We have recently shown that the proapoptotic BBC3/PUMA (p53 up-regulated modulator of apoptosis) gene is a Myc target and through recruitment to an E-box binding site on its promoter, Myc cooperates with the PI3/AKT pathway to repress FOXO3a mediated activation of PUMA expression [7
]. In the course of this study we were intrigued to observe a Myc responsive RNAPII peak in the second intron of PUMA locus in condition in which the PUMA expression was inhibited (data not shown). This finding prompted us to investigate the presence of actively expressed genes responsive to Myc activation in the region downstream of PUMA. Exploring the genomic region downstream of PUMA locus we found the presence of SUMO-activating enzyme SAE1 gene whose transcription proceeds in the opposite strand to PUMA.
To determine the expression of SAE1 in the presence of hyper-activated Myc protein, we used two Myc-inducible cell lines: the rat fibroblast RAT-MycER and the human retinal hT-RPE-MycER cell lines in which the inducible Myc-estrogen receptor fusion transgene (MycER protein) is activated upon treatment with tamoxifen (OHT) [6
As shown in , in RAT-MycER, SAE1 expression as well as the known Nucleolin (NCL) Myc target, increase in response to Myc activation while the levels of expression of PUMA are inhibited as previously described [6
]. To determine the contribution of Myc in the SAE1 activation we
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) (more ...)
silenced Myc expression by transfection of specific siRNAs in the RAT-MycER cells and we found that Myc silencing inhibits activation of SAE1 transcription (). To further determine the contribution of Myc in SAE1 activation, we used the isogenic RAT-Myc-/- cells and measured SAE1 expression levels in starved versus serum-induced cells. As shown in , SAE1 expression slightly increases upon serum addiction while the expression was significantly higher when a Myc expression vector was introduced by transfection into RAT-Myc-/- cells. Collectively these results suggest that SAE1 expression is indeed regulated by Myc.
SAE1 sequence is extremely conserved between rat and human suggesting an evolutionary fundamental role in cell physiology. In this respect we analyzed SAE1 expression in the retinal human Myc inducible cell line hT-RPE-MycER [8
] and, as shown in , Myc activation results in induction
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 (more ...)
of SAE1 expression at levels comparable to the known Nucleolin (NCL), a well defined Myc target. To explore the role of Myc in SAE1 activation we scanned its genomic sequences for putative Myc binding sites. Several E-boxes were found, , and we focalized our attention on two closely associated E-boxes in position close to the TSS site (-170 and -101). Most importantly these E-boxes are located in a region with high level of H3K4Me3, a prerequisite for Myc binding () [9
]. To assess Myc occupancy on SAE1 we carried out qChIP analysis in the RPE cells, which were growth factors-deprived for 2 days (0) and treated for 6hrs with OHT for Myc activation. Myc-immunoprecipitated chromatin was analyzed using amplicons spanning the SAE1 and NCL E-boxes and the qChIP data () show that Myc is recruited on the E-box sites at the SAE1 promoter with efficiency comparable to recruitment on NCL. Although we cannot exclude the presence of additional E-box binding sites that might contribute to Myc activation, we can conclude that SAE1 is a bona-fide positively regulated Myc target through a direct binding to the E-boxes close to SAE1 TSS site.
While our studies on Myc role on SAE1 expression were in progress it has been reported the identification of genes vital to support Myc-addicted tumors through a genome wide genetic screen for Myc-synthetic lethal (MySL) shRNAs in human mammary epithelial cells [5
]. The most significant candidates that have been isolated in this study are the SAE1 and SAE2 genes whose products associate to form the heterodimer that is a critical component of the SUMO activating enzyme needed for SUMO conjugation to proteins [4
]. The authors found that SAE was required for growth of Myc dependent tumors and that the low expression of SAE1/2 in human breast cancers with high Myc expression levels correlates with longer survival of the patients. SAE depleted cells with high levels of Myc expression were found impaired in a correct mitotic spindle formation and consequently committed to apoptosis highlighting 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. Thus, recruitment of Myc on SAE1 modulates its expression supporting Myc oncogenic program by activation of Sumoylation dependent Myc-switchers (SMS genes).
Intriguing is the fact that the PUMA and SAE1 are adjacent on the human chromosome 19 and that their expression has been always found inversely correlated. While Myc cooperates with PI3/AKT pathway to repress PUMA transcription, hyper activation of Myc activates SAE1 transcription. An extensive analysis of chromatin domains at the PUMA locus and surrounding region including SAE1, has been recently provided by the Espinosa’s laboratory [11
]. It has been identified a peculiar transcriptional organization of the locus with the presence of the insulator protein CTCF (CCCTC-binding factor) and the associated cohesin complex at intragenic PUMA as well as at intergenic regions between PUMA and SAE1 (). Since CTCF functions involve the formation of ‘chromatin loops’ and these loops seems to be regulated in a signaling specific manner [13
], it is possible that Myc binding might induce an intergenic gene looping between PUMA and SAE1 leading to the divergent Myc-mediated transcription control of SAE1 and PUMA loci. Further investigations will be necessary to validate these speculative hypotheses.
A general schematic of SAE1/PUMA locus showing CTCF binding sites and identified Myc binding sites (E-box).