GSE127212
MYC-associated Factor X, MAX, controls the clock transcriptional network [RNA-Seq]
Purpose: Quantitative RT-PCR and ChIP analyses identified MAX as a transcriptional repressor of circadian BMAL1 target genes. The goal of this study is to compare the transcriptional effect of a knockdown of either MAX and BMAL1 in a human cell line (MDA-MB-231) Methods: Polyadenylated mRNA profiles of human breast cancer MDA-MB-231 cells with knocked down MAX or BMAL1 were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000 sequencer. Cells transfected with a non-targeting siRNA sequence were used as a control. RNA sequencing counts were used to determine differentially expressed genes with DeSeq2  package included in the Galaxy web platform (usergalaxy.org). Differentially expressed genes (DEGs) were defined adopting an adjust P < 0.001 as a statistical cut-off value. Results: Transcript assembly and quantification of RNA-sequencing reads identified 4863 and 4247 differentially expressed genes (DEGs) upon knockdown of BMAL1 or MAX, respectively. The comparison of the two set of genes revealed that 2391 of siBMAL1 DEGs (almost 50%) were also differentially expressed in MAX-silenced cells. Heat map and clustering analysis of this sub-set of genes revealed that more than 90% (2241 out of 2391) were coherently altered in both conditions (i.e. their expression was altered in the same direction). Consistent with our quantitative RT-PCR experiments, genes belonging to the circadian rhythm signalling were altered by both MAX and BMAL1 silencing. Conclusion: This study indicate that MAX is a  part of the molecular clock transcriptional network. Overall design: Polyadenylated mRNA profiles of human breast cancer MDA-MB-231 cells with knocked down MAX or BMAL1 were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000 sequencer