SUMO-activating SAE1 transcription is positively regulated by Myc

Abstract

Myc protein plays a fundamental role in regulation of cell cycle, proliferation, differentiation and apoptosis by modulating the expression of a large number of targets. Here we report the transactivation ability of the human Myc protein to activate the SUMO-activating enzyme SAE1 transcription. We found that Myc activates SAE1 transcription via direct binding to canonical E-Boxes sequences located close to the SAE1 transcription start site. A recent report has highlighted the crucial role of the SAE gene expression in Myc mediated oncogenesis. Our study adds new insight in this context since we show here that Myc directly activates SAE1 transcription, suggesting that Myc oncogenic activity which depends on SAE1 is ensured by Myc itself through direct binding and transcriptional activation of SAE1 expression.

Keywords: Myc; SAE1; SUMOylation; transcription.

Figure 1

Myc activates SAE1 expression. (A) mRNA expression levels of SAE1, NCL and PUMA were quantified by qRT–PCR in quiescent cells (0) and after 4 and 6hrs of treatment with serum and OHT. (B) Myc expression was inhibited with specific siRNA (siMyc) and siRNA non-targeting (siCtl) was used as scrambled RNAs. SAE1 mRNAs expression levels were quantified by qRT–PCR in quiescent cells (0) and 6hrs of treatment with serum and OHT. The efficiency of Myc silencing by siRNA treatments measured by qRT–PCR is shown on the right. (C) RAT-Myc-/- cells as well as cells transfected with a Myc expression vector were serum deprived for 48 hrs. SAE1 mRNA levels were evaluated by qRT– PCR 6 hrs after serum induction. Values were compared to quiescent cells (0).

Figure 2

(A) hT-RPE-MycER cells were synchronized by 2 days of growth factors deprivation. SAE1, NCL and PUMA mRNAs expression levels were quantified by qRT–PCR in synchronized (0) and cells treated with growth factor + OHT (6hrs). All mRNA levels were normalized to β-glucuronidase (GUS) mRNA levels and all values represent the average of at least three independent experiments. Error bars indicate SD. Protein expression is shown in immunoblots of whole-cell extracts with anti-SAE1 and actin for loading control. (B) An adapted UCSC genome browser view of SAE1 genomic sequence displaying H3K4me3 and H3K4me1 levels and putative E-box (asterisks). Myc occupancy on SAE1 and NCL chromatin in synchronized (0) and Myc induced hT-RPE-MycER cells for 6hrs is shown. ChIP-enriched DNA was quantified by real-time PCR analysis using amplicon covering the region containing the Myc binding E-boxes as shown in Figure 2A.

Figure 3

A general schematic of SAE1/PUMA locus showing CTCF binding sites and identified Myc binding sites (E-box).