br Experimental Procedures br Author Contributions br Acknow

Experimental Procedures
Author Contributions
Acknowledgments We thank Andy Riddell and Nigel Miller for flow cytometry and FACS support and Peter Humphreys for imaging support. We also thank Martin Leeb for creating and validating the TmKO2 reporter and Masaki Kinoshita for targeting and validated the RGd2/APB1 cell line. The APB1 construct was a kind gift from Toshihiro Kobayashi and Azim Surani. We thank Jennifer Nichols, Kevin Chalut, Graziano Martello, and Harry Leitch for discussions and comments on the manuscript. This research was funded by the Wellcome Trust (091484/Z/10/Z). The Cambridge Stem Cell Institute receives core support from the Wellcome Trust and the Medical Research Council (G1100526). C.M. was funded by a BBSRC studentship (961424). A.S. is a Medical Research Council Professor.
Introduction THAP1 (THAP [Thanatos-associated protein] domain-containing, apoptosis-associated protein 1) is a member of a large family of proteins which are primarily transcription factors (Gervais et al., 2013; Roussigne et al., 2003). The THAP domain, an atypical zinc finger (CysX2-4CysX35-53CysX2His), is highly conserved and is part of the DNA-binding domain (DBD) with homology to P-transposable elements (Majumdar and Rio, 2015). THAP1 mutations cause DYT6 dystonia (Fuchs et al., 2009), and mutations are located throughout the protein, with about 50% in the DNA-binding domain. Importantly, recessive mutations have been identified (Houlden et al., 2010; Schneider et al., 2011; Xiromerisiou et al., 2012). THAP1 functions, targets, and the mechanisms by which its mutations lead to dystonia are largely unknown, including the effects of mutations on DNA binding (Campagne et al., 2012). Functional studies of THAP1 in human umbilical vein endothelial melatonin receptor agonist (HUVECs) show a role in the S phase of mitosis via modulation of pRb-E2F cell-cycle target genes, including RRM1 (Clouaire et al., 2005). In vitro, a coiled-coil domain is required for dimerization (Sengel et al., 2011). Other interactors include prostate apoptosis response-4 protein (Par-4), an effector of cell death linked to prostate cancer and neurodegenerative diseases (Roussigne et al., 2003); HCF-1, a transcriptional co-activator involved in cell-cycle regulation; and O-GlcNAc transferase (OGT), which catalyzes the addition of O-GlcNAc and thereby also participates in epigenetic regulation of gene expression with an essential function in dividing cells (Mazars et al., 2010). In mouse models of DYT6 which harbor either a disease-causing C54Y mutation in the DBD or a null allele (ΔExon2) (Ruiz et al., 2015), rare homozygous embryos survived to day 14. They were small with defects in peripheral organs and brain, which showed deficits in the number and morphology of neurons. To study the impact of the mutant alleles on stem cell maintenance and differentiation, we generated mouse embryonic stem cells (mESCs) homozygous for either the C54Y (Thap1) or ΔExon2 (Thap1) alleles. Herein, we characterize both ESCs, which are viable with intact stem cell characteristics, but with abnormalities in cell death, cell cycle, and proliferation rate, that are more severe in the ΔExon2 than in the C54Y homozygote. Furthermore, we show that during differentiation of embryoid bodies (EBs), wild-type THAP1 is required for repression of a cohort of core pluripotency-associated genes and survival in ΔExon2 cells, and apparently for terminal neuronal differentiation in C54Y homozygote surviving cells.
Results
Discussion We have identified THAP1 as a major regulator of ESC survival and, to a lesser degree, proliferation and differentiation. As THAP1 mutations result in a neurologic disease, we were particularly interested in the effects of a causative mutation, C54Y, on neuronal differentiation. We had previously posited, based on observations of homozygote mutant embryos, that THAP1 is required in multiple stages and layers during embryogenesis, organogenesis, and maturation of the nervous system, including neurogenesis and neuritogenesis, and that the C54Y variant is frequently unable to substitute for the WT (Ruiz et al., 2015).