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. 2019 Oct;213(2):685-703.
doi: 10.1534/genetics.119.302600. Epub 2019 Aug 16.

Role of the Chromosome Architectural Factor SMCHD1 in X-Chromosome Inactivation, Gene Regulation, and Disease in Humans

Chen-Yu Wang  1   2 , Harrison Brand  3   4   5   6 , Natalie D Shaw  7   8 , Michael E Talkowski  3   4   5   6 , Jeannie T Lee  9   2
Affiliations

Role of the Chromosome Architectural Factor SMCHD1 in X-Chromosome Inactivation, Gene Regulation, and Disease in Humans

Chen-Yu Wang et al. Genetics. 2019 Oct.

Abstract

Structural maintenance of chromosomes flexible hinge domain-containing 1 (SMCHD1) is an architectural factor critical for X-chromosome inactivation (XCI) and the repression of select autosomal gene clusters. In mice, homozygous nonsense mutations in Smchd1 cause female-specific embryonic lethality due to an XCI defect. However, although human mutations in SMCHD1 are associated with congenital arhinia and facioscapulohumeral muscular dystrophy type 2 (FSHD2), the diseases do not show a sex-specific bias, despite the essential nature of XCI in humans. To investigate whether there is a dosage imbalance for the sex chromosomes, we here analyze transcriptomic data from arhinia and FSHD2 patient blood and muscle cells. We find that X-linked dosage compensation is maintained in these patients. In mice, SMCHD1 controls not only protocadherin (Pcdh) gene clusters, but also Hox genes critical for craniofacial development. Ablating Smchd1 results in aberrant expression of these genes, coinciding with altered chromatin states and three-dimensional (3D) topological organization. In a subset of FSHD2 and arhinia patients, we also found dysregulation of clustered PCDH, but not HOX genes. Overall, our study demonstrates preservation of XCI in arhinia and FSHD2, and implicates SMCHD1 in the regulation of the 3D organization of select autosomal gene clusters.

Keywords: HOX genes; SMCHD1; X-chromosome inactivation; chromatin; clustered protocadherin genes; epigenetics.

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Figures

Figure 1
Figure 1
Dosage compensation is preserved in female arhinia and FSHD2 patients harboring SMCHD1 mutations. (A) A table listing the cohort of female arhinia and FSHD2 patients, and their associated SMCHD1 mutations included in this study. Arhinia patients harboring SMCHD1 mutations often exhibit normal levels of SMCHD1 protein (Shaw et al. 2017). In contrast, it has been reported that SMCHD1 mutations in FSHD2 patients are usually associated with reduced SMCHD1 protein levels (Lemmers et al. 2012), although the two FSHD2 patients listed here have not been tested for SMCHD1 levels. (B) The domain structure of human SMCHD1 protein with the locations of mutations found in patients listed in (A) overlaid. Green dots, missense mutations. Red dot, nonsense or frameshift mutations. This figure was modified from Shaw et al. (2017). (C) CDPs for fold changes of X-linked and autosomal genes in mouse cells. Note that X–A imbalance was not seen in male Smchd1−/− mouse NPCs, indicating that X–A imbalance in Smchd1−/− females originated from the inactive X chromosome. P-values given by Wilcoxon ranked sum test (unpaired, one-sided). (D) CDPs for fold changes of X-linked and autosomal genes in female arhinia patients vs. female controls. (E) CDPs for fold changes of X-linked and autosomal genes in female FSHD2 patients vs. female controls. CDP, cumulative distribution plot; Chr, chromosome; ES, embryonic stem; FSHD2, facioscapulohumeral muscular dystrophy type 2; ID, identifier; NPC, neural progenitor cell; SMC, structural maintenance of chromosomes; SMCHD1, SMC flexible hinge domain-containing 1; GHKL, Gyras, Hsp90, Histidine Kinase, MutL; WT, wild-type.
Figure 2
Figure 2
Human homologs of class I SMCHD1-sensitive Xi genes resist upregulation in heterozygous female patients. (A) CDPs comparing class I SMCHD1-sensitive genes (class I genes), other X-linked genes, and autosomal genes in mouse cells. P-values: class I genes vs. autosomal genes (black); class I genes vs. other X-linked genes (blue). P-values given by Wilcoxon ranked sum test (unpaired, one-sided). (B) CDPs comparing human homologs of class I SMCHD1-sensitive genes (class I genes), other X-linked genes, and autosomal genes in female arhinia patients vs. female controls. (C) CDPs comparing human homologs of class I SMCHD1-sensitive genes (class I genes), other X-linked genes, and autosomal genes in female FSHD2 patients vs. female controls. CDP, cumulative distribution plot; ES, embryonic stem; FSHD2, facioscapulohumeral muscular dystrophy type 2; NPC, neural progenitor cell; SMCHD1, structural maintenance of chromosomes flexible hinge domain-containing 1; WT, wild-type; Xi, inactive X chromosome.
Figure 3
Figure 3
Inactivation of facultative escapees is sensitive to Smchd1 ablation in mice. (A) Location of facultative escapees relative to various X-linked gene classes. Gray-shaded areas are regions with facultative escapees. (B) Pearson correlation analysis of the occurrence of facultative escapees relative to various X-linked gene classes on the X. (C) Testing the random likelihood of having 41 class I genes as facultative escapees. Random samplings of 126 genes from 355 genes subject to XCI in which 58 are facultative escapees were simulated 10,000 times. A probability density plot was generated for the number of facultative escapees sampled. (D) Representative examples of class I genes that either overlap with or are adjacent to reported escapees. Top track: all reported (constitutive and facultative) escapees (purple bars). Middle track: silent genes (black bars), constitutive escapees (pink bars), class I genes (red bars), unclassified genes (gray bars), and class III genes (blue bars). Bottom track: RefSeq genes. (E) Nearest neighbor analysis: box plots showing distribution of distances of class I and class III genes from each other, and to escapees. e.g., the left panel shows the distance distribution from a class I gene to the nearest escapee (pink), class I (red), or class III (blue) gene. For this analysis, the 41 facultative escapees are removed from the class I list (class I*). P-values were determined by Wilcoxon ranked sum test. NS, not significant (P > 0.05). a, P = 3.303 × 10−4. b, P = 3.221 × 10−6. c, P = 6.14 × 10−12. d, P = 8.556 × 10−15. XCI, X-chromosome inactivation.
Figure 4
Figure 4
Behavior of facultative escapees in female arhinia and FSHD2 patients. (A) CDPs comparing facultative escapees, genes subject to XCI, and autosomal genes in mouse cells. P-values: facultative escapees vs. autosomal genes (black); facultative escapees vs. genes subject to XCI (blue). P-values given by Wilcoxon ranked sum test (unpaired, one-sided). (B) CDPs comparing facultative escapees, genes subject to XCI, and autosomal genes in female arhinia patients vs. female controls. (C) CDPs comparing facultative escapees, genes subject to XCI, and autosomal genes in female FSHD2 patients vs. female controls. CDP, cumulative distribution plot; ES, embryonic stem; FSHD2, facioscapulohumeral muscular dystrophy type 2; NPC, neural progenitor cell; WT, wild-type; XCI, X-chromosome inactivation.
Figure 5
Figure 5
Smchd1 ablation alters the expression, chromatin states, and three-dimensional organization of the Pcdha gene cluster in mice. (A) RNA-seq, H3K4me3, H3K27me3, CTCF, and RAD21 ChIP-seq (GSE99991) tracks at the Pcdha cluster in WT (clone1) and Smchd1−/− (clone1) female mouse NPCs, with scales indicated in each track. +, the plus strand. −, the minus strand. (B) Dot plots showing the FPKMs of the 14 Pcdha and 22 Pcdhb genes in WT (n = 3) and Smcdh1−/− (n = 3) mouse NPC clones. (C) Hi-C contact maps at 10-kb resolution at the Pcdhα cluster in WT (clone1, bottom) and Smchd1−/− (clone1, top) female mouse NPCs (GSE99991). Also shown are CTCF ChIP-seq tracks in WT (black) and Smchd1−/− (red) female mouse NPCs. ChIP-seq, chromatin immunoprecipitation-sequencing; Chr, chromosome; CTCF, CCCTC-binding factor; FPKM, fragments per kilobase of transcript per million mapped reads; NPC, neural progenitor cell; RNA-seq, RNA-sequencing; WT, wild-type.
Figure 6
Figure 6
Dysregulation of the PCDHA gene cluster in arhinia and FSHD2 patients. (A) Dot plots showing the FPKMs of the 15 PCDHA genes in LCLs of 10 arhinia patients (red) and 10 controls (black). See Table S2 for the results of DESeq analyses, which similarly showed upregulation of PCDHA genes in patient Y1 and B1. (B) Strand-resolved RNA-seq coverage tracks at the PCDHA gene cluster of three arhinia patients (B1,Y1, and A1) and one control (D2). +, the plus strand. −, the minus strand. (C) Dot plots showing the FPKMs of the 14 PCDHA genes in muscle biopsies of nine FSHD1 patients (blue), four FSHD2 patients carrying SMCHD1 mutations (red), two FSHD2 patients without SMCHD1 mutations (green), and eight controls (black). See Table S2 for the results of DESeq analyses, which similarly showed upregulation of PCDHA genes in patient F14. (D) RNA-seq coverage tracks at the PCDHA gene cluster of one FSHD2 patients (F14) and one control (C7). +, the plus strand. −, the minus strand. FPKM, fragments per kilobase of transcript per million mapped reads; FSHD2, facioscapulohumeral muscular dystrophy type 2; LCL; lymphoblastoid cell line; RNA-seq, RNA-sequencing; SMCHD1, structural maintenance of chromosomes flexible hinge domain-containing 1.
Figure 7
Figure 7
Smchd1 ablation alters the expression, chromatin states, and three-dimensional organization of the HoxB gene cluster in mice. (A) Bar plots showing the FPKMs of all four clusters of Hox genes in WT (n = 3) and Smcdh1−/− (n = 3) mouse NPC clones. (B) RNA-seq, H3K4me3, H3K27me3, EZH2, CTCF, and RAD21 ChIP-seq (GSE99991) tracks at the HoxB cluster in WT (clone1) and Smchd1−/− (clone1) female mouse NPCs, with scales indicated in each track. +, the plus strand. −, the minus strand. (C) Hi-C contact maps at 25-kb resolution at the HoxB cluster in WT (clone1, bottom) and Smchd1−/− (clone1, top) female mouse NPCs (GSE99991). Also shown are H3K4me3 and H3K27me3 ChIP-seq tracks in WT and Smchd1−/− female mouse NPCs. (D) Differential Hi-C contact maps generated by dividing the WT with the Smchd1−/− contact map at 25-kb resolution at the HoxB cluster (GSE99991). ChIP-seq, chromatin immunoprecipitation-sequencing; CTCF, CCCTC-binding factor; FPKM, fragments per kilobase of transcript per million mapped reads; NPC, neural progenitor cell; RNA-seq, RNA-sequencing; WT, wild-type.
Figure 8
Figure 8
HOX genes are not dysregulated in LCLs of arhinia patients and muscle biopsies of FSHD2 patients. (A) Dot plots showing the FPKMs of the four clusters of HOX genes in LCLs of 10 arhinia patients (red) and 10 controls (black). (B) Dot plots showing the FPKMs of the four clusters of HOX genes in muscle biopsies of nine FSHD1 patients (blue), four FSHD2 patients carrying SMCHD1 mutations (red), two FSHD2 patients without SMCHD1 mutations (green), and eight controls (black).(C) A table listing other homeobox genes upregulated in Smchd1−/− mouse NPCs. (D) The top 20 list of the GO analysis of the 230 upregulated genes in Smchd1−/− mouse NPCs. GO terms that were not enriched with statistical significance were labeled with lighter colors. FPKM, fragments per kilobase of transcript per million mapped reads; FSHD2, facioscapulohumeral muscular dystrophy type 2; GO, gene ontology; ID, identifier; LCL; lymphoblastoid cell line; NPC, neural progenitor cell; SMCHD1, structural maintenance of chromosomes flexible hinge domain-containing 1.
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