Figure 5
Aspa knock-down leads to global transcriptional repression, reduced cytoplasmic acetyl-CoA and histone acetylation.
(a–e) Differentiated iBACs with stable knock-down of Aspa (siAspa) or non-targeting control cells (ntc) were used for Affymetrix microarray analysis, histone isolation and ChIP-qPCR (each n = 3). (a) Two hundred and fifty genes downregulated by Aspa knock-down were submitted to DAVID functional annotation using GO terms and KEGG pathways. Count shows the number of mapped genes and Benjamini-Hochberg’s correction was used for adjusting p-value for multiple testing. (b) Top 20 genes significantly downregulated by Aspa knock-down. Ucp1 and Cidea are among the strongest repressed genes. (c) Acetyl-CoA measured with HPLC/HRMS in cytoplasmic fractions. Student’s t-test; *p < 0.05 (n = 3). (d) Western blot of histone preparations using an antibody against global histone H3 acetylation. Total histone H3 antibody serves as loading control. (e) Densitometric quantification of immunoblot. Student’s t-test; **p < 0.01. (f) ChIP-qPCR fold-enrichment of Aspa knock-down vs. control cells in promoters/enhancers of transcriptionally repressed genes. Histone acetylation was assessed on H3K27 and H3K9 using acetylation-specific antibodies for immunoprecipitation. Insulin (Ins) serves as non-regulated control region. The acetylation-independent activating histone mark H3K4me1 is not significantly changed. One-sample t-test; *p < 0.05; **p < 0.01.
