Figure 3. H3K9 methyltransferases, demethylases, and methyl binders are critical for reprogramming.

A) Depletion of H3K9 HMTases and Cbx3 enhances OSK reprogramming. Oct4-GFP reporter MEFs were infected with individual retroviruses expressing Oct4, Sox2, and Klf4 (OSK) and transfected with siRNAs targeting the indicated genes every three days starting on day seven post-infection. The number of GFP-positive colonies was determined on day 25 of reprogramming: i) for the knockdown of H3K9 methyltransferases Ehmt1, Ehmt2, and Setdb1; either singly or combined (indicated as 3XHMT); ii) for the knockdown of the heterochromatin protein 1 family members Cbx1 = HP1β, Cbx3 = HP1γ, or Cbx5 = HP1α. Data from two technical replicates (Rep1 and Rep2) from one out of three representative experiments are presented,.

B) Depletion of the H3K9 HMTases or Cbx3 enhances the efficiency and kinetics of OSCK reprogramming. Oct4-GFP reporter MEFs carrying a single, doxycycline (dox)- inducible polycistronic transgene encoding Oct4, Sox2, cMyc, and Klf4 (OSCK) were induced to reprogram by the addition of dox. On days 4, 6 and 10 post-induction, control siRNAs or the siRNA cocktail targeting 3XHMT or Cbx3 were transfected. To determine when reprogramming factor-independent GFP-positive colonies can be obtained, dox was withdrawn on days 6, 7, 8, 9, and 10, respectively, and in all cases the number of GFP-positive colonies determined on day 15. Upon dox withdrawal, siRNA treatments were not continued.

C) Overexpression of the histone H3K9 demethylase Jmjd2c enhances reprogramming. Oct4-GFP reporter MEFs were reprogrammed with OSK as in (A) with the addition of Tomato (control) or Jmjd2c-expressing retroviruses. The number of GFP-positive colonies obtained was quantified on day 25. Data from two technical replicates (Rep1 and Rep 2) from one out of three representative experiments are presented.