Generation of Jmjd2c-knockout ESC lines

ESCs lines carrying a single Jmjd2c targeted allele and their wild-type (WT) counterparts, both from C57BL/6 mouse genetic background, were obtained from the EUCOMM/IKMC repository pipeline through collaboration with Dr Cynthia Fisher and Dr William Skarnes (Wellcome Trust Sanger Institute, Cambridge) (Bradley et al., 2012; Skarnes et al., 2011). The KO first allele (tm1a) was generated by gene trapping through the insertion of a cassette encoding lacZ/β-galactosidase and a neomycin resistance gene, upstream of a “critical exon” common to all predicted Jmjd2c transcripts (Fig.3.1A). The generation of homozygous knockout cell lines was then carried out by Dr Cynthia Fisher (Wellcome Trust Sanger Institute, Cambridge) through homologous recombination of a gene-trap cassette into the second allele (tm2), encoding the hygromycin resistance gene and GFP (Fig.3.1.A). Both cassettes are promoterless, hence relying on the active transcription of Jmjd2c, and contain an En2 splice acceptor (SA) signal and the SV40 polyadenylation (pA) sequences to ensure transcription is spliced into the cassette and is stopped at the inserted sites (Fig.3.1A).

Validation of the knockout was performed by Dr Cynthia Fisher (Wellcome Trust Sanger Institute, Cambridge) at the DNA level using a Long-Range PCR approach with specific primer pairs (Fig.3.1A; Table B-I – Appendix B), hence showing correct insertion of the trapping cassettes in both alleles. A 5kb and a 6.3kb fragment corresponding to the first and second mutated alleles, respectively, were successfully amplified using genomic DNA extracted from the homozygous Jmjd2c-KO ESC (ESCJmjd2c-KO_{) clones E2 and E3, as opposed to WT ESCs (ESC}WT₎

(Fig.3.1B). Additionally, the expression of GFP from the second allele was confirmed in E2 and E3 mutant clones by immunofluorescence (ESCKO-E2_{, ESC}KO-E3_{; Fig.3.1C). GFP expression, alongside}

with drug resistance to neomycin and hygromycin in culture, further indicated correct transcription of the inserted cassettes in homozygous KO clones.

The depletion of Jmjd2c was validated both at the mRNA and protein level in the two independent Jmjd2c-KO ESC lines. For this, specific primer pairs were designed spanning exons

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located upstream (exons 5 and 7; Fig3.1D) and downstream (exons 14 and 15, exons 18 and 20; Fig.3.1.D) of the “critical exon” and hence the trapping cassettes. Jmjd2c mRNA levels detected when using upstream primer pairs were unchanged in mutant cells relative to WT ESCs. In contrast, transcript levels spanning exons located downstream of the critical exon were significantly reduced by about 90% (Fig.3.1D). The residual expression of full-length transcript could arise due to alternative splicing around the inserted cassettes, but this however did not result in translation of a full-length Jmjd2c protein, as described below.

To examine whether the targeted trapping resulted in the abolishment of full-length Jmjd2c protein, western blot analysis was performed on total protein lysates extracted from WT and Jmjd2c-KO ESC clones E2 and E3 using a previously described (Loh et al., 2007) anti-Jmjd2c antibody (kindly provided by Professor Huck-Hui Ng, Genome Institute Singapore). As expected, high levels of Jmjd2c protein were detected in the WT ESCline, whereas expression of full-length Jmjd2c was completely abolished in Jmjd2c-KO ESC lines (Fig.3.1E), hence confirming that these cells are indeed constitutively depleted for Jmjd2c. Moreover, Jmjd2c depletion did not affect Jmjd2b, as Jmjd2b protein expression remained unchanged between the same WT and KO ESC lines (Fig.3.1E). The insertion of gene trapping cassettes is predicted to result in the expression of putative truncated proteins, however since the trapping cassettes are inserted upstream from the PHD and Tudor domains, this is not expected to be located in the nucleus (Pedersen et al., 2014; Shin and Janknecht, 2007). Altogether, these results validate the successful generation of homozygous Jmjd2c-knockout ESC clones.

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Figure 3.1 Validation of Jmjd2c-knockout in ESCs

A. Schematic representation of the double knockout strategy. The first and second mutated alleles carry a

cassette containing the neomycin-βgalactosidase and hygromycin-GFP resistance/expression genes, to allow for efficient selection and identification. In the first allele, the FRT sites were inserted to allow for optional reversion of homozygous to heterozygous by removal of the cassette with FLP recombinase. The loxP sites were inserted in the vicinity of the critical exon 9 to allow for the optional creation of a conditional null allele with Cre recombinase. The splice acceptor (SA) site in the cassettes promotes incorporation of the insert into the RNA transcript after RNA splicing, whereas the polyA (pA) signal site ensures that RNA polymerase transcription is stopped. Arrows indicate the location of the primers used for genotyping and the expected PCR band sizes is indicated for each primer pair. B. Genotyping of both alleles was achieved by Long Range PCR on the JM8 wild-type (ESCWT_{) cells, and homozygous Jmjd2c-knockout (ESC}Jmjd2c-KO₎

mutant clones (E2, E3). Performed in collaboration with Dr Cynthia Fisher. C. Immunofluorescent labelling of GFP in ESCWT _{and ESC}Jmjd2c-KO _{clones (KO-E2, KO-E3). DAPI staining was used to visualise nuclei. Scale,}

ES C WT ES C KO -E2 ES C KO -E3 C. _DAPI GFP B. E. ESCWT _{E2 E3} Jmjd2c Jmjd2b α-Tubulin 0.0 0.5 1.0 1.5 2.0

ex5-7 ex14-15 ex18-20

Jmjd2c locus R e lat iv e m R NA le ve ls WT KO-E2 KO-E3 D. ESCJmjd2c-KO ESCWT ESCKO-E2 ESCKO-E3 ESCWT _{E2 E3} 5 ESCJmjd2c-KO 8 5 8 Kb A. 5Kb 6.3Kb * * * * * *

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100 µm. D. Transcript levels of Jmjd2c were quantified using primers located upstream (exon5-7) and downstream (exon14-5, exon18-20) of the critical exon. Levels were normalized to 2 housekeeping genes (S17 and L19), and expressed relative to ESCWT_{. Error bars represent SEM of 3 biological replicates. P<0.05} (*), Mann-Whitney U test. E. Western Blot for Jmjd2c and Jmjd2b and on whole cell extracts from ESCWT _and ESCJmjd2c-KO _{clones as indicated. α-Tubulin was used as a loading control.}