Fig. 4. Knockout (KO) of Usp7 alters histone H2B monoubiquitination (H2Bub1) of genes involved in neuronal differentiation. (A-C) Relative expression levels of neuronal lineage cell-specific transcription factors (A) Ascl1, (B) Ngn1, (C) NeuroD1, and (D, E) marker genes (D) Map2 and (E) βIII-tubulin messenger RNA (mRNA) were analyzed with real-time polymerase chain reaction (PCR). Samples were collected on day 9 after differentiation from control and Usp7 KO P19 cells. Results were normalized with the level of GAPDH. (F-H) Samples were collected on day 9 after differentiation from control and Usp7 KO P19 cells and subjected to chromatin immunoprecipitation analysis with antibodies specific for H2Bub1 and H2B. Immunoprecipitated DNA was quantified by real-time PCR with primers specific for the promoter regions of (F) Olig2, (G) GFAP, and (H) Pax6. Error bars were derived from three independent experiments. N=3. RA: retinoic acid. *p<0.05, **p<0.01, ***p<0.001.