Smchd1-Dependent and -Independent Pathways Determine Developmental Dynamics of CpG Island Methylation on the Inactive X Chromosome

Gendrel, Anne-Valérie; Apedaile, Anwyn; Coker, Heather; Termanis, Ausma; Zvetkova, Ilona; Godwin, Jonathan; Tang, Y. Amy; Huntley, Derek; Montana, Giovanni; Taylor, Steven L.; Giannoulatou, Eleni; Heard, Edith; Stancheva, Irina; Brockdorff, Neil

doi:10.1016/j.devcel.2012.06.011

Cited by 161 publications

(201 citation statements)

References 70 publications

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“…By conventional light microscopy, Xist RNA appears as a μm-scale "cloud" in interphase cells (e.g., Fig. 1A) (6) and as broad discontinuous bands on mitotic chromosomes (16, 17), with broad overlapping staining of PRC2 and other chromatin epitopes (9,13,18,19). To push the resolving power of light microscopy, two recent studies used structured illumination microscopy (SIM) with a resolving power of ∼100 nm to examine the relationship of Xist RNA to nuclear markers (20,21).…”

mentioning

confidence: 99%

The Xist RNA-PRC2 complex at 20-nm resolution reveals a low Xist stoichiometry and suggests a hit-and-run mechanism in mouse cells

Sunwoo

Lee

2015

Proc. Natl. Acad. Sci. U.S.A.

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X-chromosome inactivation (XCI) is initiated by the long noncoding RNA Xist, which coats the inactive X (Xi) and targets Polycomb repressive complex 2 (PRC2) in cis. Epigenomic analyses have provided significant insight into Xist binding patterns and chromatin organization of the Xi. However, such epigenomic analyses are limited by averaging of population-wide dynamics and do not inform behavior of single cells. Here we view Xist RNA and the Xi at 20-nm resolution using STochastic Optical Reconstruction Microscopy (STORM) in mouse cells. We observe dynamics at the single-cell level not predicted by epigenomic analysis. Only ∼50 hubs of Xist RNA occur on the Xi in the maintenance phase, corresponding to 50-100 Xist molecules per Xi and contrasting with the chromosome-wide "coat" observed by deep sequencing and conventional microscopy. Likewise, only ∼50 hubs PRC2 are observed. PRC2 and Xist foci are not randomly distributed but showed statistically significant spatial association. Knock-off experiments enable visualization of the dynamics of dissociation and relocalization onto the Xi and support a functional tethering of Xist and PRC2. Our analysis reveals that Xist-PRC2 complexes are less numerous than expected and suggests methylation of nucleosomes in a hit-and-run model.super resolution microscopy | Xist RNA | Polycomb | X inactivation | chromosome X -chromosome inactivation (XCI) silences a whole X chromosome in the early mammalian female embryo and is maintained through life to balance gene dosage between sexes (1-4). Silencing requires the long noncoding Xist RNA (5), which is expressed only from the inactive X (Xi) and is known to "coat" the Xi in cis (6). Biochemical and genetic analyses have shown that expression of Xist RNA initiates XCI by targeting Polycomb repressive complex 2 (PRC2) to a nucleation site located at the X-inactivation center (7-9). Upon leaving the nucleation site, the Xist-PRC2 complex spreads together along the X-chromosome to establish silencing by depositing the repressive trimethylated histone H3-lysine 27 (H3K27me3) mark (9-13). Understanding how silencing factors spread in cis has been advanced by employment of next-generation sequencing techniques. For example, CHART-seq (capture hybridization analysis of RNA targets with deep sequencing) and ChIP-seq analyses have provided a first molecular examination of Xist binding patterns (10,14) and localization dynamics of various chromatin epitopes (11, 15) at a population-wide level. Developmental profiling demonstrates that Xist RNA and PRC2 initially localize to broad gene-rich, multimegabase clusters and later spread into intervening gene-poor regions.Although these epigenomic studies have yielded high-resolution views, they have also been limited by the population averaging of millions of input cells, masking the dynamics and potential variations at the level of single cells or single chromosomes. Although conventional light microscopy has provided critical single-cell perspectives over the past five decades, its resolving powe...

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confidence: 99%

The Xist RNA-PRC2 complex at 20-nm resolution reveals a low Xist stoichiometry and suggests a hit-and-run mechanism in mouse cells

Sunwoo

Lee

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…21 In the mouse, Smchd1 has a role in the establishment and maintenance of CpG methylation of a subset of genes on the inactive X-chromosome and in the expression of several autosomal gene clusters that are monoallelicly expressed. [33][34][35][36][37] Female homozygous MommeD1 mice, which completely lack Smchd1 protein, die at midgestation because of a failure in X inactivation. 38 Family Rf385 shows that having two SMCHD1 copies with a missense variant is viable, both in males and females.…”

Section: A a T T A A A G T A A G T A T C T A A T T A A A G T A A G Tmentioning

confidence: 99%

Double SMCHD1 variants in FSHD2: the synergistic effect of two SMCHD1 variants on D4Z4 hypomethylation and disease penetrance in FSHD2

et al. 2015

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Facioscapulohumeral muscular dystrophy (FSHD) predominantly affects the muscles in the face, trunk and upper extremities and is marked by large clinical variability in disease onset and progression. FSHD is associated with partial chromatin relaxation of the D4Z4 repeat array on chromosome 4 and the somatic expression of the D4Z4 encoded DUX4 gene. The most common form, FSHD1, is caused by a contraction of the D4Z4 repeat array on chromosome 4 to a size of 1-10 units. FSHD2, the less common form of FSHD, is most often caused by heterozygous variants in the chromatin modifier SMCHD1, which is involved in the maintenance of D4Z4 methylation. We identified three families in which the proband carries two potentially damaging SMCHD1 variants. We investigated whether these variants were located in cis or in trans and determined their functional consequences by detailed clinical information and D4Z4 methylation studies. In the first family, both variants in trans were shown to act synergistically on D4Z4 hypomethylation and disease penetrance, in the second family both SMCHD1 functionaffecting variants were located in cis while in the third family one of the two variants did not affect function. This study demonstrates that having two SMCHD1 missense variants that affect function is compatible with life in males and females, which is remarkable considering its role in X inactivation in mice. The study also highlights the variability in SMCHD1 variants underlying FSHD2 and the predictive value of D4Z4 methylation analysis in determining the functional consequences of SMCHD1 variants of unknown significance.

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“…11 The SMCHD1 gene product codes for protein that regulates chromatin repression in a wide variety of organisms. 10,12 Given the wider role of SMCHD1 in regulating methylation, it is possible that SMCHD1 could be a modifier in human diseases.…”

Section: Introductionmentioning

confidence: 99%

Identification of two novel SMCHD1 sequence variants in families with FSHD-like muscular dystrophy

Winston¹,

Duerden²,

Mort³

et al. 2014

Eur J Hum Genet

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Facioscapulohumeral muscular dystrophy 1 (FSHD1) is caused by a contraction in the number of D4Z4 repeats on chromosome 4, resulting in relaxation of D4Z4 chromatin causing inappropriate expression of DUX4 in skeletal muscle. Clinical severity is inversely related to the number of repeats. In contrast, FSHD2 patients also have inappropriate expression of DUX4 in skeletal muscle, but due to constitutional mutations in SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1), which cause global hypomethylation and hence general relaxation of chromatin. Thirty patients originally referred for FSHD testing were screened for SMCHD1 mutations. Twenty-nine had 411 D4Z4 repeats. SMCHD1 c.1040 þ 1G4A, a pathogenic splice-site variant, was identified in a FSHD1 family with a borderline number of D4Z4 repeats (10) and a variable phenotype (in which a LMNA1 sequence variant was previously described), and SMCHD1 c.2606 G4T, a putative missense variant (p.Gly869Val) with strong in vitro indications of pathogenicity, was identified in a family with an unusual muscular dystrophy with some FSHD-like features. The two families described here emphasise the genetic complexity of muscular dystrophies. As SMCHD1 has a wider role in global genomic methylation, the possibility exists that it could be involved in other complex undiagnosed muscle disorders. Thus far, only 15 constitutional mutations have been identified in SMCHD1, and these two sequence variants add to the molecular and phenotypic spectrum associated with FSHD.

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Smchd1-Dependent and -Independent Pathways Determine Developmental Dynamics of CpG Island Methylation on the Inactive X Chromosome

Cited by 161 publications

References 70 publications

The Xist RNA-PRC2 complex at 20-nm resolution reveals a low Xist stoichiometry and suggests a hit-and-run mechanism in mouse cells

The Xist RNA-PRC2 complex at 20-nm resolution reveals a low Xist stoichiometry and suggests a hit-and-run mechanism in mouse cells

Double SMCHD1 variants in FSHD2: the synergistic effect of two SMCHD1 variants on D4Z4 hypomethylation and disease penetrance in FSHD2

Identification of two novel SMCHD1 sequence variants in families with FSHD-like muscular dystrophy

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