Disruption of cell division cycle associated 7 (CDCA7) has been linked to aberrant DNA hypomethylation, but the impact of DNA methylation loss on transcription has not been investigated. Here, we show that CDCA7 is critical for maintaining global DNA methylation levels across multiple tissues in vivo. A pathogenic Cdca7 missense variant leads to the formation of large, aberrantly hypomethylated domains overlapping with the B genomic compartment but without affecting the deposition of H3K9 trimethylation (H3K9me3). CDCA7-associated aberrant DNA hypomethylation translated to localized, tissue-specific transcriptional dysregulation that affected large gene clusters. In the brain, we identify CDCA7 as a transcriptional repressor and epigenetic regulator of clustered protocadherin isoform choice. Increased protocadherin isoform expression frequency is accompanied by DNA methylation loss, gain of H3K4 trimethylation (H3K4me3), and increased binding of the transcriptional regulator CCCTC-binding factor (CTCF). Overall, our in vivo work identifies a key role for CDCA7 in safeguarding tissue-specific expression of gene clusters via the DNA methylation pathway.

CZ OPENSCREEN Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Department of Human Genetics Leiden University Medical Center Leiden Netherlands

Laboratory of Cell Differentiation Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Leiden Genome Technology Center Department of Human Genetics Leiden University Medical Center Leiden Netherlands

See more in PubMed

Greenberg M. V. C., Bourc'his D., The diverse roles of DNA methylation in mammalian development and disease. Nat. Rev. Mol. Cell Biol. 20, 590–607 (2019). PubMed

Janssen S. M., Lorincz M. C., Interplay between chromatin marks in development and disease. Nat. Rev. Genet. 23, 137–153 (2022). PubMed

Thijssen P. E., Ito Y., Grillo G., Wang J., Velasco G., Nitta H., Unoki M., Yoshihara M., Suyama M., Sun Y., Lemmers R. J. L. F., de Greef J. C., Gennery A., Picco P., Kloeckener-Gruissem B., Güngör T., Reisli I., Picard C., Kebaili K., Roquelaure B., Iwai T., Kondo I., Kubota T., van Ostaijen-ten Dam M. M., van Tol M. J. D., Weemaes C., Francastel C., van der Maarel S. M., Sasaki H., Mutations in CDCA7 and HELLS cause immunodeficiency-centromeric instability-facial anomalies syndrome. Nat. Commun. 6, 7870 (2015). PubMed PMC

Vukic M., Daxinger L., DNA methylation in disease: Immunodeficiency, centromeric instability, facial anomalies syndrome. Essays Biochem. 63, 773–783 (2019). PubMed PMC

Velasco G., Grillo G., Touleimat N., Ferry L., Ivkovic I., Ribierre F., Deleuze J. F., Chantalat S., Picard C., Francastel C., Comparative methylome analysis of ICF patients identifies heterochromatin loci that require ZBTB24, CDCA7 and HELLS for their methylated state. Hum. Mol. Genet. 27, 2409–2424 (2018). PubMed

Hardikar S., Ying Z., Zeng Y., Zhao H., Liu B., Veland N., McBride K., Cheng X., Chen T., The ZBTB24-CDCA7 axis regulates HELLS enrichment at centromeric satellite repeats to facilitate DNA methylation. Protein Cell 11, 214–218 (2020). PubMed PMC

Unoki M., Funabiki H., Velasco G., Francastel C., Sasaki H., CDCA7 and HELLS mutations undermine nonhomologous end joining in centromeric instability syndrome. J. Clin. Invest. 129, 78–92 (2019). PubMed PMC

Hop P. J., Luijk R., Daxinger L., van Iterson M., Dekkers K. F., Jansen R., BIOS Consortium, Heijmans B. T., ’t Hoen P. A. C., van Meurs J., Jansen R., Franke L., Boomsma D. I., Pool R., van Dongen J., Hottenga J. J., van Greevenbroek M. M. J., Stehouwer C. D. A., van der Kallen C. J. H., Schalkwijk C. G., Wijmenga C., Zhernakova S., Tigchelaar E. F., Slagboom P. E., Beekman M., Deelen J., van Heemst D., Veldink J. H., van den Berg L. H., van Duijn C. M., Isaacs A., Uitterlinden A. G., Jhamai P. M., Verbiest M., Suchiman H. E. D., Verkerk M., van der Breggen R., van Rooij J., Lakenberg N., Mei H., van Iterson M., Zhernakova D. V., van ’t Hof P., Deelen P., ’t Hoen P. A. C., Vermaat M., Luijk R., Bonder M. J., van Dijk F., Arindrarto W., Kielbasa S. M., van Zwet E. W., ’t Hoen P. B., van Meurs J. B. J., ’t Hoen P. A. C., Ikram M. A., van Greevenbroek M. M. J., Boomsma D. I., Slagboom P. E., Veldink J. H., van Zwet E. W., Heijmans B. T., Genome-wide identification of genes regulating DNA methylation using genetic anchors for causal inference. Genome Biol. 21, 220 (2020). PubMed PMC

Jenness C., Giunta S., Müller M. M., Kimura H., Muir T. W., Funabiki H., HELLS and CDCA7 comprise a bipartite nucleosome remodeling complex defective in ICF syndrome. Proc. Natl. Acad. Sci. U.S.A. 115, E876–E885 (2018). PubMed PMC

Blewitt M. E., Vickaryous N. K., Hemley S. J., Ashe A., Bruxner T. J., Preis J. I., Arkell R., Whitelaw E., An N-ethyl-N-nitrosourea screen for genes involved in variegation in the mouse. Proc. Natl. Acad. Sci. U.S.A. 102, 7629–7634 (2005). PubMed PMC

Chong S., Vickaryous N., Ashe A., Zamudio N., Youngson N., Hemley S., Stopka T., Skoultchi A., Matthews J., Scott H. S., de Kretser D., O'Bryan M., Blewitt M., Whitelaw E., Modifiers of epigenetic reprogramming show paternal effects in the mouse. Nat. Genet. 39, 614–622 (2007). PubMed PMC

Daxinger L., Harten S. K., Oey H., Epp T., Isbel L., Huang E., Whitelaw N., Apedaile A., Sorolla A., Yong J., Bharti V., Sutton J., Ashe A., Pang Z., Wallace N., Gerhardt D. J., Blewitt M. E., Jeddeloh J. A., Whitelaw E., An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse. Genome Biol. 14, R96 (2013). PubMed PMC

Youngson N. A., Epp T., Roberts A. R., Daxinger L., Ashe A., Huang E., Lester K. L., Harten S. K., Kay G. F., Cox T., Matthews J. M., Chong S., Whitelaw E., No evidence for cumulative effects in a Dnmt3b hypomorph across multiple generations. Mamm. Genome 24, 206–217 (2013). PubMed

Karimi M., Johansson S., Stach D., Corcoran M., Grandér D., Schalling M., Bakalkin G., Lyko F., Larsson C., Ekström T. J., LUMA (Luminometric Methylation Assay)—A high throughput method to the analysis of genomic DNA methylation. Exp. Cell Res. 312, 1989–1995 (2006). PubMed

Ryba T., Battaglia D., Pope B. D., Hiratani I., Gilbert D. M., Genome-scale analysis of replication timing: From bench to bioinformatics. Nat. Protoc. 6, 870–895 (2011). PubMed PMC

Hiratani I., Ryba T., Itoh M., Yokochi T., Schwaiger M., Chang C. W., Lyou Y., Townes T. M., Schübeler D., Gilbert D. M., Global reorganization of replication domains during embryonic stem cell differentiation. PLoS Biol. 6, e245 (2008). PubMed PMC

Zhou W., Dinh H. Q., Ramjan Z., Weisenberger D. J., Nicolet C. M., Shen H., Laird P. W., Berman B. P., DNA methylation loss in late-replicating domains is linked to mitotic cell division. Nat. Genet. 50, 591–602 (2018). PubMed PMC

Meuleman W., Peric-Hupkes D., Kind J., Beaudry J. B., Pagie L., Kellis M., Reinders M., Wessels L., van Steensel B., Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence. Genome Res. 23, 270–280 (2013). PubMed PMC

Berman B. P., Weisenberger D. J., Aman J. F., Hinoue T., Ramjan Z., Liu Y., Noushmehr H., Lange C. P., van Dijk C., Tollenaar R. A., van den Berg D., Laird P. W., Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat. Genet. 44, 40–46 (2011). PubMed PMC

Lu J. Y., Chang L., Li T., Wang T., Yin Y., Zhan G., Han X., Zhang K., Tao Y., Percharde M., Wang L., Peng Q., Yan P., Zhang H., Bi X., Shao W., Hong Y., Wu Z., Ma R., Wang P., Li W., Zhang J., Chang Z., Hou Y., Zhu B., Ramalho-Santos M., Li P., Xie W., Na J., Sun Y., Shen X., Homotypic clustering of L1 and B1/Alu repeats compartmentalizes the 3D genome. Cell Res. 31, 613–630 (2021). PubMed PMC

Lu J. Y., Shao W., Chang L., Yin Y., Li T., Zhang H., Hong Y., Percharde M., Guo L., Wu Z., Liu L., Liu W., Yan P., Ramalho-Santos M., Sun Y., Shen X., Genomic repeats categorize genes with distinct functions for orchestrated regulation. Cell Rep. 30, 3296–3311.e5 (2020). PubMed PMC

Bader M., Alenina N., Andrade-Navarro M. A., Santos R. A., MAS and its related G protein-coupled receptors, Mrgprs. Pharmacol. Rev. 66, 1080–1105 (2014). PubMed

Dietschi Q., Tuberosa J., Fodoulian L., Boillat M., Kan C., Codourey J., Pauli V., Feinstein P., Carleton A., Rodriguez I., Clustering of vomeronasal receptor genes is required for transcriptional stability but not for choice. Sci. Adv. 8, eabn7450 (2022). PubMed PMC

Drouin M., Saenz J., Chiffoleau E., C-type lectin-like receptors: Head or tail in cell death immunity. Front. Immunol. 11, 251 (2020). PubMed PMC

Anholt R. R., Olfactomedin proteins: Central players in development and disease. Front. Cell Dev. Biol. 2, 6 (2014). PubMed PMC

Nicetto D., Nicetto D., Donahue G., Jain T., Peng T., Sidoli S., Sheng L., Montavon T., Becker J. S., Grindheim J. M., Blahnik K., Garcia B. A., Tan K., Bonasio R., Jenuwein T., Zaret K. S., H3K9me3-heterochromatin loss at protein-coding genes enables developmental lineage specification. Science 363, 294–297 (2019). PubMed PMC

Brinkman A. B., Gu H., Bartels S. J. J., Zhang Y., Matarese F., Simmer F., Marks H., Bock C., Gnirke A., Meissner A., Stunnenberg H. G., Sequential ChIP-bisulfite sequencing enables direct genome-scale investigation of chromatin and DNA methylation cross-talk. Genome Res. 22, 1128–1138 (2012). PubMed PMC

Dunican D. S., Cruickshanks H. A., Suzuki M., Semple C. A., Davey T., Arceci R. J., Greally J., Adams I. R., Meehan R. R., Lsh regulates LTR retrotransposon repression independently of Dnmt3b function. Genome Biol. 14, R146 (2013). PubMed PMC

Reddington J. P., Perricone S. M., Nestor C. E., Reichmann J., Youngson N. A., Suzuki M., Reinhardt D., Dunican D. S., Prendergast J. G., Mjoseng H., Ramsahoye B. H., Whitelaw E., Greally J. M., Adams I. R., Bickmore W. A., Meehan R. R., Redistribution of H3K27me3 upon DNA hypomethylation results in de-repression of Polycomb target genes. Genome Biol. 14, R25 (2013). PubMed PMC

Schroeder D. I., Lott P., Korf I., LaSalle J. M., Large-scale methylation domains mark a functional subset of neuronally expressed genes. Genome Res. 21, 1583–1591 (2011). PubMed PMC

Loo L., Simon J. M., Xing L., McCoy E. S., Niehaus J. K., Guo J., Anton E. S., Zylka M. J., Single-cell transcriptomic analysis of mouse neocortical development. Nat. Commun. 10, 134 (2019). PubMed PMC

Okamoto N., Kohmoto T., Naruto T., Masuda K., Imoto I., Primary microcephaly caused by novel compound heterozygous mutations in ASPM. Hum. Genome Var. 5, 18015 (2018). PubMed PMC

Frosk P., Arts H. H., Philippe J., Gunn C. S., Brown E. L., Chodirker B., Simard L., Majewski J., Fahiminiya S., Russell C., Liu Y. P., FORGE Canada Consortium, Canadian Rare Diseases: Models & Mechanisms Network, Hegele R., Katsanis N., Goerz C., del Bigio M. R., Davis E. E., A truncating mutation in CEP55 is the likely cause of MARCH, a novel syndrome affecting neuronal mitosis. J. Med. Genet. 54, 490–501 (2017). PubMed PMC

Sano K., Tanihara H., Heimark R. L., Obata S., Davidson M., St John T., Taketani S., Suzuki S., Protocadherins: A large family of cadherin-related molecules in central nervous system. EMBO J. 12, 2249–2256 (1993). PubMed PMC

Toyoda S., Kawaguchi M., Kobayashi T., Tarusawa E., Toyama T., Okano M., Oda M., Nakauchi H., Yoshimura Y., Sanbo M., Hirabayashi M., Hirayama T., Hirabayashi T., Yagi T., Developmental epigenetic modification regulates stochastic expression of clustered protocadherin genes, generating single neuron diversity. Neuron 82, 94–108 (2014). PubMed

Esumi S., Kakazu N., Taguchi Y., Hirayama T., Sasaki A., Hirabayashi T., Koide T., Kitsukawa T., Hamada S., Yagi T., Monoallelic yet combinatorial expression of variable exons of the protocadherin-alpha gene cluster in single neurons. Nat. Genet. 37, 171–176 (2005). PubMed

Mountoufaris G., Chen W. V., Hirabayashi Y., O’Keeffe S., Chevee M., Nwakeze C. L., Polleux F., Maniatis T., Multicluster Pcdh diversity is required for mouse olfactory neural circuit assembly. Science 356, 411–414 (2017). PubMed PMC

Chen W. V., Nwakeze C. L., Denny C. A., O’Keeffe S., Rieger M. A., Mountoufaris G., Kirner A., Dougherty J. D., Hen R., Wu Q., Maniatis T., Pcdhαc2 is required for axonal tiling and assembly of serotonergic circuitries in mice. Science 356, 406–411 (2017). PubMed PMC

Katori S., Noguchi-Katori Y., Okayama A., Kawamura Y., Luo W., Sakimura K., Hirabayashi T., Iwasato T., Yagi T., Protocadherin-αC2 is required for diffuse projections of serotonergic axons. Sci. Rep. 7, 15908 (2017). PubMed PMC

Kiefer L., Chiosso A., Langen J., Buckley A., Gaudin S., Rajkumar S. M., Servito G. I. F., Cha E. S., Vijay A., Yeung A., Horta A., Mui M. H., Canzio D., WAPL functions as a rheostat of Protocadherin isoform diversity that controls neural wiring. Science 380, eadf8440 (2023). PubMed

Jiang Y., Loh Y. H. E., Rajarajan P., Hirayama T., Liao W., Kassim B. S., Javidfar B., Hartley B. J., Kleofas L., Park R. B., Labonte B., Ho S. M., Chandrasekaran S., do C., Ramirez B. R., Peter C. J., C W J. T., Safaie B. M., Morishita H., Roussos P., Nestler E. J., Schaefer A., Tycko B., Brennand K. J., Yagi T., Shen L., Akbarian S., The methyltransferase SETDB1 regulates a large neuron-specific topological chromatin domain. Nat. Genet. 49, 1239–1250 (2017). PubMed PMC

Hagelkruys A., Horrer M., Taubenschmid-Stowers J., Kavirayani A., Novatchkova M., Orthofer M., Pai T. P., Cikes D., Zhuk S., Balmaña M., Esk C., Koglgruber R., Moeseneder P., Lazovic J., Zopf L. M., Cronin S. J. F., Elling U., Knoblich J. A., Penninger J. M., The HUSH complex controls brain architecture and protocadherin fidelity. Sci. Adv. 8, eabo7247 (2022). PubMed PMC

Canzio D., Nwakeze C. L., Horta A., Rajkumar S. M., Coffey E. L., Duffy E. E., Duffié R., Monahan K., O’Keeffe S., Simon M. D., Lomvardas S., Maniatis T., Antisense lncRNA transcription mediates DNA demethylation to drive stochastic protocadherin α promoter choice. Cell 177, 639–653.e15 (2019). PubMed PMC

Guo Y., Monahan K., Wu H., Gertz J., Varley K. E., Li W., Myers R. M., Maniatis T., Wu Q., CTCF/cohesin-mediated DNA looping is required for protocadherin α promoter choice. Proc. Natl. Acad. Sci. U.S.A. 109, 21081–21086 (2012). PubMed PMC

Monahan K., Rudnick N. D., Kehayova P. D., Pauli F., Newberry K. M., Myers R. M., Maniatis T., Role of CCCTC binding factor (CTCF) and cohesin in the generation of single-cell diversity of Protocadherin-α gene expression. Proc. Natl. Acad. Sci. U.S.A. 109, 9125–9130 (2012). PubMed PMC

Kehayova P., Monahan K., Chen W., Maniatis T., Regulatory elements required for the activation and repression of the protocadherin-alpha gene cluster. Proc. Natl. Acad. Sci. U.S.A. 108, 17195–17200 (2011). PubMed PMC

Borgel J., Guibert S., Li Y., Chiba H., Schübeler D., Sasaki H., Forné T., Weber M., Targets and dynamics of promoter DNA methylation during early mouse development. Nat. Genet. 42, 1093–1100 (2010). PubMed

Yagi M., Kabata M., Tanaka A., Ukai T., Ohta S., Nakabayashi K., Shimizu M., Hata K., Meissner A., Yamamoto T., Yamada Y., Identification of distinct loci for de novo DNA methylation by DNMT3A and DNMT3B during mammalian development. Nat. Commun. 11, 3199 (2020). PubMed PMC

Hansen R. S., Wijmenga C., Luo P., Stanek A. M., Canfield T. K., Weemaes C. M. R., Gartler S. M., The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc. Natl. Acad. Sci. U.S.A. 96, 14412–14417 (1999). PubMed PMC

Jin B., Tao Q., Peng J., Soo H. M., Wu W., Ying J., Fields C. R., Delmas A. L., Liu X., Qiu J., Robertson K. D., DNA methyltransferase 3B (DNMT3B) mutations in ICF syndrome lead to altered epigenetic modifications and aberrant expression of genes regulating development, neurogenesis and immune function. Hum. Mol. Genet. 17, 690–709 (2008). PubMed

Okano M., Bell D. W., Haber D. A., Li E., DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257 (1999). PubMed

Xu G. L., Bestor T. H., Bourc'his D., Hsieh C. L., Tommerup N., Bugge M., Hulten M., Qu X., Russo J. J., Viegas-Péquignot E., Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 402, 187–191 (1999). PubMed

Dahlet T., Argüeso Lleida A., al Adhami H., Dumas M., Bender A., Ngondo R. P., Tanguy M., Vallet J., Auclair G., Bardet A. F., Weber M., Genome-wide analysis in the mouse embryo reveals the importance of DNA methylation for transcription integrity. Nat. Commun. 11, 3153 (2020). PubMed PMC

Ehrlich M., Sanchez C., Shao C., Nishiyama R., Kehrl J., Kuick R., Kubota T., Hanash S. M., ICF, an immunodeficiency syndrome: DNA methyltransferase 3B involvement, chromosome anomalies, and gene dysregulation. Autoimmunity 41, 253–271 (2008). PubMed PMC

Geiman T. M., Tessarollo L., Anver M. R., Kopp J. B., Ward J. M., Muegge K., Lsh, a SNF2 family member, is required for normal murine development. Biochim. Biophys. Acta 1526, 211–220 (2001). PubMed

Velasco G., Hubé F., Rollin J., Neuillet D., Philippe C., Bouzinba-Segard H., Galvani A., Viegas-Péquignot E., Francastel C., Dnmt3b recruitment through E2F6 transcriptional repressor mediates germ-line gene silencing in murine somatic tissues. Proc. Natl. Acad. Sci. U.S.A. 107, 9281–9286 (2010). PubMed PMC

Sun L. Q., Lee D. W., Zhang Q., Xiao W., Raabe E. H., Meeker A., Miao D., Huso D. L., Arceci R. J., Growth retardation and premature aging phenotypes in mice with disruption of the SNF2-like gene, PASG. Genes Dev. 18, 1035–1046 (2004). PubMed PMC

Eads C. A., Nickel A. E., Laird P. W., Complete genetic suppression of polyp formation and reduction of CpG-island hypermethylation in Apc(Min/+) Dnmt1-hypomorphic Mice. Cancer Res. 62, 1296–1299 (2002). PubMed

Li E., Bestor T. H., Jaenisch R., Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992). PubMed

Sharif J., Muto M., Takebayashi S. I., Suetake I., Iwamatsu A., Endo T. A., Shinga J., Mizutani-Koseki Y., Toyoda T., Okamura K., Tajima S., Mitsuya K., Okano M., Koseki H., The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 450, 908–912 (2007). PubMed

Salhab A., Nordström K., Gasparoni G., Kattler K., Ebert P., Ramirez F., Arrigoni L., Müller F., Polansky J. K., Cadenas C., Hengstler J. G., Lengauer T., Manke T., DEEP Consortium, Walter J., A comprehensive analysis of 195 DNA methylomes reveals shared and cell-specific features of partially methylated domains. Genome Biol. 19, 150 (2018). PubMed PMC

H. Funabiki, I. E. Wassing, Q. Jia, J. D. Luo, T. Carroll, Coevolution of the CDCA7-HELLS ICF-related nucleosome remodeling complex and DNA methyltransferases. bioRxiv 2023.01.30.526367 (2023). 10.1101/2023.01.30.526367. PubMed DOI PMC

Yu W., McIntosh C., Lister R., Zhu I., Han Y., Ren J., Landsman D., Lee E., Briones V., Terashima M., Leighty R., Ecker J. R., Muegge K., Genome-wide DNA methylation patterns in LSH mutant reveals de-repression of repeat elements and redundant epigenetic silencing pathways. Genome Res. 24, 1613–1623 (2014). PubMed PMC

Unoki M., Chromatin remodeling in replication-uncoupled maintenance DNA methylation and chromosome stability: Insights from ICF syndrome studies. Genes Cells 26, 349–359 (2021). PubMed PMC

Ming X., Zhang Z., Zou Z., Lv C., Dong Q., He Q., Yi Y., Li Y., Wang H., Zhu B., Kinetics and mechanisms of mitotic inheritance of DNA methylation and their roles in aging-associated methylome deterioration. Cell Res. 30, 980–996 (2020). PubMed PMC

Han M., Li J., Cao Y., Huang Y., Li W., Zhu H., Zhao Q., Han J. D. J., Wu Q., Li J., Feng J., Wong J., A role for LSH in facilitating DNA methylation by DNMT1 through enhancing UHRF1 chromatin association. Nucleic Acids Res. 48, 12116–12134 (2020). PubMed PMC

Wang S., Zhang C., Hasson D., Desai A., SenBanerjee S., Magnani E., Ukomadu C., Lujambio A., Bernstein E., Sadler K. C., Epigenetic compensation promotes liver regeneration. Dev. Cell 50, 43–56.e6 (2019). PubMed PMC

Isbel L., Prokopuk L., Wu H., Daxinger L., Oey H., Spurling A., Lawther A. J., Hale M. W., Whitelaw E., Wiz binds active promoters and CTCF-binding sites and is required for normal behaviour in the mouse. eLife 5, e15082 (2016). PubMed PMC

Justice M., Carico Z. M., Stefan H. C., Dowen J. M., A WIZ/Cohesin/CTCF complex anchors DNA loops to define gene expression and cell identity. Cell Rep. 31, 107503 (2020). PubMed PMC

Jia Z., Li J., Ge X., Wu Y., Guo Y., Wu Q., Tandem CTCF sites function as insulators to balance spatial chromatin contacts and topological enhancer-promoter selection. Genome Biol. 21, 75 (2020). PubMed PMC

Hirayama T., Tarusawa E., Yoshimura Y., Galjart N., Yagi T., CTCF is required for neural development and stochastic expression of clustered Pcdh genes in neurons. Cell Rep. 2, 345–357 (2012). PubMed

Hark A. T., Schoenherr C. J., Katz D. J., Ingram R. S., Levorse J. M., Tilghman S. M., CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405, 486–489 (2000). PubMed

Bell A. C., Felsenfeld G., Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405, 482–485 (2000). PubMed

Elmer J. L., Hay A. D., Kessler N. J., Bertozzi T. M., Ainscough E. A. C., Ferguson-Smith A. C., Genomic properties of variably methylated retrotransposons in mouse. Mob. DNA 12, 6 (2021). PubMed PMC

Chen K., Hu J., Moore D. L., Liu R., Kessans S. A., Breslin K., Lucet I. S., Keniry A., Leong H. S., Parish C. L., Hilton D. J., Lemmers R. J., van der Maarel S., Czabotar P. E., Dobson R. C., Ritchie M. E., Kay G. F., Murphy J. M., Blewitt M. E., Genome-wide binding and mechanistic analyses of Smchd1-mediated epigenetic regulation. Proc. Natl. Acad. Sci. U.S.A. 112, E3535–E3544 (2015). PubMed PMC

Almenar-Queralt A., Merkurjev D., Kim H. S., Navarro M., Ma Q., Chaves R. S., Allegue C., Driscoll S. P., Chen A. G., Kohlnhofer B., Fong L. K., Woodruff G., Mackintosh C., Bohaciakova D., Hruska-Plochan M., Tadokoro T., Young J. E., el Hajj N., Dittrich M., Marsala M., Goldstein L. S. B., Garcia-Bassets I., Chromatin establishes an immature version of neuronal protocadherin selection during the naive-to-primed conversion of pluripotent stem cells. Nat. Genet. 51, 1691–1701 (2019). PubMed PMC

Canzio D., Maniatis T., The generation of a protocadherin cell-surface recognition code for neural circuit assembly. Curr. Opin. Neurobiol. 59, 213–220 (2019). PubMed PMC

de Greef J. C., Wang J., Balog J., den Dunnen J., Frants R. R., Straasheijm K. R., Aytekin C., van der Burg M., Duprez L., Ferster A., Gennery A. R., Gimelli G., Reisli I., Schuetz C., Schulz A., Smeets D. F. C. M., Sznajer Y., Wijmenga C., van Eggermond M., van Ostaijen-ten Dam M., Lankester A. C., van Tol M., van den Elsen P., Weemaes C. M., van der Maarel S., Mutations in ZBTB24 are associated with immunodeficiency, centromeric instability, and facial anomalies syndrome type 2. Am. J. Hum. Genet. 88, 796–804 (2011). PubMed PMC

Nitta H., Unoki M., Ichiyanagi K., Kosho T., Shigemura T., Takahashi H., Velasco G., Francastel C., Picard C., Kubota T., Sasaki H., Three novel ZBTB24 mutations identified in Japanese and Cape Verdean type 2 ICF syndrome patients. J. Hum. Genet. 58, 455–460 (2013). PubMed

Unoki M., Velasco G., Kori S., Arita K., Daigaku Y., Yeung W. K. A., Fujimoto A., Ohashi H., Kubota T., Miyake K., Sasaki H., Novel compound heterozygous mutations in UHRF1 are associated with atypical immunodeficiency, centromeric instability and facial anomalies syndrome with distinctive genome-wide DNA hypomethylation. Hum. Mol. Genet. 32, 1439–1456 (2023). PubMed

Lefebvre J. L., Kostadinov D., Chen W. V., Maniatis T., Sanes J. R., Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature 488, 517–521 (2012). PubMed PMC

Thu C. A., Chen W. V., Rubinstein R., Chevee M., Wolcott H. N., Felsovalyi K. O., Tapia J. C., Shapiro L., Honig B., Maniatis T., Single-cell identity generated by combinatorial homophilic interactions between α, β, and γ protocadherins. Cell 158, 1045–1059 (2014). PubMed PMC

El Hajj N., Dittrich M., Haaf T., Epigenetic dysregulation of protocadherins in human disease. Semin. Cell Dev. Biol. 69, 172–182 (2017). PubMed

Jia Z., Wu Q., Clustered protocadherins emerge as novel susceptibility loci for mental disorders. Front. Neurosci. 14, 587819 (2020). PubMed PMC

Pilsner J. R., Lazarus A. L., Nam D.-H., Letcher R. J., Sonne C., Dietz R., Basu N., Mercury-associated DNA hypomethylation in polar bear brains via the Luminometric Methylation Assay: A sensitive method to study epigenetics in wildlife. Mol. Ecol. 19, 307–314 (2010). PubMed

Bock C., Reither S., Mikeska T., Paulsen M., Walter J., Lengauer T., BiQ Analyzer: Visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 21, 4067–4068 (2005). PubMed

Kumaki Y., Oda M., Okano M., QUMA: Quantification tool for methylation analysis. Nucleic Acids Res. 36, W170–W175 (2008). PubMed PMC

Krishnaswami S. R., Grindberg R. V., Novotny M., Venepally P., Lacar B., Bhutani K., Linker S. B., Pham S., Erwin J. A., Miller J. A., Hodge R., McCarthy J. K., Kelder M., McCorrison J., Aevermann B. D., Fuertes F. D., Scheuermann R. H., Lee J., Lein E. S., Schork N., McConnell M. J., Gage F. H., Lasken R. S., Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nat. Protoc. 11, 499–524 (2016). PubMed PMC

Brind'Amour J., Liu S., Hudson M., Chen C., Karimi M. M., Lorincz M. C., An ultra-low-input native ChIP-seq protocol for genome-wide profiling of rare cell populations. Nat. Commun. 6, 6033 (2015). PubMed

Krueger F., Andrews S. R., Bismark: A flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011). PubMed PMC

Akalin A., Kormaksson M., Li S., Garrett-Bakelman F. E., Figueroa M. E., Melnick A., Mason C. E., methylKit: A comprehensive R package for the analysis of genome-wide DNA methylation profiles. Genome Biol. 13, R87 (2012). PubMed PMC

Yu G., Wang L. G., He Q. Y., ChIPseeker: An R/Bioconductor package for ChIP peak annotation, comparison and visualization. Bioinformatics 31, 2382–2383 (2015). PubMed

Quinlan A. R., Hall I. M., BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010). PubMed PMC

Langmead B., Salzberg S. L., Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012). PubMed PMC

Zhang Y., Liu T., Meyer C. A., Eeckhoute J., Johnson D. S., Bernstein B. E., Nusbaum C., Myers R. M., Brown M., Li W., Liu X. S., Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008). PubMed PMC

Xu S., Grullon S., Ge K., Peng W., Spatial clustering for identification of ChIP-enriched regions (SICER) to map regions of histone methylation patterns in embryonic stem cells. Methods Mol. Biol. 1150, 97–111 (2014). PubMed PMC

Ramírez F., Ryan D. P., Grüning B., Bhardwaj V., Kilpert F., Richter A. S., Heyne S., Dündar F., Manke T., deepTools2: A next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 44, W160–W165 (2016). PubMed PMC

Dobin A., Davis C. A., Schlesinger F., Drenkow J., Zaleski C., Jha S., Batut P., Chaisson M., Gingeras T. R., STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013). PubMed PMC

Broad Institute, Picard Tools. GitHub repository (v2.17); http://broadinstitute.github.io/picard (n.d.).

Anders S., Pyl P. T., Huber W., HTSeq—A Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015). PubMed PMC

Love M. I., Huber W., Anders S., Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014). PubMed PMC

Kernfeld E. M., Genga R. M. J., Neherin K., Magaletta M. E., Xu P., Maehr R., A single-cell transcriptomic atlas of thymus organogenesis resolves cell types and developmental maturation. Immunity 48, 1258–1270.e6 (2018). PubMed PMC

Stuart T., Butler A., Hoffman P., Hafemeister C., Papalexi E., Mauck W. M. III, Hao Y., Stoeckius M., Smibert P., Satija R., Comprehensive integration of single-cell data. Cell 177, 1888–1902.e21 (2019). PubMed PMC

T. Stuart, W. W. Kretzschmar, sinto. GitHub (v0.7.5); https://timoast.github.io/sinto/ (n.d.).

Shen Y., Yue F., McCleary D. F., Ye Z., Edsall L., Kuan S., Wagner U., Dixon J., Lee L., Lobanenkov V. V., Ren B., A map of the cis-regulatory sequences in the mouse genome. Nature 488, 116–120 (2012). PubMed PMC

Liu T., Ortiz J. A., Taing L., Meyer C. A., Lee B., Zhang Y., Shin H., Wong S. S., Ma J., Lei Y., Pape U. J., Poidinger M., Chen Y., Yeung K., Brown M., Turpaz Y., Liu X. S., Cistrome: An integrative platform for transcriptional regulation studies. Genome Biol. 12, R83 (2011). PubMed PMC

W. Zhou, H. Q. Dinh, Z. Ramjan, D. J. Weisenberger, C. M. Nicolet, H. Shen, P. W. Laird, B. P. Berman, TCGA WGBS, PMD and Solo-WCGW CpGs. GitHub; https://zwdzwd.github.io/pmd [accessed 14 April 2023].

Zhou-lab, ImprintingAnno. GitHub; https://github.com/zhou-lab/ImprintingAnno [accessed 8 June 2023].