Inducible TDG knockout models to study epigenetic regulation

Abstract

Mechanistic and functional studies by gene disruption or editing approaches often suffer from confounding effects like compensatory cellular adaptations generated by clonal selection. These issues become particularly relevant when studying factors directly involved in genetic or epigenetic maintenance. To provide a genetic tool for functional and mechanistic investigation of DNA-repair mediated active DNA demethylation, we generated experimental models in mice and murine embryonic stem cells (ESCs) based on a minigene of the thymine-DNA glycosylase (TDG). The loxP-flanked miniTdg is rapidly and reliably excised in mice and ESCs by tamoxifen-induced Cre activation, depleting TDG to undetectable levels within 24 hours. We describe the functionality of the engineered miniTdg in mouse and ESCs (TDGiKO ESCs) and validate the pluripotency and differentiation potential of TDGiKO ESCs as well as the phenotype of induced TDG depletion. The controlled and rapid depletion of TDG allows for a precise manipulation at any point in time of multistep experimental procedures as presented here for neuronal differentiation in vitro. Thus, we provide a tested and well-controlled genetic tool for the functional and mechanistic investigation of TDG in active DNA (de)methylation and/or DNA repair with minimal interference from adaptive effects and clonal selection.

Keywords: Active DNA Demethylation; Base Excision Repair; Cre/loxP; Embryonic Stem Cells; Minigene; Neuronal Differentiation; TDG; Tamoxifen.

Figure 1.. miniTdg complementation of Tdg ^-/- mice and mESCs.

( A) Synthetic miniTdg gene on the plasmid integrated to a random genomic locus by pronuclear injection. miniTdg consisting of 6 kb endogenous Tdg promoter, the Tdg coding sequence (CDS) including a chimeric splice donor ( Tdg exon 1 of transcript variant 2 NM_172552.4, rabbit β-globin intron), to allow for alternative splicing, 3 kb of the 3’ untranslated region (3’ UTR) and terminator (term) of Tdg, and the bovine growth hormone terminator sequence (BGH!). loxP sites are indicated in red. ( B) Expected and obtained genotype distribution in offspring (n=116) from crosses of heterozygous mice. ( C) Top: Immunoblot for TDG of whole-cell SDS-extracts from derived ESCs with different genotype. Bottom: Loading control by Ponceau staining of the membrane. ( D) Accumulation of oxidized 5-mC derivates measured by HPLC-MS/MS in ESCs with the indicated genotype. For each genotype, means and standard deviations of two independent clones with 1 or 2 repeated measurements are shown. Asterisks indicate p-values of Student’s t-test, compared to TDG WT: *** p-value < 0.001. ( E) Scheme of the targeting construct pROSA26-ERT2CreERT2 for the ROSA26 locus with the tamoxifen-inducible Cre recombinase ( Matsuda & Cepko, 2007). ERT2-Cre-ERT2 expression is under the control of the synthetic cytomegalo-virus/chicken-actin/beta-globin promoter (CAG). Homology arms for ROSA26 targeting are indicated in light grey. A blasticidin resistance cassette (BlaR) for positive selection is under the control of the SV40 promoter and terminator. ( F) Predicted restriction pattern of ROSA26 WT and integration events (top) and fragments detected by Southern blotting (bottom). Left: The hybridization probe (P1) locating to the left ROSA26 homology arm detected a genomic fragment of 6.9 kb. The 4.4 kb fragment represents the wild-type ROSA26 allele. Right: Detection of possible off-target events using a second probe (P2) locating to the blasticidin selection marker within the targeting construct. See Underlying data for the raw data behind this figure ( Schwarz, 2020).

Figure 2.. Cre induction for two hours results in the depletion of TDG within 24 hours without affecting cell viability.

( A) Scheme of the miniTdg cassette before (top) and after (bottom) Cre-mediated recombination. Position of PCR primers are indicated with green arrows. ( B) Quantitation of miniTdg excision upon OHT administration in TDGiKO1, TDGiKO4 and parental ESCs without the Cre recombinase (Mm01). Genomic DNA of cells was extracted two days after OHT treatment with indicated concentrations for 2 hours. Copy number of the miniTdg is measured by qPCR using primers 2 and 3, normalized to the nearby terminator region (primers 4/5) and a control locus on chromosome two. Shown are means and standard error from technical quadruplicates per clone. Asterisks indicate p-values of Student’s t-test, compared to DMSO control: ** p-value < 0.01, *** p-value < 0.001. ( C) Quantitation of miniTdg excision as in ( B), but Cre induction executed in ES medium containing 15% FCS. ( D) Phase-contrast images of TDGiKO1 ESCs in 2i medium, two days after OHT treatment for two hours at indicated concentrations. ( E) Detection of Cre recombination events (158 bp) by PCR using primers 1 and 3 ( A) before and after addition of OHT. ( F) Time-course assessment of TDG protein levels expressed from the miniTdg gene. Immunodetection with an anti-TDG antibody was performed with western blotted NP-40 cell extracts of 2i cultivated TDGiKO1 ES cells after Cre induction by 1 µM OHT for 2 h. See Underlying data for the raw data behind this figure ( Schwarz, 2020).

Figure 3.. TDGiKO ESCs express a functional TDG and are proficient for cell lineage commitment.

( A) Measurement of oxidized derivates of 5-mC in selected ESCs by HPLC-MS/MS, cultivated in 2i medium + LIF, one week after TDG depletion by 1 µM of OHT for 2 h. Shown are the means and standard deviation of 2-3 biological replica per condition. ( B) Scheme of the neural differentiation protocol ( Bibel et al., 2007), adapted with a 16 h priming step in ESC medium for ESCs grown in 2i medium. Representative phase-contrast images of TDGiKO1 cells with (+OHT) and without (mock) TDG depletion at the differentiation stages indicated (arrows). ( C) Expression of pluripotency and germ layer marker genes were assessed by qRT-PCR in TDGiKO1 cells at the stages of differentiation as marked in ( B). Expression was normalized to the housekeeping genes Rps13 and Eef1a1. Shown are means and standard errors of 3 biological replicates. Asterisks indicate p-values of Student’s t-test, compared to the non-induced condition: * p-value < 0.05, *** p-value < 0.001. See Underlying data for the raw data behind this figure ( Schwarz, 2020).