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. 2016 Oct 15;143(20):3674–3685. doi: 10.1242/dev.139360

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© 2016. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 1. — Generation of H3K27me3-deficient hESCs by ectopic JMJD3c expression. (A) Structures of the JMJD3f and JMJD3c proteins. JMJD3c was designed to contain the JmjC domain (amino acids 1376-1484). HA was added to JMJD3f and JMJD3c and an NLS was added to the N terminus of JMJD3c. (B) hESCs were transfected with the synthetic mRNAs for HA-JMJD3f or HA-JMJD3c and were stained with HA and H3K27me3 antibodies. Non-transfected hESCs were used as a control. The arrowheads indicate the transfected cells. The mean fluorescent intensities of H3K27me3 in the transfected cells were 0.65 for HA-JMJD3f and 0.26 for HA-JMJD3c compared with the non-transfected cells. More than 20 nuclei from three independent experiments were examined. Scale bar: 20 μm. (C) The effects of transfection of the HA-JMJD3c and HA-JMJD3f mRNAs on the H3K27me3 levels were analyzed by immunoblotting. Emerald mRNA (Em) was transfected as a control. The H3 antibody was used as a loading control. The average signal intensities of H3K27me3 from two independent biological replicates were 0.92 for Em, 0.65 for HA-JMJD3f, and 0.36 for HA-JMJD3c compared with the non-transfected cells. (D) Construct used for Tet-On induction of JMJD3c expression in hESCs (JMJD3c-hESCs). pA, polyA signal; PB, piggyBac repeat. (E) JMJD3c-hESCs were stained with X-Gal 3 days after Dox treatment. Scale bar: 500 μm. (F) HA-JMJD3c-induced H3K27me3 demethylation was detected 1 to 3 days after Dox treatment. The average signal intensities of H3K27me3 from two independent biological replicates were 0.70 on Day 1, 0.52 on Day 2, and 0.24 on Day 3 compared with Day 0. (G) A point mutation in JMJD3c (mut) was inserted at aa 1390 for catalytic inactivation. (H) Effects of HA-JMJD3c and the HA-JMJD3c mut on the H3K27me3 levels. The average signal intensities of H3K27me3 from two independent biological replicates were 0.97 for Em, 0.23 for JMJD3c, and 1.0 for the JMJD3c mut compared with the non-transfected cells.