De Novo Mutation in RING1 with Epigenetic Effects on Neurodevelopment

De novo mutation in RING1 with epigenetic effects on neurodevelopment

Sarah B. Piercea, Mikaela D. Stewartb, Suleyman Gulsunera, Tom Walsha, Abhinav Dhallc, Jon M. McClelland, Rachel E. Klevitb, and Mary-Claire Kinga,e,1

aDepartment of Medicine, University of Washington, Seattle, WA 98195; bDepartment of Biochemistry, University of Washington, Seattle, WA 98195; cDepartment of Chemistry, University of Washington, Seattle, WA 98195; dDepartment of Psychiatry, University of Washington, Seattle, WA 98195; and eDepartment of Genome Sciences, University of Washington, Seattle, WA 98195

Contributed by Mary-Claire King, January 6, 2018 (sent for review December 7, 2017; reviewed by H. Robert Horvitz and Matthew State) RING1 is an E3-ubiquitin ligase that is involved in epigenetic control of Results transcription during development. It is a component of the polycomb Clinical Characterization. The patient was born at 40 wk gestation repressive complex 1, and its role in that complex is to ubiquitylate with mild intrauterine growth retardation and microcephaly. Her histone H2A. In a 13-year-old girl with syndromic neurodevelopmental early motor and language development were normal, but after disabilities, we identified a de novo mutation, RING1 p.R95Q, which the first year of life, she showed delayed acquisition of additional alters a conserved arginine residue in the catalytic RING domain. In language and cognitive skills and delayed adaptive social skills. vitro assays demonstrated that the mutant RING1 retains capacity to At age 13 y, she developed a skin vasculitis and mild scoliosis. catalyze ubiquitin chain formation, but is defective in its ability to Psychotic symptoms also began at approximately age 13, in- ubiquitylate histone H2A in nucleosomes. Consistent with this in vitro cluding hearing multiple voices, belief she was being poisoned, effect, cells of the patient showed decreased monoubiquitylation of and confused disorganized thinking. Cognitive testing showed a histone H2A. We modeled the mutant RING1 in Caenorhabditis ele- verbal IQ of 55 and a visual performance IQ of 63. Electroen- gans by editing the comparable amino acid change into spat-3, cephalography, brain magnetic resonance imaging, and brain the suggested RING1 ortholog. Animals with either the missense computed tomography scans were normal. Chromosome analysis

mutation or complete knockout of spat-3 were defective in mono- at the 550-band level was normal. Additional details of the pa- GENETICS ubiquitylation of histone H2A and had defects in neuronal migration tient’s clinical profile are provided in SI Appendix. and axon guidance. Relevant to our patient, animals heterozygous for either the missense or knockout allele also showed neuronal Identification of a de Novo Missense Mutation in RING1. Genomic defects. Our results support three conclusions: mutation of RING1 DNA extracted from peripheral blood from the patient and isthelikelycauseofahumanneurodevelopmental syndrome, her parents was evaluated by whole exome sequencing. Vari- mutation of RING1 can disrupt histone H2A ubiquitylation with- ants were filtered to identify homozygous, compound heterozy- out disrupting RING1 catalytic activity, and the comparable mu- gous, or de novo alleles potentially damaging to gene function. tation in C. elegans spat-3 both recapitulates the effects on The only candidate allele fulfilling these criteria was RING1 histone H2A ubiquitylation and leads to neurodevelopmental c.284G > A, p.R95Q (NM_002931.3) at chr6:33,177,736 (hg19). abnormalities. This role for RING1 adds to our understanding of The mutation was heterozygous in the patient and absent from the importance of aberrant epigenetic effects as causes of human both parents, both by exome sequencing and by Sanger se- neurodevelopmental disorders. quencing (Fig. 1A); hence, de novo in the patient. The mutation was not present in >120,000 exomes in the reference public RING1 | histone H2A | psychosis | ubiquitin | CRISPR/Cas9 database gnomAD (5), nor had we ever encountered it in any other exome or whole genome sequences in our laboratory. The evelopment of the mammalian nervous system requires Dprecise temporal and spatial control of gene expression. Epi- Significance genetic mechanisms, including histone modifications and chromatin remodeling, maintain chromatin in active, repressed, or poised As part of the polycomb repressive complex 1, RING1 contributes to states and cooperate with transcriptional regulatory mechanisms to epigenetic regulation of gene expression during development. We control gene expression during development. Mutations that disrupt identified a de novo mutation in RING1 in a patient with neuro- the function of genes involved in these processes are increasingly logical, skeletal, and immunological abnormalities. The mutation being identified as the causes of neurodevelopmental disorders in disrupts the ability of RING1 to ubiquitylate histone H2A in nucle- humans (1). osomes, without disrupting its ability to catalyze ubiquitin chain Among the most critical epigenetic regulators are polycomb formation. The comparable mutation in Caenorhabditis elegans group proteins, which combine in polycomb repressive com- causes defects in neuronal development that are as severe as de- plexes (PRCs) to initiate and maintain transcriptional repres- fects caused by a complete loss of gene function. These results identify RING1 as critical to neurodevelopment via its involvement sion during normal development (2). Loss of PRC1 function in in chromatin modification and epigenetic transcriptional regulation. either vertebrates or invertebrates leads to axial patterning defects. Mutations in the genes encoding PRC1 subunits PHC1 Author contributions: S.B.P., M.D.S., S.G., T.W., J.M.M., R.E.K., and M.-C.K. designed re- (polyhomeotic homolog 1) and PCGF2 (polycomb group ring search; S.B.P., M.D.S., S.G., T.W., A.D., J.M.M., R.E.K., and M.-C.K. performed research; A.D. finger 2) lead to human neurodevelopmental disorders char- contributed new reagents/analytic tools; S.B.P., M.D.S., S.G., T.W., A.D., J.M.M., R.E.K., and M.-C.K. analyzed data; and S.B.P., M.D.S., J.M.M., R.E.K., and M.-C.K. wrote the paper. acterized by microcephaly, learning disabilities, and dysmor- Reviewers: H.R.H., Massachusetts Institute of Technology; and M.S., University of California, phic features (3, 4). PRC1 complexes each include one of the San Francisco. E3 ubiquitin ligases RING1 or RING2, which catalyze mono- The authors declare no conflict of interest. ubiquitylation of histone H2A. In this report, we examine the Published under the PNAS license. consequences for transcriptional repression in neurodevelopment 1To whom correspondence should be addressed. Email: [email protected]. of a damaging mutation in RING1, detected in a 13-year-old girl This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with neuropsychiatric and other developmental abnormalities. 1073/pnas.1721290115/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1721290115 PNAS Latest Articles | 1of6 Downloaded by guest on September 23, 2021 A S K R S L S K R S L S K R/Q S L

Father Mother Patient RING1: c.284G>A (p.R95Q) B Species Protein Residues ...... H. sapiens RING1 48-101 C P I C L D M L K N T M T T K E C L H R F C S D C I V T A - L R S G N K E C P T C R K K L V S K R SLRPDP H. sapiens RING2 51-104 C P I C L D M L K N T M T T K E C L H R F C A D C I I T A - L R S G N K E C P T C R K K L V S K R SLRPDP G. galus RING2 51-104 C P I C L D M L K N T M T T K E C L H R F C A D C I I T A - L R S G N K E C P T C R K K L V S K R SLRPDP D. rerio RING2 53-106 C P I C L D M L K N T M T T K E C L H R F C A D C I I T A - L R S G N K E C P T C R K K L V S K R SLRPDP D. melanogaster ESC 46-99 C P I C L D M L K K T M T T K E C L H R F C S D C I V T A - L R S G N K E C P T C R K K L V S K R SLRADP C. elegans SPAT-3 162-215 C D V C Q E L I Q G S I M T K K C G H R F C D Q C I L V A F M R S G N T - C P T C R Q N L G S K R ELQQDP

Fig. 1. Identification of a de novo RING1 missense mutation. (A) Sanger sequence traces indicate that RING1 c.284G > A (p.R95Q) is heterozygous in the patient and absent from the father and mother. (B) Protein sequence alignments of the RING domains of RING1 orthologs. The altered arginine residue is indicated in red. Conserved zinc-coordinating residues are highlighted in yellow. Residues that contact the nucleosome in the RING2 3D structure (PDB ID code 4R8P) are indicated by dots above the alignment.

mutation was predicted by in silico tools to be damaging to Fig. S1), indicating that R95Q does not disrupt the ability of RING1 protein function (Polyphen-2 score 0.999; SIFT P = RING1tobindtooractivatetheE2∼ubiquitin conjugate. We 0.00). RING1 (also known as RING1A) is highly intolerant to conclude that regardless of the PCGF partner, RING1 p.R95Q truncating mutations (pLI = 0.92) and to missense mutations perturbs ubiquitylation of nucleosomal histone H2A in vitro without (Z = 3.42) (5). RING1 encodes an E3 ubiquitin ligase that acts disrupting RING1 catalytic activity. That is, the H2A ubiquitylation in PRC1 complexes to monoubiquitylate histone H2A (6). The and catalytic activities of RING domains can be separated and patient’s mutation alters an arginine residue in the catalytic mutation may affect one function but not the other. RING domain that is conserved from worms through humans Given that RING1 p.R95Q specifically disrupts ubiquitylation (Fig. 1B). of histone H2A in nucleosomes, we hypothesized that the mutation disrupts RING1 interaction with the nucleosomes them- RING1 p.R95Q Disrupts Histone H2A Ubiquitylation in Nucleosomes. selves. RING2 (also known as RING1B or RNF2) is a paralog of RING1 is known to ubiquitylate histone H2A. To determine RING1, for which a structure has been determined in complex whether the patient’s mutation alters this activity, we evaluated with BMI1, nucleosomes, and E2 enzyme (Fig. 2C) (8). The RING1 activity in vitro using recombinant enzymes and purified RING domains of RING1 and RING2 are virtually identical, mononucleosomes. Ubiquitin transfer is accomplished by an including at RING2 positions shown to contact the nucleosome enzymatic cascade in which ubiquitin is first activated by an (Fig. 1B), and are therefore likely to form comparable structures. E1 enzyme, in an ATP-dependent reaction, then transferred to In the RING2/BMI1 complex, RING2 p.R98, homologous to the active site of an E2 enzyme. The E3 enzyme, in this case RING1 p.R95, interacts with the acidic patch on nucleosomes RING1, then binds to an E2∼ubiquitin conjugate and activates it and is distant from the PCGF and E2 interaction interfaces (Fig. to transfer ubiquitin onto a substrate. RING1 must form a het- 2B and SI Appendix, Fig. S2). Mutation of arginine to alanine at erodimer with one of six partners from the polycomb group ring residue 98 in RING2 results in a loss of nucleosome ubiquitylation finger (PCGF) family to display full catalytic activity. For our assay, and binding in vitro (8). Together, these results suggest that argi- RING domains of RING1 and a PCGF were coexpressed and nines at residue 95 of RING1 and at residue 98 of RING2 play copurified and combined with purified, recombinant ubiquitin, E1, similar roles in nucleosome interaction and that substitution of E2, mononucleosomes, and ATP. Formation of ubiquitylated his- glutamine for arginine at RING1 residue 95 is likely responsible for tone H2A was followed by Western blot analysis. When partnered disruption of nucleosome interaction. with either BMI1 (the canonical PCGF) or PCGF1 (the most active PCGF) (7), wild-type RING1 ubiquitylated histone H2A. In con- Ubiquitylated Histone H2A Is Reduced in Cells of the Patient. To trast, RING1 p.R95Q failed to ubiquitylate histone H2A when determine whether RING1 p.R95Q affects histone H2A ubiq- partnered with either BMI1 or PCGF1 (Fig. 2A). uitylation in vivo, we measured the levels of ubiquitylated histone To determine whether the failure of RING1 p.R95Q to ubiq- H2A in lymphoblast cell lines from the patient and from control uitylate nucleosomal histone H2A resulted from a defect in catalytic individuals (Fig. 3A). Ubiquitylated histone H2A was signifi- activity, we compared the ability of wild-type RING1 and RING1 cantly reduced in cells of the patient (P = 0.0002). Similar levels p.R95Q to catalyze the formation of ubiquitin chains. RING1/ of RING1 protein were detected in patient and control cells, PCGF heterodimers were combined with all components needed to suggesting that the reduction in ubiquitylated histone H2A was transfer ubiquitin in the absence of a nucleosome substrate. When due to a change in protein function and not in protein level (Fig. partnered with either BMI1 or PCGF1, both wild-type and mutant 3B). Because changes in the amount of RING2 protein could RING1 catalyzed formation ofpolyubiquitinchains(SI Appendix, potentially compensate for loss of RING1 protein function, we

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1721290115 Pierce et al. Downloaded by guest on September 23, 2021 similar (Fig. 1B). The residue equivalent to R95 of RING1 is R209 of SPAT-3. Using CRISPR/Cas9 gene editing technology, we engineered the SPAT-3(R209Q) mutation, designated as spat-3(mgw14[R209Q]) (SI Appendix,Fig.S3). To characterize the null phenotype of spat-3, we also engineered an allele in which the entire 9,698 bp spat-3 coding region is deleted, designated as spat-3(mgw27[KO]). SPAT-3(R209Q) and SPAT-3 null animals had no obvious morphological or behavioral phenotypes, although adult hermaphrodites appeared somewhat bloated, consistent with an egg-laying defect. SPAT-3 is known to be important for neuronal migration and axon guidance (9). We investigated the effects of SPAT-3(R209Q) and the SPAT-3 null mutation on neuronal migration and axon guidance. During C. elegans embryogenesis, the two HSN neurons, which innervate the egg-laying muscles, migrate from the tail to their final midbody positions near the vulva (Fig. 4A) (10). However, in more than 80% of SPAT-3(R209Q) animals, HSN neurons did not reach the correct midbody position (Fig. 4 B and G). Notably, HSN neurons failed to reach the correct position in a similar fraction of SPAT-3 null animals (Fig. 4 C and G). Furthermore, in more than 20% of animals with either mutation, both HSN neurons completely failed to migrate and remained in the tail. In contrast, in all wild-type animals, at least one HSN migrated to the correct position, with the second HSN failing to fully migrate in only 3% of animals. To investigate whether SPAT-3(R209Q) or SPAT-3 null mutations affected axon guidance, we examined the outgrowth of axons from two different neurons, HSN and PVQ. In wild-type GENETICS animals, axons of the HSN neurons extend ventrally from the cell Fig. 2. RING 1 p.R95Q causes a defect in ubiquitylation of histone H2A bodies near the vulva to the ventral nerve cord and then grow specifically on nucleosomes. (A) In vitro nucleosome ubiquitylation assay of ∼ wild-type RING1 or RING1 p.R95Q heterodimers with BMI1 or PCGF1. All anteriorly to the head (Fig. 4A) (10). In 30% of animals with proteins and nucleosomes were expressed and purified, and protein con- either the SPAT-3(R209Q) or null mutation, the HSN axons centrations in samples were matched using UV absorbance at 280 nm. His- failed to reach the head, whereas in all wild-type animals, the tone H2A was detected by immunoblotting. The RING1 p.R95Q mutant is HSN axons reached the head (Fig. 4 B, C, and H). Defects in deficient at ubiquitylating H2A in nucleosomes. (B) Structure of the RING2/ HSN axon morphology are known to be associated with defects BMI1 complex, showing BMI1 (tan), RING2 (green), and E2 (gold) bound to a in HSN migration and therefore may not reflect a specific effect nucleosome (gray) (PDB ID code 4R8P). The structure highlights the contact on axon outgrowth (11). We therefore also evaluated PVQ between the conserved positive arginine residue (blue spheres) and the nucleosome acidic patch (shown in red). neurons, whose axon outgrowth requires MIG-32, the hetero- dimer partner of SPAT-3 (9). The two PVQ interneurons in the tail extend processes anteriorly to the head in the parallel left also measured levels of RING2 protein. Similar levels of RING2 and right tracts of the ventral nerve cord (Fig. 4D) (12). In all protein were detected in cells from the patient and controls (Fig. 3C). animals, the PVQ cell bodies were properly positioned in the tail. In ∼30% of animals with either the SPAT-3(R209Q) or null Neuronal Migration and Axon Guidance Are Disrupted in a C. elegans mutation, the PVQ axons failed to remain in their parallel tracts Model of RING1 p.R95Q. To investigate the impact of RING1 p.R95Q and exhibited inappropriate crossing of the midline (Fig. 4 E, F, on development, we studied the effect of the mutation on protein and H). In contrast, in only 10% of wild-type animals was in- function in C. elegans. spat-3 has been suggested as the C. elegans appropriate midline crossing of the PVQ axons observed. ortholog of RING1 and RING2 (9). Amino acid sequences of the Our patient is heterozygous for RING1 p.R95Q, so we tested RING domains of SPAT-3 and RING1 are 50% identical and 72% whether animals heterozygous for SPAT-3(R209Q) exhibited

A B C C1 P C2 C3 C1 P C2 C3 293T C1 P C2 C3 293T H2Aub1 RING1 RING2 HPRT H2A HPRT

1.2(#$" 3.0'" 18'&" 16'%" 1.0(" 2.5&#$" levels 14'$" 1 0.8!#'" 2.0&" 12'#" 10'!" 0.6!#&" 1.5%#$" 8&" 0.4!#%" 1.0%" 6%" 4$" 0.2!#$" 0.5!#$" 2#" 0!" !"0 0!" )(" *" )$" )+" Relative H2Aub Relative RING1 levels C1 C1 P C2 C3 C1 P C2 C3 293T Relative RING2 levels P C2 C3 293T

Fig. 3. Reduced histone H2A ubiquitylation in patient-derived lymphoblasts. Whole-cell lysates from lymphoblasts of the patient (P) and three unrelated female controls (C1, C2, C3) and from HEK293T cells were analyzed by immunoblotting. Upper are representative immunoblots; Lower are means ± SD of

three independent experiments. (A) Levels of ubiquitylated histone H2A (H2A-Ub1) relative to H2A. (B) Levels of RING1 relative to HPRT. (C) Levels of RING2 relative to HPRT.

Pierce et al. PNAS Latest Articles | 3of6 Downloaded by guest on September 23, 2021 A D control 100µm 25 µm Ptph-1::GFP vulva HSNs Psra-6::GFP B E

vulva HSNs

100µm 25 µm Ptph-1::GFP Psra-6::GFP C F

vulva

µ µ

SPAT-3(KO) SPAT-3(R209Q) SPAT-3(R209Q) SPAT-3(KO) 100 m 25 m Ptph-1::GFP HSNs Psra-6::GFP G H I

100 50 100 control SPAT-3(R209Q) 80 40 80 SPAT-3(KO) 60 30 60

40 20 40 % of animals 20 10 % of animals 20

0 0 0 full partial no HSN PVQ full partial no

migration migration migration animals with axon defect (%) migration migration migration

Fig. 4. Neuronal phenotypes in C. elegans model of RING1 p.R95Q. Analysis of control, SPAT-3(R209Q), and SPAT-3 knockout (SPAT-3 KO) animals expressing

GFP reporters. (A) Ptph-1::GFP is expressed in HSN neurons of an otherwise wild-type adult animal. An open arrowhead indicates the position of the two HSN cell bodies. Small white arrowheads indicate the HSN axons, which extend in the ventral nerve cord to the head. A large white arrowhead marks the position

of the vulva. (B and C) Ptph-1::GFP is expressed in the HSN neurons of spat-3 mutant animals. HSN cell bodies and axons are indicated as in A.(D) Psra-6::GFP is expressed in the PVQ neurons of an otherwise wild-type L1 larva. (E and F) Psra-6::GFP is expressed in the PVQ neurons of spat-3 mutant animals. Arrows indicate inappropriate midline crossing by the PVQ axons. Anterior is to the left in all images. (G) Control or mutant animals were scored for full, partial, or no migration of the HSN cell body from the tail to the vulva. For each animal, the HSN with least defective migration was scored. (H) Control or mutant animals were scored for HSN or PVQ axon defects. (I) Control or heterozygous mutant animals were scored for full, partial, or no migration of the HSN cell body. For each animal, the HSN cell body with the most defective migration was scored. Values represent mean ± SD for three independent experiments. At least 100 total animals were scored per strain.

neuronal abnormalities. We crossed wild-type hermaphrodites or SPAT-3 knockout animals, the H2Aub1 band was far weaker. homozygous spat-3 mutant hermaphrodites with wild-type males. Levels of unmodified histone H2A were similar in all samples. To identify progeny unambiguously, we crossed to males carrying These results indicate that mutation of the SPAT-3 residue an unrelated fluorescent reporter and scored migration of the comparable to RING1 p.R95 is sufficient to eliminate ubiq- HSN neurons in the heterozygous fluorescent progeny. In more uitylation of the histone H2A C-terminal tail. than 35% of animals heterozygous for either the SPAT-3(R209Q) A previously characterized allele of spat-3, spat-3(gk22),harborsa or the null mutation, at least one HSN neuron failed to migrate to 2,318-bp deletion that is predicted to cause a frame shift and termi- the correct position (Fig. 4I). In contrast, in fewer than 8% of wild- nation (9, 13). The predicted truncated protein produced in type animals did an HSN neuron fail to reach the correct position. spat-3(gk22) animals, if stable, would retain 1,625 amino acids These results indicate that heterozygosity for the mutant allele is of the 2,476-amino acid full-length protein, including the cat- sufficient to cause a neuronal migration defect. alytic RING domain. Animals with the spat-3(gk22) allele have detectable but somewhat lower levels of H2Aub1 compared Ubiquitylated Histone H2A Is Reduced in C. elegans Model of RING1 p.R95Q. The in vitro nucleosome ubiquitylation assay indicated that RING1 p.R95Q disrupts the ability of RING1 to ubiq- WT R209Q KO DEL 1.0(" uitylate histone H2A on nucleosomes. The reduction of ubiq- !#'" H2Aub1 0.8 levels

uitylated H2A in the patient’s cells further suggests that RING1 1 p.R95Q disrupts the ubiquitylation of histone H2A in vivo. To !#&"0.6 further evaluate this effect, we investigated whether in C. ele- !#%"0.4 H2A gans SPAT-3(R209Q) disrupted the ubiquitylation of histone !#$"0.2 H2A. We extracted histones from wild-type and spat-3 mutant !"0 larvae and analyzed the ubiquitylation of histone H2A by Relative H2Aub WT R209Q KO DEL Westernblot.Usinganantibodyspecific for the C-terminal tail of human histone H2A that is monoubiquitylated on lysine 119, Fig. 5. Defective ubiquitylation of histone H2A in C. elegans model of RING1 p.R95Q. (Left) Immunoblot analysis of acid-extracted histones from wild- we detected a band in wild-type animals that is the appropriate type larvae (WT) or larvae carrying the SPAT-3 (R209Q), knockout (KO) or partial

size for ubiquitylated histone H2A (Fig. 5 and SI Appendix,Fig. deletion (DEL) mutations. (Right) Levels of ubiquitylated histone H2A (H2A-Ub1) S4,bandH2Aub1). In contrast, in either SPAT-3(R209Q) or relative to H2A were quantified from two independent experiments.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1721290115 Pierce et al. Downloaded by guest on September 23, 2021 with wild-type animals (Fig. 5 and SI Appendix,Fig.S4). This to ensure adequate precursor proliferation and proper neuronal observation is consistent with a previous report of this allele differentiation, and that tissue-specific requirements stem from (9) and with the suggestion that spat-3(gk22) animals produce temporal and spatial differences in their expression (2, 28). a truncated SPAT-3 protein with reduced stability and/or Studies of schizophrenia, autism, and intellectual disability have reduced ubiquitylation activity. led to identification of critical de novo mutations in genes involved in chromatin remodeling and histone modification (14, 29–31). Of Discussion particular relevance to RING1, de novo mutations in AUTS2 have We suggest that de novo mutation RING1 p.R95Q is the cause of been identified in all these disorders (32). AUTS2 is part of a the neurodevelopmental phenotype of our patient. Our evidence noncanonical PRC1 that activates, rather than represses, transcrip- for this causal relationship is based on genomic analysis and tion during development (33). AUTS2 and RING2 colocalize at functional studies. RING1 p.R95Q was the only candidate allele promoters of neuronal genes in mouse brain. In mouse, brain-specific revealed by genomic analysis of the patient and her parents. This loss of Auts2 leads to developmental phenotypes consistent with analysis included whole exome sequencing (including all coding AUTS2 disruption in humans. Disruption of RING1 may impact sequences, UTRs, and noncoding RNAs genome-wide) and a AUTS2-dependent transcriptional activation, which could contribute genome-wide screen of gene disrupting copy number variants to the patient’s neurodevelopmental anomalies. (CNVs). Genomic analysis yielded no damaging homozygous or Our patient developed mild scoliosis, but the consequences of compound heterozygous genotypes, no damaging CNVs, and only mutation in RING1 and other genes encoding PRC1 subunits are one de novo mutation, RING1 p.R95Q. De novo mutations, both primarily neurological, with minor or no skeletal manifestations point mutations and CNVs, have been established as causes of (3, 4). In contrast, loss-of-function mutations in the corre- neurodevelopmental disorders including autism, developmental sponding mouse genes have prominent skeletal phenotypes (25, delay, and schizophrenia (14–17). No mutations of RING1 have 34, 35). The difference in phenotypes may be explained by the been associated with human disorders by any mode of inheritance, fact that the human RING1, PHC1, and PCGF2 mutations are all and RING1 p.R95Q has never been previously observed. Having missense substitutions, in contrast to the mouse null mutations. thus established this allele as a unique candidate by genetic cri- Alternatively, the degree of functional redundancy among the teria, we then tested the biological link between the patient’s PRC1 paralogs may differ between human and mouse. RING1 genotype and her phenotype with experiments based on Our patient also developed vasculitis, often observed in autoim-

biochemistry and on C. elegans biology. mune or immunodeficiency syndromes (36). PRC1 activity is im- GENETICS The RING1 site that is altered in our patient is conserved as portant for immune system homeostasis. PRC1 proteins regulate arginine in all sequenced species. The defects in neuronal mi- hematopoietic stem cell self-renewal and differentiation (37) as well gration and axon guidance of C. elegans SPAT-3(R209Q) mu- as T-cell proliferation and survival. T cell-specific deletion of Ring1 tants demonstrate that arginine at this site is essential in vivo for and Ring2 converts T cell precursors to B cells (38, 39). Silencing of neuronal cell behavior. Indeed, the effects of SPAT-3(R209Q) PCGF6 enhances dendritic cell activation, suggesting a role for on neuronal migration and axon guidance were equivalent to PRC1 in inflammatory processes (40). The vasculitis phenotype of removing the entire SPAT-3 gene. The neurological phenotypes the patient may reflect dysregulation of the immune system induced and decreased ubiquitylated histone H2A in mutant animals by disruption of RING1 activity. suggest that ubiquitylation of nucleosomes contributes to the Neurodevelopmental disorders are highly heterogeneous both role of PRC1 in neuronal development. phenotypically and genetically. Many critical mutations in genes Identification of RING1 p.R95Q in a patient with a neuro- leading to these disorders, including the RING1 mutation of our developmental disorder provides a glimpse into the role of patient, are de novo. Among the clinical features of our patient RING1 in human neuronal development. The consequences of are those characteristic of intellectual disability and schizo- mutation of RING1 are similar to the consequences of mutations phrenia. RING1 provides an additional example of a gene in- in PHC1, a component of the polycomb repressor complex, and volved in chromatin modification and epigenetic transcriptional in ASXL3, a component of the polycomb-repressive deubiquiti- regulation for which damaging mutations are implicated in nation complex, which also disrupt ubiquitylation of histone H2A neurodevelopmental disorders. and result in neurodevelopmental disorders (3, 18). PRC1 components RING1, RING2, and BMI1 promote self- Materials and Methods renewal and prevent premature differentiation of neural stem – Detailed methods, including genomics, cloning and protein purification, cells (19 21). RING2 and BMI1 are also involved in neuronal ubiquitylation assays, protein structure visualization, preparation and im- subtype specification, and RING2 is involved in the transition munoblotting of human cell extracts, C. elegans strains, CRISPR/Cas9 gene from neuron to astrocyte production (22–24), but the role of editing, microscopy, and histone extraction and analysis are provided in SI RING1 has remained unclear. RING1 and RING2 are catalyti- Appendix. The study was approved by the institutional review boards of the cally equivalent (2) and are similarly intolerant to loss of function University of Washington (protocol 29501) and Seattle Children’s Hospital mutations (5). However, RING1 and RING2 are not fully re- (protocol 11106). All subjects provided written informed consent. dundant during development. Mice lacking Ring1 exhibit skeletal patterning and eye defects, whereas complete loss of Ring2 ACKNOWLEDGMENTS. We thank C. Chatterjee for purified recombinant – mononucleosomes and M. Ailion, I. Topalidou, and M. K. Lee for technical causes embryonic lethality (25 27). RING1 and RING2 may advice. This work was supported by National Institutes of Health Grants carry out specific activities in addition to their shared enzymatic R01MH083989 (to M.-C.K., J.M.M., S.B.P., and T.W.), R01GM088055 (to R.E.K.), activity. It is also possible that their combined activity is critical andT32CA080416(toM.D.S.).

1. Fahrner JA, Bjornsson HT (2014) Mendelian disorders of the epigenetic machinery: 6. Cao R, Tsukada Y, Zhang Y (2005) Role of Bmi-1 and Ring1A in H2A ubiquitylation and Tipping the balance of chromatin states. Annu Rev Genomics Hum Genet 15:269–293. Hox gene silencing. Mol Cell 20:845–854. 2. Chittock EC, Latwiel S, Miller TC, Müller CW (2017) Molecular architecture of poly- 7. Taherbhoy AM, Huang OW, Cochran AG (2015) BMI1-RING1B is an autoinhibited RING comb repressive complexes. Biochem Soc Trans 45:193–205. E3 ubiquitin ligase. Nat Commun 6:7621. 3. Awad S, et al. (2013) Mutation in PHC1 implicates chromatin remodeling in primary 8. McGinty RK, Henrici RC, Tan S (2014) Crystal structure of the PRC1 ubiquitylation microcephaly pathogenesis. Hum Mol Genet 22:2200–2213. module bound to the nucleosome. Nature 514:591–596. 4. Deciphering Developmental Disorders Study (2015) Large-scale discovery of novel 9. Karakuzu O, Wang DP, Cameron S (2009) MIG-32 and SPAT-3A are PRC1 homologs that genetic causes of developmental disorders. Nature 519:223–228. control neuronal migration in Caenorhabditis elegans. Development 136:943–953. 5. Lek M, et al.; Exome Aggregation Consortium (2016) Analysis of protein-coding ge- 10. Desai C, Garriga G, McIntire SL, Horvitz HR (1988) A genetic pathway for the devel- netic variation in 60,706 humans. Nature 536:285–291. opment of the Caenorhabditis elegans HSN motor neurons. Nature 336:638–646.

Pierce et al. PNAS Latest Articles | 5of6 Downloaded by guest on September 23, 2021 11. Garriga G, Desai C, Horvitz HR (1993) Cell interactions control the direction of out- 26. Lorente M, et al. (2006) Homeotic transformations of the axial skeleton of growth, branching and fasciculation of the HSN axons of Caenorhabditis elegans. YY1 mutant mice and genetic interaction with the polycomb group gene Ring1/ Development 117:1071–1087. Ring1A. Mech Dev 123:312–320. 12. White JG, Southgate E, Thomson JN, Brenner S (1986) The structure of the nervous 27. Voncken JW, et al. (2003) Rnf2 (Ring1b) deficiency causes gastrulation arrest and cell system of the nematode Caenorhabditis elegans. Philos Trans R Soc Lond B Biol Sci cycle inhibition. Proc Natl Acad Sci USA 100:2468–2473. 314:1–340. 28. Miller JA, et al. (2014) Transcriptional landscape of the prenatal human brain. Nature 13. Labbé JC, Pacquelet A, Marty T, Gotta M (2006) A genomewide screen for suppressors 508:199–206. of par-2 uncovers potential regulators of PAR protein-dependent cell polarity in 29. McCarthy SE, et al. (2014) De novo mutations in schizophrenia implicate chromatin – Caenorhabditis elegans. Genetics 174:285 295. remodeling and support a genetic overlap with autism and intellectual disability. Mol 14. Gulsuner S, et al.; Consortium on the Genetics of Schizophrenia (COGS); PAARTNERS Psychiatry 19:652–658. Study Group (2013) Spatial and temporal mapping of de novo mutations in schizo- 30. De Rubeis S, et al.; DDD Study; Homozygosity Mapping Collaborative for Autism; – phrenia to a fetal prefrontal cortical network. Cell 154:518 529. UK10K Consortium (2014) Synaptic, transcriptional and chromatin genes disrupted in 15. de Ligt J, et al. (2012) Diagnostic exome sequencing in persons with severe intellectual autism. Nature 515:209–215. disability. N Engl J Med 367:1921–1929. 31. Hamdan FF, et al. (2014) De novo mutations in moderate or severe intellectual dis- 16. Xu B, et al. (2011) Exome sequencing supports a de novo mutational paradigm for ability. PLoS Genet 10:e1004772. schizophrenia. Nat Genet 43:864–868. 32. Oksenberg N, Ahituv N (2013) The role of AUTS2 in neurodevelopment and human 17. O’Roak BJ, et al. (2014) Recurrent de novo mutations implicate novel genes un- evolution. Trends Genet 29:600–608. derlying simplex autism risk. Nat Commun 5:5595. 33. Gao Z, et al. (2014) An AUTS2-polycomb complex activates gene expression in the 18. Bainbridge MN, et al. (2013) De novo truncating mutations in ASXL3 are associated CNS. Nature 516:349–354. with a novel clinical phenotype with similarities to Bohring-Opitz syndrome. Genome 34. Takihara Y, et al. (1997) Targeted disruption of the mouse homologue of the Dro- Med 5:11. sophila polyhomeotic gene leads to altered anteroposterior patterning and neural 19. Molofsky AV, et al. (2003) Bmi-1 dependence distinguishes neural stem cell self- crest defects. Development 124:3673–3682. renewal from progenitor proliferation. Nature 425:962–967. 35. Akasaka T, et al. (1996) A role for mel-18, a polycomb group-related vertebrate gene, 20. Fasano CA, et al. (2007) shRNA knockdown of Bmi-1 reveals a critical role for p21-Rb pathway in NSC self-renewal during development. Cell Stem Cell 1:87–99. during theanteroposterior specification of the axial skeleton. Development 122: – 21. Román-Trufero M, et al. (2009) Maintenance of undifferentiated state and self- 1513 1522. renewal of embryonic neural stem cells by polycomb protein Ring1B. Stem Cells 27: 36. Relan M, Lehman HK (2014) Common dermatologic manifestations of primary im- 1559–1570. mune deficiencies. Curr Allergy Asthma Rep 14:480. 22. Golden MG, Dasen JS (2012) Polycomb repressive complex 1 activities determine the 37. Radulovic V, de Haan G, Klauke K (2013) Polycomb-group proteins in hematopoietic columnar organization of motor neurons. Genes Dev 26:2236–2250. stem cell regulation and hematopoietic neoplasms. Leukemia 27:523–533. 23. Morimoto-Suzki N, et al. (2014) The polycomb component Ring1B regulates the timed 38. Onodera A, Nakayama T (2015) Epigenetics of T cells regulated by polycomb/trithorax termination of subcerebral projection neuron production during mouse neocortical molecules. Trends Mol Med 21:330–340. development. Development 141:4343–4353. 39. Ikawa T, et al. (2016) Conversion of T cells to B cells by inactivation of polycomb- 24. Hirabayashi Y, et al. (2009) Polycomb limits the neurogenic competence of neural mediated epigenetic suppression of the B-lineage program. Genes Dev 30:2475–2485. precursor cells to promote astrogenic fate transition. Neuron 63:600–613. 40. Boukhaled GM, et al. (2016) The transcriptional repressor polycomb group factor 6, 25. del Mar Lorente M, et al. (2000) Loss- and gain-of-function mutations show a poly- PCGF6, negatively regulates dendritic cell activation and promotes quiescence. Cell comb group function for Ring1A in mice. Development 127:5093–5100. Rep 16:1829–1837.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1721290115 Pierce et al. Downloaded by guest on September 23, 2021