Fig. 2
MYC occupies the promoters of lysosomal genes and that of TFEB and TFE3. a Motif analysis using HDAC2-binding sites present in lysosomal genes. b Left, silencing of HDAC2 downregulated MYC protein expression in HeLa cells. Right, Coomassie stained immunoblot used as the loading control. c Quantification of MYC levels in HDAC2 silenced HeLa cells normalized to loading control (n = 3 independent experiments). d Co-immunoprecipitation of MYC with HDAC2 from lysates obtained from HeLa cells. HDAC2 band is labeled with an asterisk. Heavy chain IgG is labeled with an arrow. e Immunoprecipitation of MYC followed by immunoblot of MYC protein. MYC band is labeled with an asterisk. Heavy chain IgG is labeled with an arrow. f Graph represents HDAC2 binding to the promoter of MYC (n = 5 biologically independent samples) in HeLa cells treated with DMSO and SAHA (20 μM for 24 h); IgG control (n = 3 biologically independent samples). g–i Myc binding to the promoters of (g) Neu1, Ctsa, (h) Lamp1, Mcoln1 and i Tfeb and Tfe3 was analyzed in ChIP-seq datasets performed with anti-Myc antibody in mouse group 3 medulloblastoma cells overexpressing Myc (Trp53–/– overexpressing Myc). Input DNA (Inp) serves as reference. j, k Histograms represent MYC binding to the promoters of (j) NEU1 (n = 3 biologically independent samples), LAMP1 (n = 3 biologically independent samples), MCOLN1 (n = 3 biologically independent samples), TFEB (n = 6 biologically independent samples), TFE3 (n = 3 biologically independent samples) and (k) MYC (n = 3 biologically independent samples) in HeLa cells treated with SAHA (20 μM for 24 h) or DMSO. NEU1 INT oligos were used as negative control for non-specific antibody binding (n = 3 biologically independent samples); IgG control (n = 3 biologically independent samples). l Sequential ChIP experiments were performed from HeLa cells with anti-MYC antibody followed by anti-HDAC2 antibody and analyzed by RT-qPCR at the promoter region of TFEB and TFE3 (n = 3 biologically independent samples); IgG control (n = 3 biologically independent samples). All the graphs are presented as mean ± SD. Statistical analysis was performed using the Student t-test. *p < 0.05, **p < 0.01, ****p < 0.0001
