WIZ (Widely Interspaced Zinc Finger) is associated with the G9a-GLP protein complex, a key H3K9 methyltransferase suggesting a role in transcriptional repression. However, its role in embryonic development is poorly described. In order to assess the loss of function of WIZ, we generated CRISPR/Cas9 WIZ knockout mouse model with 32 nucleotide deletion. Observing the lethality status, we identified the WIZ knockouts to be subviable during embryonic development and non-viable after birth. Morphology of developing embryo was analyzed at E14.5 and E18.5 and our findings were supported by microCT scans. Wiz KO showed improper development in multiple aspects, specifically in the craniofacial area. In particular, shorter snout, cleft palate, and cleft eyelids were present in mutant embryos. Palatal shelves were hypomorphic and though elevated to a horizontal position on top of the tongue, they failed to make contact and fuse. By comparison of proliferation pattern and histone methylation in developing palatal shelves we brought new evidence of importance WIZ dependent G9a-GLP methylation complex in craniofacial development, especially in palate shelf fusion.

Animal Research Centre Ulm University Ulm Germany

Laboratory of Transgenic Models of Diseases and the Czech Centre of Phenogenomics Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czechia

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Alvizi L., Ke X., Brito L. A., Seselgyte R., Moore G. E., Stanier P., et al. (2017). Differential methylation is associated with non-syndromic cleft lip and palate and contributes to penetrance effects. Sci. Rep. 7:2441. 10.1038/s41598-017-02721-0 PubMed DOI PMC

Anchlia S., Rao K. S., Bonanthaya K., Anupama B., Nayak I. V. (2011). Ophthalmic considerations in cleft lip and palate patients. J. Maxillofac. Oral Surg. 10 14–19. 10.1007/s12663-010-0058-z PubMed DOI PMC

Banck M. S., Li S., Nishio H., Wang C., Beutle A. S., Walsh M. J. (2009). The ZNF217 oncogene is a candidate organizer of repressive histone modifiers. Epigenetics 4 100–106. 10.4161/epi.4.2.7953 PubMed DOI PMC

Bian C., Chen Q., Yu X. (2015). The zinc finger proteins ZNF644 and WIZ regulate the G9a/GLP complex for gene repression. Elife 4:e05606. 10.7554/eLife.05606 Erratum in: Elife 4. 10.7554/eLife.08168 PubMed DOI PMC

Bush J. O., Jiang R. (2012). Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development. Development 139 231–243. 10.1242/dev.067082 Erratum in: Development 139:828 PubMed DOI PMC

Cox J., Hein M. Y., Luber C. A., Paron I., Nagaraj N., Mann M. (2014). Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 13 2513–2526. PubMed PMC

Cox J., Mann M. (2008). MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26 1367–1372. PubMed

Daxinger L., Harten S. K., Oey H., Epp T., Isbel L., Huang E., et al. (2013). An ENU mutagenesis screen identifies novel and known genes involved in epigenetic processes in the mouse. Genome Biol. 14:R96. 10.1186/gb-2013-14-9-r96 PubMed DOI PMC

Dickinson M. E., Flenniken A. M., Ji X., Teboul L., Wong M. D., White J. K., et al. (2016). High-throughput discovery of novel developmental phenotypes. Nature 537 508–514. 10.1038/nature19356 Erratum in: Nature 551:398 PubMed DOI PMC

Du J., Johnson L. M., Jacobsen S. E., Patel D. J. (2015). DNA methylation pathways and their crosstalk with histone methylation. Nat. Rev. Mol. Cell Biol. 16 519–532. 10.1038/nrm4043 PubMed DOI PMC

Enomoto H., Nelson C. M., Somerville R. P., Mielke K., Dixon L. J., Powell K., et al. (2010). Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation. Development 137 4029–4038. 10.1242/dev.050591 PubMed DOI PMC

Gonseth S., Shaw G. M., Roy R., Segal M. R., Asrani K., Rine J., et al. (2019). Epigenomic profiling of newborns with isolated orofacial clefts reveals widespread DNA methylation changes and implicates metastable epiallele regions in disease risk. Epigenetics 14 198–213. 10.1080/15592294.2019.1581591 PubMed DOI PMC

Gritli-Linde A. (2007). Molecular control of secondary palate development. Dev. Biol. 301 309–326. 10.1016/j.ydbio.2006.07.042 PubMed DOI

Gyory I., Wu J., Fejér G., Seto E., Wright K. L. (2004). PRDI-BF1 recruits the histone H3 methyltransferase G9a in transcriptional silencing. Nat. Immunol. 5 299–308. 10.1038/ni1046 PubMed DOI

Hebert A. S., Richards A. L., Bailey D. J., Ulbrich A., Coughlin E. E., Westphall M. S., et al. (2014). The one hour yeast proteome. Mol. Cell. Proteomics 13 339–347. PubMed PMC

Isbel L., Prokopuk L., Wu H., Daxinger L., Oey H., Spurling A., et al. (2016). Wiz binds active promoters and CTCF-binding sites and is required for normal behaviour in the mouse. Elife 5:e15082. 10.7554/eLife.15082 PubMed DOI PMC

Ito Y., Yeo J. Y., Chytil A., Han J., Bringas P., Jr., Nakajima A., et al. (2003). Conditional inactivation of Tgfbr2 in cranial neural crest causes cleft palate and calvaria defects. Development 130 5269–5280. 10.1242/dev.00708 PubMed DOI

Jin J. Z., Li Q., Higashi Y., Darling D. S., Ding J. (2008). Analysis of Zfhx1a mutant mice reveals palatal shelf contact-independent medial edge epithelial differentiation during palate fusion. Cell Tissue Res. 333 29–38. 10.1007/s00441-008-0612-x PubMed DOI PMC

Justice M., Carico Z. M., Stefan H. C., Dowen J. M. (2020). A WIZ/Cohesin/CTCF complex anchors DNA loops to define gene expression and cell identity. Cell Rep. 31:107503. 10.1016/j.celrep.2020.03.067 PubMed DOI PMC

Kim S., Prochazka J., Bush J. O. (2017). Live imaging of mouse secondary palate fusion. J. Vis. Exp. 125:e56041. 10.3791/56041 PubMed DOI PMC

Kuriyama M., Udagawa A., Yoshimoto S., Ichinose M., Sato K., Yamazaki K., et al. (2008). DNA methylation changes during cleft palate formation induced by retinoic acid in mice. Cleft Palate Craniofac. J. 45 545–551. 10.1597/07-134.1 PubMed DOI

Lan Y., Jiang R. (2009). Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth. Development 136 1387–1396. 10.1242/dev.028167 PubMed DOI PMC

Livak K. J., Schmittgen T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25 402–408. 10.1006/meth.2001.1262 PubMed DOI

Nakajima A., Shuler C. F., Gulka A. O. D., Hanai J. I. (2018). TGF-β signaling and the epithelial-mesenchymal transition during palatal fusion. Int. J. Mol. Sci. 19:3638. 10.3390/ijms19113638 PubMed DOI PMC

Okano J., Suzuki S., Shiota K. (2007). Involvement of apoptotic cell death and cell cycle perturbation in retinoic acid-induced cleft palate in mice. Toxicol. Appl. Pharmacol. 221 42–56. 10.1016/j.taap.2007.02.019 PubMed DOI

Rice R., Spencer-Dene B., Connor E. C., Gritli-Linde A., McMahon A. P., Dickson C., et al. (2004). Disruption of Fgf10/Fgfr2b-coordinated epithelial-mesenchymal interactions causes cleft palate. J. Clin. Invest. 113 1692–1700. 10.1172/JCI20384 PubMed DOI PMC

Shaw G. M., Carmichael S. L., Laurent C., Rasmussen S. A. (2006). Maternal nutrient intakes and risk of orofacial clefts. Epidemiology 17 285–291. 10.1097/01.ede.0000208348.30012.35 PubMed DOI

Tachibana M., Matsumura Y., Fukuda M., Kimura H., Shinkai Y. (2008). G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription. EMBO J. 27 2681–2690. 10.1038/emboj.2008.192 PubMed DOI PMC

Tachibana M., Sugimoto K., Nozaki M., Ueda J., Ohta T., Ohki M., et al. (2002). G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev. 16 1779–1791. 10.1101/gad.989402 PubMed DOI PMC

Tachibana M., Ueda J., Fukuda M., Takeda N., Ohta T., Iwanari H., et al. (2005). Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. Genes Dev. 19 815–826. 10.1101/gad.1284005 PubMed DOI PMC

Tang Q., Li L., Lee M. J., Ge Q., Lee J. M., Jung H. S. (2016). Novel insights into a retinoic-acid-induced cleft palate based on Rac1 regulation of the fibronectin arrangement. Cell Tissue Res. 363 713–722. 10.1007/s00441-015-2271-z PubMed DOI

Tyanova S., Temu T., Sinitcyn P., Carlson A., Hein M. Y., Geiger T., et al. (2016). The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13 731–740. PubMed

Ueda J., Tachibana M., Ikura T., Shinkai Y. (2006). Zinc finger protein Wiz links G9a/GLP histone methyltransferases to the co-repressor molecule CtBP. J. Biol. Chem. 281 20120–20128. 10.1074/jbc.M603087200 PubMed DOI

Wilcox A. J., Lie R. T., Solvoll K., Taylor J., McConnaughey D. R., Abyholm F., et al. (2007). Folic acid supplements and risk of facial clefts: national population based case-control study. BMJ 334:464. 10.1136/bmj.39079.618287.0B PubMed DOI PMC

Wilkinson D. G., Nieto M. A. (1993). Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol. 225 361–373. 10.1016/0076-6879(93)25025-w PubMed DOI

Willemsen M. H., Vulto-van Silfhout A. T., Nillesen W. M., Wissink-Lindhout W. M., van Bokhoven H., Philip N., et al. (2012). Update on Kleefstra Syndrome. Mol. Syndromol. 2 202–212. 10.1159/000335648 PubMed DOI PMC

Zhang Z., Song Y., Zhao X., Zhang X., Fermin C., Chen Y. (2002). Rescue of cleft palate in Msx1-deficient mice by transgenic Bmp4 reveals a network of BMP and Shh signaling in the regulation of mammalian palatogenesis. Development 129 4135–4146. PubMed