Corresponding author. In-Jung Lee: ijlee@knu.ac.kr Fax: +82-53-958-6880 2 7 2 8 †Equal contributors: these authors equally contributed to this research work. 9The data of submission:The number of tables and figures: . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/252312 doi: bioRxiv preprint first posted online Jan. 23, 2018; 3 Abstract 3 4

Modulation of Wnt target gene expression via the TCF/LEFs remains poorly understood. We employ proximity-based biotin labeling (BioID) to examine GSK-3 inhibitor effects on the TCF7L1 interactome in mouse ESCs. We generated ESC lines with biotin ligase BirA* fused to TCF7L1 by knocking it into the endogenous TCF7L1 locus or by inserting a doxinducible BirA*-TCF7L1 transgene into the Rosa26 locus. Induction yielded BirA*-TCF7L1 levels 3-fold higher than in the endogenous system, but substantial overlap in biotinylated proteins with high peptide counts were detected by each method. Known TCF7L1 interactors TLE3/4 and β-catenin, and numerous proteins not previously associated with TCF7L1, were identified in both systems. Despite reduced BirA*-TCF7L1 levels, the number of hits identified with both BioID approaches increased after GSK-3 inhibition. We elucidate the network of TCF7L1 proximal proteins regulated by GSK-3 inhibition, validate the utility of endogenous BioID, and provide mechanistic insights into TCF7L1 target gene regulation. BioID | GSK-3 | TCF7L1 | mESCCorrespondence: dobleb@mcmaster.ca

The genome-wide chromatin occupancy of the TCF/LEF factors and its modulation by Wnt pathway activation remain poorly defined. Here, we describe mouse ES cell (mESC) lines expressing a single copy knock-in of the 3xFLAG epitope at the N-terminus of TCF7L1 and TCF7, the two most-highly expressed TCF/LEF factors in mESCs. TCF7L1 protein levels, detected by immunoblotting with a FLAG antibody, were much higher than TCF7 in mESCs maintained in standard serum-and LIF-supplemented medium, even in the presence of the GSK-3 inhibitor, CHIR99021 (CHIR). We used FLAG antibody-mediated ChIP-seq to determine TCF7 and TCF7L1 chromatin occupancy in mESCs cultured in standard medium with or without CHIR for 14 hours. TCF7 and TCF7L1 displayed very few overlapping ChIP peaks across the genome, with TCF7L1 binding significantly more genes than TCF7 in both culture conditions. Despite a reduction in total TCF7L1 protein after CHIR treatment, the TCF7L1 ChIP peak profiles were not uniformly attenuated. Our data demonstrate that TCF7L1 chromatin occupancy upon short-term CHIR treatment is modulated in a target-specific manner. Our findings also suggest that Wnt target genes in mESCs are not regulated by TCF/LEF switching, and TCF7L1, although often called a constitutive repressor, may serve as a transcriptional activator of certain target genes in CHIR-treated mESCs. TCF7L1 | TCF7 | GSK-3 | ChIP-seq | mESC | 3XFLAG-epitope | Knock-in | Correspondence: dobleb@mcmaster.ca Highlights• ChIP and cytometry data suggest that TCF7L1 does not directly regulate mESC Nanog expression.• TCF7L1 remains associated with β-catenin in the presence of CHIR99021.• TCF7 and TCF7L1 display different chromatin occupancies in mESCs.• TCF7L1 binding at specific genomic sites is variably altered by CHIR99021.

Binaural Auditory Beat (BAB) is known to induce frequency-specific health effects. Theta frequency BAB, for instance, triggers a meditative state whereas alpha frequency BAB may function as an anxiolytic. Findings upon BAB exposure demonstrate mixed results and thus require further academic investigation, for the beneficial effect of a BAB in question may become detrimental if applied inappropriately. There are, unfortunately, hundreds of publicly available commercial BABs (C-BABs) today that are neither scientifically verified nor clinically safe. Virtually anyone can construct/distribute any kinds of BAB mixes at any given time via freely available BAB generators. This means customers may be exposed to unknown or even prematurely released C-BAB, not to mention that its neurophysiological mechanism is not yet fully understood. Considering a dearth of research and a pile of anecdotal reports on its side effects, C-BAB customers are exposing themselves to medically unrecognized health risks. The health effects of C-BABs will be reviewed and the perils of its clinical implementation and recreational applications will be discussed.

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