mechanism of CHD7 function in normal and pathological states, highlighting

ease phenotypes in humans remains

in vivo

results from biochemical and studies. The emerging picture from this work unproven.

suggests that the mechanisms by which CHD7 fine-tunes gene expression are

CHARGE-associated CHD7 mutations

context specific, consistent with the pleiotropic nature of CHARGE syndrome.

can affect its nucleosome remodelling

activity in vitro.

Molecular Origins of CHARGE Syndrome

Advances in genomics over the past few years have led to impressive achievements in the The role of CHD7 in regulating nucleo-

some positioning in vivo and the effects

identification of mutations responsible for rare genetic diseases, especially those with simple,

of this activity on gene expression

monogenetic aetiologies [1]. The identification of the genetic cause of a disease represents a

require further study.

major milestone because biochemical and gene targeting approaches can then be used to

explore the biological functions of the gene in question and investigate the molecular mecha- CHD7 pleiotropy may result from its

interactions with cell type-specific tran-

nisms that underlie the disease. Typically, the insights gained from these studies have wide-

scription factors.

reaching implications by revealing fundamental biological principles that control development

and homeostasis. CHARGE syndrome (MIM 214800) is a good example of such a case. This

CHD7 recruitment to cell type-specific

syndrome is diagnosed in 1/10 000 live births and is characterised by dysmorphic features and enhancers may underlie context-spe-

cific roles.

congenital anomalies in multiple organs. The development of the nervous system (especially

cranial nerves), ears and vestibular organs, eyes, heart, urogenital, and endocrine systems can

be affected and growth retardation is often observed. The presence and severity of these

anomalies show remarkable variation between patients.

1

King's College London, Department

In 2004, shortly before the next-generation sequencing era, mutations in the CHD7 gene (MIM of Craniofacial Development and Stem

Cell Biology and MRC Centre for

608892) were identified in patients with CHARGE syndrome [2]. CHD7 encodes a chromatin-

Developmental Neurobiology, Floor

remodelling factor (CRF), which implied an epigenetic aetiology for CHARGE syndrome. CRFs

27, Guy's Hospital Tower Wing,

have evolved to deal with the additional complexity and hindrance that the tight association of London, SE1 9RT, UK

2

University of Groningen, University

eukaryotic DNA with histone proteins imposes on replication and transcription (Box 1). CRFs

Medical Center Groningen,

perform several essential functions. For instance, the clearance of nucleosomes from promoters

Department of Genetics, PO Box

and other regulatory elements is necessary for the recruitment of some transcription factors (TFs) 30.001, 9700RB Groningen,

The Netherlands

and the core transcriptional machinery, and nucleosome eviction is essential for efficient

transcriptional elongation. Conversely, the maintenance of heterochromatin is an important *Correspondence:

mechanism for stable gene repression. Thus, different classes of CRF can have opposing [email protected] (M.A. Basson).

600 Trends in Genetics, October 2015, Vol. 31, No. 10 http://dx.doi.org/10.1016/j.tig.2015.05.009

© 2015 Elsevier Ltd. All rights reserved.

Box 1. The Concept of Chromatin Remodelling Glossary

Chromatin remodelling refers to the active process of changing the structure and organisation of chromatin. Histone

22q11del syndrome: most common

subunits, termed H2A, H2B, H3, and H4, that comprise the core nucleosomal particles around which DNA is wound, are

microdeletion (1.5-3MB) syndrome,

globular proteins with exposed N-terminal ‘tails’ that contain several amino acid residues that are accessible to modifying

also known as velocardiofacial or

enzymes that catalyse the covalent addition of chemical moieties. These modifications include acetylation, methylation,

DiGeorge syndrome.

phosphorylation, ubiquitination, and ADP ribosylation, among others [66]. The linker histone H1 is also subject to covalent

Absent, small, or homeotic-like 1

modification. Histone modifications can alter the chemical properties of the nucleosome and its interaction with DNA [67].

(ASH1): a member of the trithorax

The combination of histone modifications carried by a nucleosomal array on chromatin creates unique binding sites,

group and a histone-lysine N-

which can affect CRF recruitment to this region. The most direct and dramatic changes in chromatin structure are methyltransferase.

catalysed by chromatin-remodelling enzymes. These CRFs are ATP-dependent molecular machines that can alter the

Choanal atresia: anatomical

position of nucleosomes on chromatin. Repositioning nucleosomes, or even evicting them completely from chromatin at

obstruction of the back of the nasal

a given locus, changes the accessibility of specific DNA sequences. As a consequence, TF binding sites may either

passage, interfering with the ability to

become available for, or be obscured from, binding by their cognate TF (Figure 2, main text). These changes may affect breathe.

chromatin architecture on a larger scale if the DNA–protein interactions that are altered upon the action of CRFs are

Chromodomain: protein structural

involved in maintaining higher order chromatin organisation, such as those mediated by insulating factors, including

domain of approximately 40–50

CTCF.

amino acid residues commonly found

in proteins associated with chromatin

fi

ATP-dependent CRFs are classi ed in three different groups: SWI/SNF, ISWI, and CHD. CRFs typically function in large, remodelling.

multi-subunit protein complexes. The composition of these complexes may differ between different cell types, thereby

Coloboma: an embryonic closure

altering the nature of the complex, as exemplified by the BAF complex [68]. The observation that active gene promoters

defect of the eye (e.g., affecting the

and enhancer elements can be identified empirically by nuclease-sensitivity assays as a proxy for DNA accessibility

iris and/or the retina). In CHARGE

implicates CRFs in the establishment and maintenance of chromatin structure at regulatory elements.

syndrome, retinal coloboma results in

partial or complete loss of sight.

Germline mosaicism: a situation

activities and functions, and local chromatin context can determine the effects of a CRF at a

where germ cells in an individual

specific locus [3].

differ with respect to a specific

genetic feature, usually caused by a

de novo mutation in one germ cell

In this review, we summarise some of the most recent studies that link CHD7 to specific

progenitor during development or

developmental genes and pathways. We discuss in vitro experiments that provide insights into

expansion. The chance of a disease-

CHD7 mechanism and highlight the significant gaps in our understanding of how CHD7 acts in

causing mutation being transmitted

vivo to fine-tune gene expression and control development. However, we begin with the clinical to offspring depends on the

proportion of mutated germ cells.

data obtained from patients and summarise our current knowledge of CHD7 mutations that

H3K27me3: Lysine 27 tri-methylation

cause CHARGE syndrome.

of histone 3, a post-translational

modification typically associated with

The Phenotypic Spectrum of CHARGE Syndrome repressed regulatory regions.

H3K36me3: Lysine 36 tri-methylation

CHARGE syndrome is characterised by a multitude of congenital aberrations that can vary in

of histone 3, a post-translational

both severity and presence. The most frequent features seen in patients with a CHD7 mutation

modification typically associated with

are: external ear malformations (97%); cranial nerve dysfunction (99%, causing facial palsy in gene bodies undergoing active

66%); semicircular canal anomalies (94%); coloboma (81%; see Glossary); choanal atresia transcription, used as an indicator of

transcriptional elongation.

(55%); cleft lip and/or palate (48%); anosmia (80%); genital hypoplasia (81%); congenital heart

H3K4me3: modification typically

defects (76%); and tracheoesophageal anomalies (29%). In addition, severe feeding problems

associated with active promoter

(82%), delayed motor development milestones (99%), intellectual disability (74%), and growth regions.

Imitation switch (ISWI): a class of

retardation (37%) are observed [4–8] (percentages according to [8]).

ATP-dependent CRFs.

Kallmann syndrome: a syndrome

The clinical diagnosis of CHARGE syndrome is based on major and minor diagnostic criteria

characterised by hypogonadism and

formulated by Blake [9], which were later refined by Verloes [10]. Major criteria are the presence anosmia (inability to smell), due to

failure of gonadotropin-releasing

of coloboma, choanal atresia, characteristic ear malformations, and cranial nerve dysfunction for

hormone (GnRH) and olfactory

Blake, and the presence of coloboma, choanal atresia, and hypoplastic semicircular canals for

neurons to migrate from the olfactory

Verloes. To reach a clinical diagnosis of typical CHARGE syndrome, at least three out of four

placode to the hypothalamus and

major criteria should be met according to Blake and two out of three for Verloes. Given that up to bulbus olfactorius during

development, respectively.

17% of patients with a CHD7 mutation do not fulfil these strict diagnostic criteria [5,8], molecular

H3K4me1: Lysine 4 mono-

genetic testing of CHD7 is important to confirm a diagnosis of CHARGE syndrome and enable

methylation of histone 3, a post-

appropriate health guidance and genetic counselling. translation covalent histone

modification typically associated with

distal enhancer regions.

The CHD7 Mutation Spectrum in CHARGE Syndrome

CHARGE syndrome is typically a sporadic condition and almost all CHD7 mutations occur de

novo. However, familial CHARGE syndrome has been reported [11]. Due to germline mosaicism,

Trends in Genetics, October 2015, Vol. 31, No. 10 601

Mixed lineage leukaemia (MLL):

(A) a

member of the trithorax group and a

histone-lysine N-methyltransferase. (B)

Multiplex ligation-dependent

probe amplification (MLPA): a

(C) multiplex PCR method for

simultaneously detecting copy

(D) number variation across several loci.

Orthodenticle homeobox 2

(E) (OTX2): a homeobox protein.

Polybromo, Brg1-Associated

(F) Factors (PBAF): an ATP-dependent

CRF complex of the SWI/SNF family.

Remodel the Structure of

Chromatin (RSC): a 17 subunit

ATP-dependent chromatin-

Figure 1. Overview of Reported Chromodomain Helicase DNA Binding Protein 7 (CHD7) Mutations. Mutations remodelling complex of the SWI/SNF

reported in CHD7 aligned along a schematic representation of the CHD7 protein (E): splice site (A), missense (B), frame shift family, involved in DNA replication.

(C) and nonsense mutations (D), and partial gene deletions [red lines in (F)] and duplication [green line in (F)]. The mutations SANT-like ISWI domain/

are spread throughout the CHD7 gene, but missense mutations occur only in the middle of the gene. Key: chromodomain; switching-defective protein 3

helicase N; DEXDc; Helicase C; SANT domain; BRK domain. Adapted from [13]. An overview of mutations (Swi3), adaptor 2 (Ada2), nuclear

i

and polymorphic variants of the CHD7 gene can be found in the CHD7 locus-specific database . receptor co-repressor (N-CoR),

TFIIIB (SLIDE/SANT): functional

protein domains associated with

nucleosome recognition and

endowing chromatin remodellers with

the recurrence risk is only 2–3% for parents of children with a de novo mutation [11,12].

the ability to generate ordered

However, patients with CHD7 mutations have a 50% chance of transmitting the dominant

nucleosome arrays.

CHD7 mutation to their offspring [12]. Semaphorin-3A (SEMA3A): a

secreted, guidance molecule,

involved in olfactory development and

Over 500 different pathogenic CHD7 alterations have been identified [13,14]. A schematic

GnRH neuron migration, and

representation of CHD7 and the positions of mutations within the gene are shown in Figure 1.

mutations associated with Kallmann

The distribution of types of mutation is summarised in Table 1. Nonsense mutations (44%) and syndrome.

frame shift deletions or insertions (34%) are the most prevalent. These mutations are distributed SMAD: vertebrate homologues of

mothers against decapentaplegic

throughout the entire coding region, suggesting that premature termination of the protein, even

(MAD), intracellular signalling

at the C terminus, is detrimental to protein function. Splice site (11%) and missense mutations

molecules that transduce BMP and

(8%) are predominantly located in the middle of the protein, in or near coding regions for the transforming growth factor (TGF)-b

signals, by translocating to the

known functional domains of CHD7, where they are predicted to be pathogenic (Figure 1).

nucleus and functioning as TFs upon

Overall, approximately 30% of mutations are located within regions coding for a functional

BMP receptor activation.

domain. Whole-exon deletions rarely occur (<1%) and deletions encompassing the complete

Switch/Sucrose NonFermentable

CHD7 gene have been described in only ten cases in the literature. Translocations at chromo- (SWI/SNF): an ATP-dependent CRF

complex.

some 8q12 that disrupt CHD7 have been identified in three cases, with one resulting in an

T-box 1 (TBX1): a TF that controls

additional deletion of a portion of CHD7 (reviewed in [13]).

the development of multiple organs

affected in 22q11del syndrome.

Treacher Collins syndrome: a

i

developmental syndrome caused by

Table 1. Types of Pathogenic Variant in CHD7, Based on CHD7 data

mutations in the TCOF1 gene,

Mutation type Number of patients Percentage

characterised by craniofacial

Nonsense 352 43.9 anomalies. Increased apoptosis and

reduced proliferation of neural crest

Frame shift indels 273 34.0

cells are caused by upregulated p53

Splice site 90 11.2 activity in response to reduced rRNA

synthesis.

Missense 66 8.2

WD repeat-containing protein 5

In-frame deletions 2 0.2 (WDR5): forms a complex with MLL

to regulate histone-lysine tri-

Whole-exon deletions and duplications 7 0.9

methylation.

Whole-gene deletions 10 1.2

Translocations 2 0.2

Total 802

602 Trends in Genetics, October 2015, Vol. 31, No. 10

Genotype–phenotype Correlations in CHARGE Syndrome

Carriers of a CHD7 mutation display a striking phenotypic variability, even within the rare familial

cases, where family members carry an identical mutation [8,11].

In general, missense mutations are associated with a milder phenotype and a lower prevalence

of choanal atresia, cleft lip and/or palate, and congenital heart defects [5,15]. In families with

parent-to-child transmission, the CHARGE phenotype is usually atypical. In four out of seven

(57%) two-generation families, a missense mutation in CHD7 was found, contrasting with the

8% missense mutations in the general CHARGE population [8]. With the exception of this

association of a milder phenotype with missense mutations, there is no clear genotype–

phenotype correlation.

What about Patients with CHARGE Syndrome without CHD7 Mutations?

In patients with clinically typical CHARGE syndrome, the detection rate of CHD7 mutations is

over 90%. In research studies, detection rates range from 33% to 100%, depending on the

inclusion criteria (reviewed in [13]). In a routine clinical setting, sequencing of CHD7 reveals a

mutation in 32–41% of the patients suspected of having CHARGE syndrome [13,16]. Thus, the

chance of finding a mutation is critically dependent on the accuracy of the initial clinical diagnosis.

In the remaining 5–10% of patients with clinically typical CHARGE syndrome, the apparent lack

of CHD7 mutations may be explained by alterations in CHD7 that are not detected with routine

genotyping strategies. These may include intragenic rearrangements or mutations in intronic or

promoter regions, and whole-gene or whole-exon deletions on one allele, which are not always

screened for. The application of next-generation sequencing techniques, especially whole-

genome sequencing in combination with analysis for deletions and rearrangements, is expected

to solve a major proportion of the 5–10% of patients with unexplained typical CHARGE

syndrome. It should also be kept in mind that mutations within sequenced regions can be

missed by conventional techniques. For example, whole-exome sequencing of all coding exons

including at least 20-base pair (bp) intronic DNA to capture intron–exon boundaries, of seven

patients with clinically typical CHARGE syndrome, for whom standard Sanger sequencing failed

to identify a CHD7 mutation, identified two intronic (c.5051-15T>A and c.5405-17G>A) muta-

tions in the CHD7 gene in two patients (N. Corsten-Janssen, unpublished).

Despite most patients carrying mutations in CHD7, it is still possible that other genes are involved in

CHARGE syndrome. In 2004, the gene encoding sema domain, immunoglobulin domain (Ig), short

basic domain, secreted, (semaphorin) 3E (SEMA3E) was shown to be interrupted by a de novo

balanced translocation [t(2;7)(p14;q21.11)] in a patient with CHARGE syndrome [17]. However,

there is little additional evidence that SEMA3E has a major role: only one de novo missense

mutation in SEMA3E was found in a cohort of 24 patients with CHARGE syndrome, and Sema3e

mutant mice do not phenocopy any of the cardinal features of CHARGE syndrome [18].

Other genetic alterations that can phenocopy CHARGE syndrome, resulting in a clinical diag-

nosis of CHARGE syndrome, include chromosomal aberrations [e.g., 22q11.2 deletion syn-

drome, involving the gene encoding T-box 1 (TBX1) [19], 3p13-p21 deletions [20], and 5q11.2

deletion syndrome [21]], teratogen exposure (e.g., retinoic acid or antithyroid drugs [22]), or

maternal diabetes.

CHD7 Mechanism of Action

CHD7 is a member of the chromodomain helicase DNA-binding family of ATP-dependent

chromatin remodelling enzymes. These proteins share a conserved Snf2 helicase-like ATPase

domain that catalyses the translocation of nucleosomes along DNA in chromatin [23]. Mecha-

nisms that maintain specific nucleosome positioning at regulatory regions are critical for normal

Trends in Genetics, October 2015, Vol. 31, No. 10 603

gene regulation, because tight nucleosome–DNA interactions can preclude the productive

association of DNA with TFs and core transcription machinery (Figure 2). Furthermore, nucleo-

some eviction downstream of gene promoters might be required for efficient transcriptional

elongation. For example, CHD1-mediated nucleosome depletion downstream of active gene

(A) CHD7-mediated chroman remodelling at poised enhancers

TSS

CHD7

pTF Nucleosome translocaon

Enhancer (B) Acve enhancer–promotor complex(C) Inacve, repressed enhancer conformaons

CHD1 (i) mRNA Transcriponal Pol II elongaon TSS

TFIID

Mediator CoR pTF TF pTF TF eRNA Pol II

(ii)

TSS

CHD7

Figure 2. Proposed Model of Reported Chromodomain Helicase DNA Binding Protein 7 (CHD7) Action at

Enhancers. (A) CHD7 is recruited to poised cell type-specific enhancers through interactions with cell type-specific pioneer

transcription factors (pTF) and histone modifications, such as H3 mono methyl K4 (H3K4me1) (yellow dot). pTFs can access

their DNA-binding sites without a requirement for additional chromatin remodelling. Upon recruitment, CHD7 catalyses

nucleosome translocation along the DNA, thereby revealing additional TF-binding sites (green rectangle). (B) Recruitment

of additional TFs, perhaps associated with co-activators such as p300 (not shown), results in further modification (H3K27ac,

green dot) and remodelling of chromatin, recruitment of RNA Polymerase II (Pol II) and transcription. Enhancer-associated RNA

transcripts (eRNA), together with interactions with TFs, may facilitate association with subunits of the Mediator complex and

chromatin looping to allow long-range enhancer–promoter interactions. Mediator association with core transcriptional

machinery (such as TFIID) facilitates transcriptional initiation at the transcriptional start site (TSS). Additional interactions,

for example the recruitment of CHD1 by Mediator and Pol II, enhance transcriptional elongation by remodelling chromatin in

front of the elongating Pol II. (C) CHD7-mediated chromatin remodelling may also result in inhibition of enhancer activity and

reduced gene expression. (i) Nucleosome repositioning might promote the association of TFs that are unable to recruit

transcriptional co-activators, or TFs complexed with co-repressors (CoR) leading to changes in chromatin modification

(e.g., H3K27me3, red dot) and structure. (ii) Alternatively, CHD7 might remodel chromatin to interfere with pTF recruitment,

thereby effectively shutting off enhancers in specific contexts. The net effect will be the failure to initiate effective long-range

enhance–promoter association and enhancement of gene expression. Future experiments will be required to test this model.

604 Trends in Genetics, October 2015, Vol. 31, No. 10

promoters was recently shown to be important to overcome a nucleosomal barrier to transcrip-

tional elongation [24].

In a recent study, it was shown that CHD7 has ATP-dependent nucleosome remodelling

activity in in vitro assays [25]. The authors confirmed that CHD7 remodelling activity was

dependent upon the presence of extranucleosomal, free DNA, a feature shared by remod-

ellers belonging to the imitation switch (ISWI) and CHD classes [26–28]. In an in vitro ‘sliding’

assay, CHD7 primarily translocated nucleosomes from one end of a DNA fragment to the

centre, an activity shared with SNF2H [25]. This observation is consistent with the notion that

these remodellers require contact with both DNA and nucleosomes to catalyse nucleosomal

repositioning for maintaining regularly spaced nucleosomal arrays on DNA. By contrast,

Switch/Sucrose NonFermentable (SWI/SNF) and chromatin structure remodelling complex

(RSC) remodellers can also destabilise nucleosome–DNA interactions leading to nucleosome

eviction from chromatin [29]. These observations predict that CHD7 might preferentially

remodel poised enhancer and/or promoter regions with already exposed DNA [30,31]

(Figure 2). However, CHD7 can also associate with the polybromo-associated BAF (PBAF)

chromatin remodelling complex in neural crest cells [32]. This mega-complex containing both

CHD and SWI/SNF components might be capable of both nucleosome sliding and eviction,

which could account for the synergistic action of these factors on neural crest gene

expression [32]. A recent study showed that CHD7 is required for the maintenance of

an open, accessible chromatin state at the promoters of CHD7 target genes in neural stem

cells [33]. Further experiments will be necessary to define whether these effects are due to

nucleosome translocation or eviction and whether a more open chromatin state at

enhancers is always associated with enhanced gene expression.

The CHD7 domains necessary for ATP-dependent remodelling have been defined. In addition

to the critical function of the DExD helicase domains, the C-terminal SANT-like ISWI domain/

switching-defective protein 3 (Swi3), adaptor 2 (Ada2), nuclear receptor co-repressor (N-CoR),

TFIIIB (SLIDE/SANT) and N-terminal chromodomains were shown to have important roles in

nucleosome remodelling activity [25]. A CHARGE syndrome-associated mutation in the first

chromodomain (S834F) completely abolished CHD7 activity and mutations in the second

chromodomain (K907T and T917M) significantly affected remodelling activity, with K907T

being the most severe. The tandem chromodomains in CHD7 are thought to mediate the

recruitment of CHD7 to enhancer regions through interactions with specific histone modifi-

cations. Indeed, ChIP-seq experiments have found that CHD7 is recruited preferentially to

genomic regions that carry the H3 mono methyl K4 (H3K4me1) modification [30,31] (Figure 2).

Furthermore, CHD7 redistribution during cell differentiation correlates with changes in

H3K4me1 and H3K4me2, providing further evidence that H3K4me status directly influences

CHD7 recruitment [30]. It is worth noting that Bouazone et al. used histones purified from HeLA

cells in their assays, which presumably still contained an array of covalent modifications, such

as H3K4me1. Thus, whether the reduced remodelling activity of chromodomain mutants is

partly due to their reduced affinity for H3K4me1-modified histones or more direct effects on

CHD7 remodelling activity has not been fully resolved. In support of the latter possibility, the

chromodomains of CHD1 have been shown to directly contact the ATPase domain and

regulate substrate recognition [34].

These important in vitro studies suggest that most CHD7 mutations found in CHARGE syn-

drome affect ATP-dependent chromatin remodelling activity. A significant remaining question is

how these defects in nucleosome remodelling affect gene expression. Genome-wide gene

expression studies in model systems have suggested that CHD7 functions as both an activator

and repressor [35]. One possibility is that the effects of CHD7 depletion at a particular regulatory

element are dependent on local DNA context (Figure 2). Alternatively, CHD7 may function

Trends in Genetics, October 2015, Vol. 31, No. 10 605

exclusively as an enhancer of gene expression and increased gene expression in CHD7-deficient

cells may be secondary to downregulated expression of transcriptional repressors. Further

studies are required to answer these questions.

CHD7 belongs to subgroup III of the mammalian CHD proteins, which includes CHD6,

CHD7, CHD8, and CHD9 [36]. The sole Drosophila homologue of this family, Kismet, has

been studied extensively and the results of these studies may provide important clues as to

the in vivo functions of CHD7. Surprisingly, no direct evidence that Kismet remodels

nucleosomes has been reported yet. Furthermore, Kismet recruitment to chromatin does

not appear to be mediated by H3K4 methylation [37]. By contrast, Kismet is required for the

efficient recruitment of the absent, small, or homeotic discs 1 (ASH1) and Trithorax (TRX)

histone methyltransferases to chromatin. No change in the levels of H3K4 methylation was

observed in Kismet mutants, but increased H3K27me3 was observed. A recent study from

the same group found that H3K36me2 and H3K36me3 histone modifications are reduced in

Kismet flies. Given that ASH1 can also catalyse the formation of these modifications, this

observation is consistent with a function for Kismet in facilitating ASH1 recruitment to

chromatin [38]. In addition, because the H3K36me2 modification is antagonistic to the

repressive H3K27me3 mark, this finding suggests a potential mechanism whereby loss

of Kismet might lead to an increase in H3K27me3 levels and gene repression.

These observations suggest that Kismet functions primarily as an enhancer of gene

expression.

Thus far, however, no evidence that CHD7 interacts with ASH1 has been reported; neither

has any evidence for H3K27me3 changes been reported in CHD7-deficient cells. Intriguingly,

TBX1, the gene mutated in 22q11del syndrome, can interact with ASH2-like (ASH2L) [39],

suggestive of a mechanism whereby TBX1-ASH2L recruitment to chromatin might enhance

subsequent CHD7 recruitment to these TBX1-controlled enhancer elements (Figure 3).

H3K4me3 is reduced at CHD7-regulated promoters in neural stem cells [33], but it remains

to be seen whether these changes are the result of a direct effect of CHD7 depletion on

recruitment of a histone methyltransferase to these promoters, or just a consequence of

reduced gene expression. Intriguingly, the highly related factor CHD8 has been found to

interact with the ASH2L–WD repeat-containing protein 5 (WDR5)– retinoblastoma binding

protein 5 (RbBP5) complex and loss of CHD8 was also associated with reduced H3K4me3

at gene promoters [40,41]. These findings suggest that the recruitment of histone meth-

yltransferase activity of Kismet is conserved in CHD8. Recently, CHD6 has been shown to

interact with the Facilitates chromatin transcription (FACT) complex, implicating it in tran-

scriptional elongation [42]. This study also identified a role for CHD6 and CHD8 in regulating

long-range chromatin interactions, presumably through interaction with CCCTC-binding

factor (CTCF). By contrast, CHD7 does not appear to be recruited to CTCF-associated

insulators in the genome [30]. Taken together, these studies suggest that members of the

CHD6–9 family function primarily as positive regulators of gene expression, through inter-

acting with complexes with H3K4 methyl transferase, transcriptional elongation, or chroma-

tin remodelling activities. Current evidence also suggests that different family members have

distinct intrinsic activities and interaction partners. In addition, family members appear to also

differ in their recruitment mechanisms. For example, whereas CHD7 appears to be primarily

recruited to distal enhancers [30,31], CHD8 is found at active gene promoters [43,44].

Discoveries that link CHD6–9 with gene activation contrast with activities of the CHD3–5

family, which is primarily associated with gene repression. These proteins can form part

of the Nucleosome remodelling deacetylase (NURD) complex that represses gene expres-

sion through histone deacetylase activity [45,46]. In addition, CHD5 can be recruited

to chromatin marked by H3K27me3 and is associated with Polycomb-mediated gene

repression [47].

606 Trends in Genetics, October 2015, Vol. 31, No. 10

CHD7 Is a Key Regulator of Developmental TF Genes and Signalling

Pathways

The reason for the dramatic phenotypic variation in CHARGE syndrome is unknown. A prevailing

hypothesis is that genes regulated by and/or in an epistatic relationship with CHD7 may

represent ‘modifier’ genes. Mutations or polymorphisms in these genes could conceivably

be responsible for much of the clinical variation that is so typical of CHARGE syndrome by

altering the penetrance and/or expressivity of specific disease traits in the context of CHD7

haploinsufficiency. In this section, we outline some of the genetic pathways recently associated

with CHD7 (Figure 3).

Key Figure

Developmental and Disease-associated Pathways Regulated by Chro-

modomain Helicase DNA Binding Protein 7 (CHD7).

Neurocristopathies Neurocristopathies 22q11del syndrome Olfactory development TBX1 target genes? Kallmann syndrome Kabuki syndrome

Sema3a KMT2D and KDM6A target genes rRNA synthesis H3K4me1

SMAD1 CHD7 BMP target genes Heart development Treacher Collins syndrome Apoptosis p53

SOX2 Otx2, Fgf10, Ngn1 Ear development Eye, ear, oesophageal phenotypes, Sox2 target genes hypogonadotropic hypogonadism Jag1, Gli3, MycN Alagile, Feingold, Renoic acid Otx2 Pallister-Hall syndromes signalling Ear, pharyngeal and Fgf8 Cerebellar vermis development heart development, Gbx2

somitogenesis

Figure 3. CHD7 can affect the activity of several signalling pathways that control development. Established connections are

in black and hypothetical interactions or associations are in blue. CHD7 regulates bone morphogenetic protein (Bmp4)

expression [69] or interacts with SMAD1 to control BMP pathway genes implicated in heart development [55]. CHD7 can

either positively regulate orthodenticle homeobox 2 (Otx2) expression during ear development [70], or repress Otx2

expression during early cerebellar development [52]. The latter results in reduced fibroblast growth factor 8 (Fgf8)

expression and cerebellar vermis hypoplasia. CHD7 can antagonize retinoic acid signalling in neuronal progenitors,

and retinoic acid pathway inhibition can rescue inner ear defects, implying hyperactive retinoic acid signalling as a cause

of inner ear defects [71]. CHD7 interacts with SRY (sex determining region Y)-box 2 (SOX2) in neural stem cells, and co-

regulates the expression of the genes encoding Jagged 1 (Jag1), GLI family zinc finger 3 (Gli3), and v-myc avian

myelocytomatosis viral oncogene neuroblastoma derived homolog (Mycn) [56], which might explain some overlapping

phenotypes with Alagile, Feingold, and Pallister Hall syndromes, although this has not been proven. Noteworthy, autosomal

dominant de novo mutations in SOX2 result in a phenotype that shares features with CHARGE syndrome such as eye and

outer ear malformations, oesophageal atresia, hearing loss, and hypogonadotropic hypogonadism [72]. CHD7 loss is

associated with p53 pathway hyperactivation [57]. The underlying mechanisms might include both direct effects of CHD7

on p53 gene expression and effects on rDNA transcription, leading to defects in ribosome biogenesis [14]. CHD7 controls

sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3E (sema3e) expression, which

may underlie defects in neural crest cell migration and olfactory development as well as being responsible for the clinical

overlap with Kallmann syndrome [35]. CHARGE and 22q11del syndromes show phenotypic overlap [49]. The presumed

shared developmental pathways have not been identified, but are likely to include shared CHD7 and T-box 1 (TBX1) target

genes. CHARGE and Kabuki syndromes also show significant clinical overlap. The genes mutated in Kabuki syndrome,

encoding lysine (K)-specific methyltransferase 2D (KMT2D) and lysine (K)-specific demethylase 6A (KDM6A), encode

histone modification enzymes that together might control CHD7 recruitment to H3K4me1-marked enhancer regions.

Therefore, CHD7, KMT2D, and KDM6A are expected to regulate the same target genes [73].

Trends in Genetics, October 2015, Vol. 31, No. 10 607

CHD7 and Fibroblast Growth Factor Signalling

The identification of CHD7 mutations in patients with Kallmann (MIM308700; MIM147950) or

22q11del (MIM192430) syndrome led to the hypothesis that a shared pathway might account

for the clinical overlap between these syndromes [48,49]. Given that both Kallmann and

22q11del syndromes have been linked to deregulated fibroblast growth factor (FGF) signalling

[50,51], the interaction between Chd7 and Fgf8 loss-of-function mutations was examined in a

mouse model. While no significant interaction was observed during aortic arch development

+/À

[49], a striking interaction was found in development of the cerebellum [52]. Neither Chd7 nor

+/À +/À +/À

Fgf8 animals exhibited clear cerebellar abnormalities, whereas Chd7 ;Fgf8 animals

presented with cerebellar vermis aplasia. Fgf8 expression was downregulated in the isthmus

organiser (IsO), the embryonic signalling centre that instructs cerebellar development, but

unaffected in other regions of the embryo. This context-specific effect on Fgf8 expression

and signalling appeared to be the result of CHD7 loss affecting Fgf8 expression indirectly,

through increased expression of the orthodenticle homeobox 2 (Otx2) homeobox gene. OTX2 is

a potent repressor of Fgf8 gene expression in the IsO [53], providing a molecular explanation for

the reduced Fgf8 expression in this region [52]. These findings indicate that tissue-specific

effects on TF expression, leading to functionally important alterations in TF networks, could

account for some of the organ-specific defects in CHARGE syndrome. Although directed gene

sequencing in a small cohort of patients with CHARGE syndrome and cerebellar defects did not

reveal any FGF8 mutations, coding or noncoding mutations, or polymorphisms in FGF pathway

or homeobox genes remain strong candidate genetic modifiers of the cerebellar phenotypes in

CHARGE syndrome [54].

CHD7 and BMP Signalling

CHD7 has been shown to directly interact with the BMP signalling mediator SMAD1 [55]. CHD7

recruitment to cardiogenic enhancers with SMAD-binding sites was significantly enhanced by

bone morphogenetic protein (BMP) stimulation. Although not directly tested in this study, these

observations suggest that CHD7 is recruited to certain gene regulatory elements through its

interaction with SMAD1. Defects in heart development in Chd7-deficient embryos correlated

with reduced expression of BMP-regulated cardiogenic genes, such as NK2 homeobox 5

(Nkx2.5), Gata4 and Tbx20. Finally, downregulated gene expression was associated with

reduced levels of H3K4me2 and H3K4me3-modified histones at the Nkx2.5 enhancer and

promoter. It is not known whether these changes in histone modification are caused directly by

CHD7 deficiency or merely indicative of less transcriptional activity as a result of reduced

enhancer activity in CHD7-deficient cells.

This study shows that CHD7 recruitment to developmentally important enhancers may be

regulated by developmental signals. Another study found that CHD7 recruitment to chromatin in

neural stem cells is partly mediated by direct interaction with the TF SRY (sex determining region

Y)-box 2 (SOX2) [56]. Taken together, these observations suggest that the ability of CHD7 to

interact with TFs that are only present or active in certain cell types or under specific conditions

contributes to context-specific CHD7 functions. Mutations in these TFs represent potential

phenotype-specific disease modifiers (Figure 3).

CHD7 and p53

A recent study found that heterozygous expression of a stabilised, transcriptionally inactive

variant of p53 during mouse development resulted in a constellation of phenotypes typical of

CHARGE syndrome [57]. The authors presented further evidence that CHD7 can repress p53

gene expression, and that Chd7-null mouse neural crest cells and fibroblasts from patients with

CHARGE syndrome showed increased p53 signalling. The authors interpreted these findings as

evidence that p53 hyperactivation might underlie some aspects of CHARGE syndrome. To test

À/À +/À À/À

this idea, Chd7 ;p53 embryos were generated and compared with Chd7 embryos at

608 Trends in Genetics, October 2015, Vol. 31, No. 10

À/À

embryonic day (E)10.5, a stage where Chd7 embryos are severely retarded and tissues are Outstanding Questions

degenerating, presumably due to rampant cell death. p53 heterozygosity can partially rescue In addition to CHD7, what other genes

are involved in CHARGE syndrome?

some of these severe phenotypes. Intriguingly, p53 deletion was also sufficient to temporarily

For approximately 5–10% of patients

overcome a delay in the development of Chd1-null embryos [58] and early developmental arrest

with a clinical diagnosis of typical

caused by p53-mediated apoptosis in Chd8-null embryos [59]. p53 reduction can also rescue CHARGE syndrome, the genetic basis

neural tube defects in other genetic contexts [60]. It is also interesting to note that phenotypic is unknown. Do these patients have

mutations in: (i) noncoding regions that

effects of TBX1 deficiency can be rescued by reducing p53 levels [61]. This work raises several

control CHD7 expression; (ii) genes

intriguing questions. Does the ability of p53 reduction to rescue abnormal phenotypes or

that encode proteins that interact with

fi

processes associated with CHD gene de ciency and phenotypes associated with CHARGE CHD7 and/or regulate CHD7 activity;

or (iii) different genes that result in a

syndrome point towards a common, underlying mechanism? Does the ability of p53 heterozy-

similar constellation of developmental

gosity to rescue a developmental phenotype necessarily imply a direct role for p53 in the

phenotypes?

phenotype, or does reducing p53 gene dosage improve embryonic growth and viability in

general, perhaps explaining the growing number of genetic mutations associated with devel- Can ‘modifier’ genes that alter expres-

sivity or penetrance of specific pheno-

opmental retardation that respond to p53 dosage reduction? Can mutations that hyperactivate

types be identified in patients?

the p53 pathway [e.g., in Mouse double minute 2 homolog (MDM2)] modify phenotypic

penetrance or expressivity in CHARGE syndrome?

Are different organ defects in CHARGE

syndrome caused by shared mecha-

nisms? Is the hypothesis that CHD7

It is not yet known whether reducing p53 activity will be able to rescue specific CHARGE

controls central developmental path-

syndrome phenotypes in an appropriate Chd7 heterozygous mouse model. If this proves to be

ways or processes and that deregula-

possible, pharmacological inhibition of p53 in utero might represent an experimental approach tion of one common pathway is

for preventing certain CHARGE syndrome phenotypes, akin to observations with Treacher responsible for several major develop-

mental phenotypes correct? Perhaps a

Collins syndrome [62]. Of course, near-complete inhibition of p53 during embryogenesis is

model whereby CHD7 has a unique,

unlikely to be a clinically viable approach for preventing CHARGE syndrome phenotypes, given

context-specific function in each cell

the strong association between p53 loss and tumorigenesis. However, a demonstration that type and developmental stage where

p53 inhibition can rescue certain phenotypes in Chd7 heterozygous embryos will significantly it acts, through facilitating or repressing

the activity of enhancer elements, is

advance our understanding of the pathological mechanisms underlying CHARGE syndrome.

more accurate (Figure 2, main text).

In summary, studies in mouse models have suggested that CHD7 functions in a context- How is CHD7 recruited to chromatin?

Are interactions with histone modifica-

dependent manner by interacting with, and affecting the expression or activation of, tissue-

tions or TFs by themselves sufficient or

specific transcriptional regulators and signalling molecules. The application of next-generation

does efficient, stable CHD7 recruitment

sequencing approaches to screen for putative disease-modifying mutations or polymorphisms

and chromatin association require

in these CHD7-interacting pathways is an important next step in understanding the pathophysi- cooperative interactions?

ology of CHARGE syndrome (see Outstanding Questions Box).

What is the primary mechanism of

CHD7 action on the chromatin tem-

Concluding Remarks

plate? Once recruited, does CHD7

Since the first identification of CHD7 mutations in patients with CHARGE syndrome just over 10 alter DNA accessibility by destabilising

DNA–histone interactions leading to

years ago, clinical genetic, animal, and biochemical studies have led to important insights into

‘indiscriminate’ nucleosome remodel-

the causes of CHARGE syndrome phenotypes. These studies have also led to the identification

ling so that the final effect of nucleo-

of novel roles for CHD7 in development and disease. If we want to understand exactly how some remodelling is determined by

CHD7 functions in vivo, new experimental approaches will need to be used. For instance, local DNA context (Figure 2, main text)?

genome-wide analysis of H3K27me3 or H3K36me3 histone modifications might reveal altered

What is the contribution, if any, of

patterns in CHD7-deficient mammalian cells. DNA accessibility assays should be used to

CHD7-interacting proteins on CHD7

identify functionally relevant changes in nucleosome organisation at key regulatory regions in action? For example, to what extent

CHD7-deficient cells. Such experiments will not only be relevant to understanding CHARGE is the interaction of CHD7 with chro-

matin-modifying enzymes or other CRF

syndrome, but will also provide critical insights into how CRFs of the CHD family fine-tune gene

such as PBAF essential for some of its

expression in vivo. Loss-of-function mutations in several CHD genes are associated with functions?

intellectual disability and neurodevelopmental disorders [63–65], and future studies should

reveal whether haploinsufficiency of the different CHD genes disrupts neural development by

similar or distinct mechanisms. Furthermore, all CHD proteins (including CHD7) have been

implicated in cancer and much work is needed to decipher the underlying mechanisms, which

are likely to involve mechanisms of deregulated developmental signalling and genomic insta-

bility (reviewed in [36]).

Trends in Genetics, October 2015, Vol. 31, No. 10 609

Acknowledgments

We wish to thank Mohi Ahmed, Christa de Geus, Clemens Kiecker, Pete Scambler, and our laboratory colleagues for

comments on the manuscript, and Mohi Ahmed for assistance with production of the figures. Our research is supported by

the Medical Research Council (MR/K022377/1).

Supplemental Information ihttp://www.CHD7.org

References

1. Boycott, K.M. et al. (2013) Rare-disease genetics in the era of developmental anomalies to human methimazole embryopathy.

next-generation sequencing: discovery to translation. Nat. Rev. Birth Defects Res. B 98, 222–229

Genet. 14, 681–691

23. Petty, E. and Pillus, L. (2013) Balancing chromatin remodeling and

2. Vissers, L.E. et al. (2004) Mutations in a new member of the histone modifications in transcription. Trends Genet. 29, 621–629

chromodomain gene family cause CHARGE syndrome. Nat.

24. Skene, P.J. et al. (2014) The nucleosomal barrier to promoter

Genet. 36, 955–957

escape by RNA polymerase II is overcome by the chromatin

3. Klement, K. et al. (2014) Opposing ISWI- and CHD-class chroma- remodeler Chd1. Elife 3, e02042

tin remodeling activities orchestrate heterochromatic DNA repair.

25. Bouazoune, K. and Kingston, R.E. (2012) Chromatin remodeling

J. Cell Biol. 207, 717–733

by the CHD7 protein is impaired by mutations that cause human

4. Lalani, S.R. et al. (2012) CHARGE syndrome. In GeneReviews developmental disorders. Proc. Natl. Acad. Sci. U.S.A. 109,

(Pagon, R.A. et al., eds), University of Washington 19238–19243

5. Jongmans, M.C. et al. (2006) CHARGE syndrome: the phenotypic 26. Brehm, A. et al. (2000) dMi-2 and ISWI chromatin remodelling

spectrum of mutations in the CHD7 gene. J. Med. Genet. 43, factors have distinct nucleosome binding and mobilization prop-

306–314 erties. EMBO J. 19, 4332–4341

6. Lalani, S.R. et al. (2006) Spectrum of CHD7 mutations in 110 27. Whitehouse, I. et al. (2003) Evidence for DNA translocation by the

individuals with CHARGE syndrome and genotype-phenotype ISWI chromatin-remodeling enzyme. Mol. Cell. Biol. 23, 1935–

correlation. Am. J. Hum. Genet. 78, 303–314 1945

7. Zentner, G.E. et al. (2010) Molecular and phenotypic aspects of 28. McKnight, J.N. et al. (2011) Extranucleosomal DNA binding directs

CHD7 mutation in CHARGE syndrome. Am. J. Med. Genet. 152A, nucleosome sliding by Chd1. Mol. Cell. Biol. 31, 4746–4759

674–686

29. Yang, J.G. et al. (2006) The chromatin-remodeling enzyme ACF is

8. Bergman, J.E. et al. (2011) CHD7 mutations and CHARGE syn- an ATP-dependent DNA length sensor that regulates nucleosome

drome: the clinical implications of an expanding phenotype. J. spacing. Nat. Struct. Mol. Biol. 13, 1078–1083

Med. Genet. 48, 334–342

30. Schnetz, M.P. et al. (2009) Genomic distribution of CHD7 on

9. Blake, K.D. et al. (1998) CHARGE association: an update and chromatin tracks H3K4 methylation patterns. Genome Res. 19,

review for the primary pediatrician. Clin. Pediatr. 37, 159–173 590–601

10. Verloes, A. (2005) Updated diagnostic criteria for CHARGE syn- 31. Schnetz, M.P. et al. (2010) CHD7 targets active gene enhancer

drome: a proposal. Am. J. Med. Genet. 133A, 306–308 elements to modulate ES cell-specific gene expression. PLoS

Genet. 6, e1001023

11. Jongmans, M.C. et al. (2008) Familial CHARGE syndrome and the

CHD7 gene: a recurrent missense mutation, intrafamilial recur- 32. Bajpai, R. et al. (2010) CHD7 cooperates with PBAF to control

rence and variability. Am. J. Med. Genet. 146A, 43–50 multipotent neural crest formation. Nature 463, 958–962

12. Pauli, S. et al. (2009) Proven germline mosaicism in a father of two 33. Feng, W. et al. (2013) The chromatin remodeler CHD7 regulates

children with CHARGE syndrome. Clin. Genet. 75, 473–479 adult neurogenesis via activation of SoxC transcription factors. Cell

Stem Cell 13, 62–72

13. Janssen, N. et al. (2012) Mutation update on the CHD7 gene

involved in CHARGE syndrome. Hum. Mutat. 33, 1149–1160 34. Hauk, G. et al. (2010) The chromodomains of the Chd1 chromatin

remodeler regulate DNA access to the ATPase motor. Mol. Cell

14. Zentner, G.E. et al. (2010) CHD7 functions in the nucleolus as a

39, 711–723

positive regulator of ribosomal RNA biogenesis. Hum. Mol. Genet.

19, 3491–3501 35. Schulz, Y. et al. (2014) CHD7, the gene mutated in CHARGE

syndrome, regulates genes involved in neural crest cell guidance.

15. Bergman, J.E. et al. (2012) A novel classification system to predict

Hum. Genet. 133, 997–1009

the pathogenic effects of CHD7 missense variants in CHARGE

syndrome. Hum. Mutat. 33, 1251–1260 36. Li, W. and Mills, A.A. (2014) Architects of the genome: CHD

dysfunction in cancer, developmental disorders and neurological

16. Bartels, C.F. et al. (2010) Mutations in the CHD7 gene: the experi-

syndromes. Epigenomics 6, 381–395

ence of a commercial laboratory. Genet. Test. Mol. Biomarkers 14,

881–891 37. Srinivasan, S. et al. (2008) Drosophila Kismet regulates histone H3

lysine 27 methylation and early elongation by RNA polymerase II.

17. Lalani, S.R. et al. (2004) SEMA3E mutation in a patient with

PLoS Genet. 4, e1000217

CHARGE syndrome. J. Med. Genet. 41, e94

38. Dorighi, K.M. and Tamkun, J.W. (2013) The trithorax group pro-

18. Gu, C. et al. (2005) Semaphorin 3E and plexin-D1 control vascular

teins Kismet and ASH1 promote H3K36 dimethylation to counter-

pattern independently of neuropilins. Science 307, 265–268

act Polycomb group repression in Drosophila. Development 140,

19. Corsten-Janssen, N. et al. (2013) More clinical overlap between

4182–4192

22q11.2 deletion syndrome and CHARGE syndrome than often

39. Stoller, J.Z. et al. (2010) Ash2l interacts with Tbx1 and is required

anticipated. Mol. Syndromol. 4, 235–245

during early embryogenesis. Exp. Biol. Med. 235, 569–576

20. Moustafa-Hawash, N. et al. (2012) CHARGE syndrome with del(3)

40. Subtil-Rodriguez, A. et al. (2014) The chromatin remodeller CHD8

(p13p21): expanding the genotype. Isr. Med. Assoc. J. 14,

133–134 is required for E2F-dependent transcription activation of S-phase

genes. Nucleic Acids Res. 42, 2185–2196

21. Snijders Blok, C. et al. (2014) Definition of 5q11.2 microdeletion

41. Yates, J.A. et al. (2010) Regulation of HOXA2 gene expression by

syndrome reveals overlap with CHARGE syndrome and 22q11

the ATP-dependent chromatin remodeling enzyme CHD8. FEBS

deletion syndrome phenotypes. Am. J. Med. Genet. 164A,

–

2843–2848 Lett. 584, 689 693

42. Sancho, A. et al. (2015) CHD6 regulates the topological arrange-

22. Komoike, Y. et al. (2013) Potential teratogenicity of methimazole:

ment of the CFTR locus. Hum. Mol. Genet. 24, 2724–2732

exposure of zebrafish embryos to methimazole causes similar

610 Trends in Genetics, October 2015, Vol. 31, No. 10

43. Cotney, J. et al. (2015) The autism-associated chromatin modifier 59. Nishiyama, M. et al. (2009) CHD8 suppresses p53-mediated

CHD8 regulates other autism risk genes during human neuro- apoptosis through histone H1 recruitment during early embryo-

development. Nat. Commun. 6, 6404 genesis. Nat. Cell Biol. 11, 172–182

44. Sugathan, A. et al. (2014) CHD8 regulates neurodevelopmental 60. Pani, L. et al. (2002) Rescue of neural tube defects in Pax-3-deficient

pathways associated with autism spectrum disorder in neural embryos by p53 loss of function: implications for Pax-3- dependent

progenitors. Proc. Natl. Acad. Sci. U.S.A. 111, E4468–E4477 development and tumorigenesis. Genes Dev. 16, 676–680

45. Kolla, V. et al. (2015) The tumor suppressor CHD5 forms a NuRD- 61. Caprio, C. and Baldini, A. (2014) p53 suppression partially rescues

type chromatin remodeling complex. Biochem. J. 468, 345–352 the mutant phenotype in mouse models of DiGeorge syndrome.

Proc. Natl. Acad. Sci. U.S.A. 111, 13385–13390

46. Denslow, S.A. and Wade, P.A. (2007) The human Mi-2/NuRD

complex and gene regulation. Oncogene 26, 5433–5438 62. Jones, N.C. et al. (2008) Prevention of the neurocristopathy

Treacher Collins syndrome through inhibition of p53 function.

47. Egan, C.M. et al. (2013) CHD5 is required for neurogenesis and

Nat. Med. 14, 125–133

has a dual role in facilitating gene expression and polycomb gene

repression. Dev. Cell 26, 223–236 63. Bernier, R. et al. (2014) Disruptive CHD8 mutations define a

subtype of autism early in development. Cell 158, 263–276

48. Marcos, S. et al. (2014) The prevalence of CHD7 missense versus

truncating mutations is higher in patients with Kallmann syndrome 64. O’Roak, B.J. et al. (2014) Recurrent de novo mutations implicate

than in typical CHARGE patients. J. Clin. Endocrinol. Metab. 99, novel genes underlying simplex autism risk. Nat. Commun. 5,

E2138–E2143 5595

49. Randall, V. et al. (2009) Great vessel development requires biallelic 65. Pinto, D. et al. (2014) Convergence of genes and cellular pathways

expression of Chd7 and Tbx1 in pharyngeal ectoderm in mice. J. dysregulated in autism spectrum disorders. Am. J. Hum. Genet.

Clin. Invest. 119, 3301–3310 94, 677–694

50. Miraoui, H. et al. (2013) Mutations in FGF17, IL17RD, DUSP6, 66. Tessarz, P. and Kouzarides, T. (2014) Histone core modifications

SPRY4, and FLRT3 are identified in individuals with congenital hypo- regulating nucleosome structure and dynamics. Nat. Rev. Mol.

gonadotropic hypogonadism. Am. J. Hum. Genet. 92, 725–743 Cell Biol. 15, 703–708

51. Vitelli, F. et al. (2002) A genetic link between Tbx1 and fibroblast 67. Hong, L. et al. (1993) Studies of the DNA binding properties of

growth factor signaling. Development 129, 4605–4611 histone H4 amino terminus. Thermal denaturation studies reveal

that acetylation markedly reduces the binding constant of the H4

52. Yu, T. et al. (2013) Deregulated FGF and homeotic gene expres-

‘tail’ to DNA. J. Biol. Chem. 268, 305–314

sion underlies cerebellar vermis hypoplasia in CHARGE syndrome.

Elife 2, e01305 68. Son, E.Y. and Crabtree, G.R. (2014) The role of BAF (mSWI/SNF)

complexes in mammalian neural development. Am. J. Med.

53. Heimbucher, T. et al. (2007) Gbx2 and Otx2 interact with the

Genet. 166C, 333–349

WD40 domain of Groucho/Tle corepressors. Mol. Cell. Biol. 27,

340–351 69. Jiang, X. et al. (2012) The mutation in Chd7 causes misexpression

of Bmp4 and developmental defects in telencephalic midline. Am.

54. Basson, M.A. (2014) Epistatic interactions between Chd7 and

J. Pathol. 181, 626–641

Fgf8 during cerebellar development: Implications for CHARGE

syndrome. Rare Dis. 2, e28688 70. Hurd, E.A. et al. (2010) The ATP-dependent chromatin remodeling

enzyme CHD7 regulates pro-neural gene expression and neuro-

55. Liu, Y. et al. (2014) CHD7 interacts with BMP R-SMADs to epi-

genesis in the inner ear. Development 137, 3139–3150

genetically regulate cardiogenesis in mice. Hum. Mol. Genet. 23,

2145–2156 71. Micucci, J.A. et al. (2014) CHD7 and retinoic acid signaling coop-

erate to regulate neural stem cell and inner ear development in

56. Engelen, E. et al. (2011) Sox2 cooperates with Chd7 to regulate

mouse models of CHARGE syndrome. Hum. Mol. Genet. 23,

genes that are mutated in human syndromes. Nat. Genet. 43, –

607–611 434 448

72. Numakura, C. et al. (2010) Supernumerary impacted teeth in a

57. Van Nostrand, J.L. et al. (2014) Inappropriate p53 activation during

patient with SOX2 anophthalmia syndrome. Am. J. Med. Genet.

development induces features of CHARGE syndrome. Nature

152A, 2355–2359

514, 228–232

73. Schulz, Y. et al. (2014) CHARGE and Kabuki syndromes: a phe-

58. Guzman-Ayala, M. et al. (2015) Chd1 is essential for the high

notypic and molecular link. Hum. Mol. Genet. 23, 4396–4405

transcriptional output and rapid growth of the mouse epiblast.

Development 142, 118–127

Trends in Genetics, October 2015, Vol. 31, No. 10 611