Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth

The gene desert upstream of the MYC oncogene on chromosome 8q24 contains susceptibility loci for several major forms of human cancer. The region shows high conservation between human and mouse and contains multiple MYC enhancers that are activated in tumor cells. However, the role of this region in normal development has not been addressed. Here we show that a 538 kb deletion of the entire MYC upstream super-enhancer region in mice results in 50% to 80% decrease in Myc expression in multiple tissues. The mice are viable and show no overt phenotype. However, they are resistant to tumorigenesis, and most normal cells isolated from them grow slowly in culture. These results reveal that only cells whose MYC activity is increased by serum or oncogenic driver mutations depend on the 8q24 super-enhancer region, and indicate that targeting the activity of this element is a promising strategy of cancer chemoprevention and therapy. DOI: http://dx.doi.org/10.7554/eLife.23382.001


Introduction
Deregulated expression of the MYC oncogene is associated with many cancer types (Reviewed in Albihn et al., 2010;Dang, 2012;Evan, 2012). MYC acts primarily as a transcriptional activator that increases expression of many genes required for RNA and protein synthesis above the level that is required in resting cells. In cancer cells, aberrantly elevated levels of MYC drive global amplification of transcription rates, providing the cells with necessary resources for rapid proliferation (see, for example Brown et al., 2008;van Riggelen et al., 2010;Ji et al., 2011;Lin et al., 2012;Sabò et al., 2014;Walz et al., 2014).
Transcription of the MYC gene is regulated by a diverse array of regulatory elements located both upstream and downstream of the MYC transcription start site (TSS). Variants in the MYC upstream region contribute to inherited susceptibility to most major forms of human cancer, and account for a very large number of cancer cases at the population level (Amundadottir et al., 2006;Gudmundsson et al., 2007;Yeager et al., 2007;Al Olama et al., 2009;Yeager et al., 2009). For example, the polymorphism rs6983267 linked to colorectal (Tomlinson et al., 2007) and prostate (Yeager et al., 2007) cancers contributes more to cancer morbidity and mortality than any other known inherited variant or mutation, including the inherited mutations in classic tumor suppressors such as RB, TP53 and APC. Through computational and experimental analyses, we and others have shown that the risk allele G of rs6983267 creates a strong binding site for the colorectal-cancer associated transcription factor Tcf7l2 (Pomerantz et al., 2009;Tuupanen et al., 2009). This binding site is located within the Myc-335 enhancer element that is dispensable for mouse viability, but required for efficient Tcf7l2-driven intestinal tumorigenesis (Sur et al., 2012b).
More recently, another enhancer element, located 1.47 Mb downstream of Myc was shown to be required for formation of acute lymphoblastic leukemia (ALL) in mice (Herranz et al., 2014). However, in contrast to the Myc-335 element, this element is also required for normal T-cell development. Thus, the mechanism by which individual Myc enhancer elements contribute to normal development and tumorigenesis is still unclear.
Several studies have shown that the 8q24 region contains a large number of additional enhancer elements (see, for example [Hallikas et al., 2006;Ahmadiyeh et al., 2010;Yan et al., 2013;Yao et al., 2014]) and super-enhancers that are active in many different types of human cancer (Hnisz et al., 2013;Lovén et al., 2013;Zhou et al., 2015). The MYC-associated super-enhancers are activated during the process of tumorigenesis (Hnisz et al., 2013), and downregulation of superenhancer activity leads to selective inhibition of MYC expression (Lovén et al., 2013). Thus, MYCassociated super-enhancer activity is required for tumorigenesis, but the role of these elements in normal tissue morphogenesis and homeostasis has been unclear.
To address this problem, we have in this work generated multiple mouse strains deficient of regulatory elements upstream of the Myc promoter. Since this region contains multiple tumor type and tissue -specific enhancers and super-enhancers, for the sake of clarity we refer to the deleted region here as the 'super-enhancer region´. By analysis of the mice, we found that the entire super-enhancer eLife digest Our cells each contain close to 20,000 genes, which provide the instructions needed to build our bodies and keep us alive. At any one time in the life of the cell, only some of these genes are active. The activity of each gene is constantly regulated to allow the cell to respond to changes in its environment. Enhancers are sections of DNA, outside of the genes, that act as molecular switches controlling the activity of genes. A gene can have many such enhancers; each enhancer is linked to a particular set of signals and having multiple enhancers allows the same gene to be activated by different signals in different tissues in the body.
Changes to enhancers can have serious consequences. By altering the activity of genes, an enhancer can have widespread effects on the health and behavior of a cell, including transforming it from healthy to cancerous. The small differences in enhancers also make some people more susceptible to cancers than others. If we can identify enhancers whose activity is commonly altered in cancers, it could be possible to target them through treatment. Yet, it is not clear whether targeting enhancers in this way could be effectively used to treat cancer without damaging healthy cells. Now, Dave, Sur et al. have examined a large enhancer region with known links to several different cancers -including prostate, breast and colon cancers -to uncover whether it also plays a critical role in healthy cells and if it could be safely targeted for treatment. The region has multiple enhancers for a cancer-linked gene called MYC and is implicated in many cancer-associated deaths every year. This particular enhancer region is found in both humans and mice, which share many genes in common. Using genetic engineering, Dave, Sur et al. removed this enhancer region from the genetic information of a group of mice. The experiment showed that mice without the enhancer region were completely healthy. Also, when tested for cancer development, these mice were much less susceptible to several major types of cancer.
This investigation reveals that it may be possible to create drugs to shut down or inhibit certain enhancers to prevent or treat cancer without damaging healthy cells. However, this is currently just one example in mice under laboratory conditions. Further research is needed to determine if a similar approach can be developed to treat patients in the clinic. region conferring multi-cancer susceptibility contributes to MYC expression in vivo, yet is not required for mouse embryonic development and viability. However, this region is required for the growth of normal cells in culture and cancer cells in vivo. As cultured cells are exposed to serum, which is a signal of tissue damage, this finding suggests that tumor cells and cells responding to damage signals share regulatory mechanisms that are dispensable for normal physiological growth control.

Results
Functional mapping of the super-enhancer region upstream of Myc To dissect functional significance of the 8q24 region during normal development, we generated series of Myc alleles in mice using homologous recombination in ES cells. These include the Myc-335 enhancer deletion allele we have described previously (Sur et al., 2012b), and deletions of two additional conserved enhancer elements, Myc-196 and Myc-540, both of which are active in mouse intestine and colorectal cancer cells. In addition, we generated a point mutation that inactivates a conserved CCCTC-Binding factor (CTCF) site 2 kb upstream of the Myc TSS. This site has previously been reported to be required for MYC expression (Gombert and Krumm, 2009), and to have insulator activity (Gombert et al., 2003) (Figure 1a). Each allele contained loxP site(s) in the same orientation to allow conditional knockouts of the enhancers, and to facilitate generation of large deletions and duplications by interallelic recombination (Wu et al., 2007). All alleles were bred to homozygosity, and resulted in generation of viable mice. Expression of Myc in the colon of Myc-196 À/À and Myc-540 À/À mice was not markedly altered, suggesting that these elements have little effect on regulation of Myc in the intestine under normal laboratory conditions ( Figure 1b). Myc expression level was also normal in Myc-CTCF mut/mut mouse colon despite loss of CTCF and cohesin (Rad21) binding to the region proximal to the Myc promoter ( Figure 1c).

Mice lacking the Myc super-enhancer region are viable and fertile
As the individual mutations and deletions had limited effect, we next decided to generate two large deletions in the Myc locus using interallelic recombination between the Myc-CTCF mut loxP site and the loxP sites at Myc-335 À and Myc-540 À , yielding deletions of 365 kb (GRCm38/mm10 chr15:61618287-61983375) and 538 kb (chr15:61445326-61983375), respectively ( Figure 2a). The resulting alleles, Myc 42-367 and Myc 42-540 , were then segregated out from the corresponding duplications, and bred to homozygosity. Given the very large regions that were deleted (Figure 2b), we expected to see a strong phenotype. However, no overt phenotype was identified in the Myc 42-367/ 42-367 mice. The mice were born at the expected mendelian ratio, and both males and females were viable and fertile. Analysis of Myc expression, however, revealed a strong decrease in Myc expression in the colon and ileum of the mice (not shown).
The larger deletion, Myc 42-540 , could also be bred to homozygosity, and both males and females were viable. Given that the entire Myc regulatory region spans more than 2 Mb of DNA and is located on both sides of the Myc coding region (Rosenbloom et al., 2013;Sloan et al., 2016), the deletion is not expected to be equivalent to deletion of the Myc gene itself. Still, the viability of the mice is striking, since the region deleted contains regions linked to risk for myeloma, chronic lymphocytic leukemia and pancreatic, thyroid, bladder, prostate, breast, and colon cancers (Chung and Chanock, 2011;Sahasrabudhe et al., 2015;Mitchell et al., 2016;Zhang et al., 2016). To characterize the mice further, we analyzed histology and MYC expression in the tissues where these tumors originate from. This analysis revealed normal morphology of mammary gland, spleen, bladder, prostate and colon in Myc 42-540/42-540 mice ( Figure 2c).

Loss of the super-enhancer region leads to tissue-specific changes in Myc expression
Although the Myc 42-540/42-540 mice exhibited a normal phenotype, Myc expression was altered in a tissue-specific manner in these mice. This is expected since this region contains individual tissue specific regulatory elements. The expression of Myc was strongly decreased in colon, small intestine and prostate of these mice (Figure 3a  Binding of CTCF and Rad21 at a control Actb locus is not affected. Red and black arrowheads denote binding sites at Myc and Actb loci, respectively; green: Myc-CTCF mut/mut , blue: wild-type. The gene body for Myc and Actb is shown below the respective panels. The qPCR analysis reveals that despite loss of CTCF/cohesin binding, the expression of Myc mRNA is not altered in the colon (for qPCR, Myc-CTCF mut/mut n = 4, wild-type n = 3). See Figure 1-source data 1 for details. Error bars denote one standard deviation. DOI: 10.7554/eLife.23382.003 The following source data is available for figure 1:    The following source data is available for figure 3: compartment, we performed immunohistochemistry (IHC) for the proliferation marker Ki-67. Both the wild-type and Myc 42-540/42-540 had similar proliferative activity in the intestinal crypts ( Figure 3b). In contrast to colon and prostate, Myc expression was not markedly affected in the bladder, and was elevated in the spleen (Figure 3a). To analyze the cellular composition of the spleen, we performed flow cytometric analysis of markers for hematopoietic stem cells and lymphoid lineage cells. Myc 42-540/42-540 mice had a near normal hematopoietic compartment ( Figure 2d). The only observed difference was a small reduction of B cells in the Myc 42-540/42-540 mice compared to the wild-type mice both in the spleen and the bone marrow. In contrast to the decrease in B-cells, the T cell numbers were not affected by the deletion (Figure 2-figure supplement 1a). This finding is consistent with the published data that regulatory elements controlling T-cell development and T-cell acute lymphoblastic leukemia are located 1.47 Mb downstream of the Myc ORF (Herranz et al., 2014).
To identify regulatory elements that could explain the effect in B-cells, we performed ChIP-seq analysis of chromatin from LSK-Flt3 neg hematopoietic stem cells and mature B-cells isolated from wild-type mice. This analysis identified two B-cell specific regulatory elements. The Myc 2-540 deletion results in loss of one of the elements, and moves the other element very close to the Myc TSS ( Figure 2-figure supplement 1b). Although the exact regulatory mechanism is not clear and requires further study, the above data is consistent with a role of the super-enhancer region in development of chronic lymphocytic leukemia, which is primarily a B-cell malignancy. However, the decrease in B-cell number does not affect viability, and the Myc 42-540/42-540 mice are healthy and do not display an immune-deficient phenotype under normal 'clean' mouse housing conditions in the absence of known pathogenic microorganisms.
To compare the role of the 8q24 super-enhancer region in growth of cells in vivo and in cell culture, we isolated fibroblasts from the skin of adult Myc 42-540/42-540 and wild-type mice. Based on presence of active histone marks, and undermethylation of focal elements, the super-enhancer region is active in fibroblasts from both humans and mice ( Figure 4a and

Deletion of the Myc super-enhancer region affects MYC target gene expression only under culture conditions
To understand the mechanism by which the deletion of the 8q24 super-enhancer region has a differential effect on growth during normal tissue homeostasis and growth under culture conditions, we subjected both the mouse tissues and cultured cells to RNA-seq analysis. Analysis of mouse tissues confirmed the changes in Myc expression observed by qPCR (Figure 5a and Figure 5-figure supplement 1). Surprisingly, despite more than 80% decrease of Myc expression in the colon, very few genes were downregulated in the tissues, and none of the significantly altered genes were known MYC targets (Supplementary file 1). These results suggest that expression of canonical MYC target genes is not sensitive to decreases in MYC protein level during normal tissue homeostasis. In contrast to the in vivo situation, where Myc is downregulated but key target genes are not affected, in cultured Myc 42-540/42-540 fibroblasts that grew slowly in culture, the downregulation of Myc lead to a loss of expression of key target genes that drive cell growth and division. Upstream regulator analysis performed using Ingenuity Pathway Analysis revealed that the highest-ranked potential regulator for the identified gene set was MYC (Figure 5b).
Measured by FPKM values, the cultured wild-type fibroblasts had higher Myc mRNA levels than normal tissues, whereas the cultured null fibroblasts had Myc levels that were comparable to or lower than those of normal wild-type tissues. The elevated Myc levels in cultured cells are caused by serum stimulation, as Myc mRNA levels are low in serum-starved fibroblasts and strongly induced by serum (Ref. [Dean et al., 1986] and our unpublished data). These results indicate that the 8q24 super-enhancer region is dispensable for normal tissue homeostasis under conditions where MYC activity is relatively low. However, the region is required for induction of MYC activity to levels that are high enough to drive the expression of MYC target genes above their basal levels during pathological growth.
The Myc super-enhancer region is required for tumorigenesis in mice We have shown earlier that deletion of a 1.7 kb Myc-335 enhancer sequence located at the 8q24 super-enhancer region is required for intestinal tumorigenesis in mice (Sur et al., 2012b). As the super-enhancer region deleted in Myc 42-540/42-540 mice carries risk also for other cancer types, including breast and bladder cancer, we tested the susceptibility of the Myc 42-540/42-540 mice to carcinogen induced bladder and mammary tumorigenesis. The Myc 42-540/42-540 mice were not resistant to N-Butyl-N(4-hydroxybutyl) nitrosamine (BBN) induced bladder tumors. Both wild-type (n = 8)  and Myc 42-540/42-540 (n = 8) mice developed urothelial changes ranging from hyperplasia to high grade invasive urothelial carcinoma after 5 months of BBN treatment. In contrast, comparison of median tumor-free survival times of wild-type and Myc 42-540/42-540 mice exposed to mammarytumor inducing dimethylbenz[a]anthracene/ medroxypregesterone (DMBA/MPA) regimen revealed that the Myc 42-540/42-540 mice were partially resistant to mammary tumorigenesis ( Figure 6a). The median tumor-free survival time for the wild-type and Myc 42-540/42-540 mice was 88 and >120 days, respectively. Although we cannot pinpoint the specific regions that contribute to breast tumorigenesis by analysis of the Myc 42-540/42-540 mice, our work is consistent with the presence of a breast cancer susceptibility locus in humans at a region syntenic to the deletion. The region is distinct from the colon cancer susceptibility locus that harbors Myc-335.
To determine whether additional elements outside of the Myc-335 region are playing a role in tumorigenesis, we crossed the Myc 42-540/42-540 mice with the Apc min mouse that is susceptible to intestinal tumors. The Myc 42-540/42-540 mice had fewer polyps than the Myc-335 À/À mice in the Apc min background. In this study the wild-type mice had on an average 56 polyps at around 4 months of age (n = 5) when they had to be euthanized for ethical reasons similar to what we reported previously. The Apc min ; Myc 42-540/42-540 looked healthy and had on an average 2.4 polyps even at 6 months of age (n = 5) compared to an average of 14.33 polyps reported for the Apc min ; Myc-335 À/À null mice at 4 months of age (Figure 6b). Together with our earlier findings, these results indicate that loss of the 8q24 super-enhancer region makes mice resistant to both genetically and chemically induced tumors. We further tested the requirement of this region for the proliferation of cancer cell lines in cultures. We found that the corresponding region (hg19: chr8:128226490-128746456) was also required for GP5d colon cancer cell growth, as indicated by progressive loss of cells bearing a CRISPR/Cas9 induced deletion of the region during co-culture with unedited cells in the population (Figure 6c).

Discussion
The region around the MYC gene carries inherited risk towards multiple major forms of cancer. On aggregate, this region contributes more to inherited cancer than any other locus in the human genome. The risk alleles for different cancer types are located in multiple distinct linkage disequilibrium blocks, indicating that different variants contribute to different cancer types. Several of these regions containing risk variants have been implicated in regulation of MYC expression (Hallikas et al., 2006;Sur et al., 2012b;Herranz et al., 2014;Uslu et al., 2014), suggesting that a large number of enhancers within this region can drive tumorigenesis. Some of the identified elements have also been shown to have roles in normal development (Herranz et al., 2014;Uslu et al., 2014).
To study the role of the 8q24 region more systematically, we have in this work deleted several individual enhancer elements, and also analyzed the effect of larger deletions on normal development and carcinogenesis in mice. Our analysis of mice lacking a 538 kb region upstream of the Myc gene suggests that enhancer elements within this region cooperatively enhance Myc expression. Deletion of individual enhancers in this region has only a weak (Sur et al., 2012b) or no effect on Myc expression in the mouse intestine in contrast to the deletion of the entire super-enhancer region, which leads to severe decrease in Myc expression in multiple tissues.
MYC deficient mouse embryos die due to placental defect at E9.5. The embryos are also smaller in size than wild-type embryos (Davis et al., 1993). However, when Myc is deleted only in the epiblast, the embryos grow normally and survive until E11.5, when they die due to defects in hematopoiesis (Dubois et al., 2008). None of these defects are observed in mice homozygous for the deletion of super-enhancer region. The 8q24 super-enhancer region is thus dispensable for MYC function both in the placenta and during early hematopoiesis. In our mouse colony, the superenhancer region deficient mice also do not display the size or weight differences reported for Myc heterozygous mice that have a 50% reduction in MYC activity (Trumpp et al., 2001). These results indicate that tissue-specific enhancers that reside outside of the deleted regions drive sufficient MYC expression in the tissues that contribute to the phenotypes observed in Myc +/À and Myc À/À mice. Consistently with this, several hematopoietic enhancers have been identified in the region 3' of the MYC ORF (Hnisz et al., 2013;Shi et al., 2013;Herranz et al., 2014).
Myc heterozygous mice also display increased longevity and enhanced healthspan (Hofmann et al., 2015). Although the deletion of the super-enhancer region that contains tissuespecific enhancers regulating MYC expression is not equivalent to a heterozygous deletion of the Myc gene in the whole body, the Myc 42-540/42-540 mice could be an interesting model for identification of the tissues that contribute to the longevity phenotype.
Despite decreased levels of MYC in multiple adult tissues, the mice lacking the super-enhancer region are viable, fertile and display normal tissue morphology in all the tissues we investigated. They display no overt phenotype and do not have marked defects in cell proliferation. The mice are, however, resistant to intestinal tumorigenesis, and DMBA-induced mammary tumors, indicating that this region is important for tumorigenesis also in mice. Our data thus shows that despite the central role of this region in tumorigenesis (Sur et al., 2012b;Lovén et al., 2013), it is dispensable for normal tissue development and homeostasis under laboratory conditions. Whereas this result may appear very surprising, it is consistent with the original identification of this region using genomewide association studies (GWAS). GWAS has a high power to identify common variants, and most variants that are common have only a limited effect on physiological functions. This is because a variant that has strong positive or negative effect is rapidly fixed or lost, respectively. Thus, GWAS are specifically biased to find variants that have a relatively large effect on disease, but a small effect on fitness.
Most genes in mammals do not have haploinsufficient phenotypes. Such buffering could be due to mechanisms that maintain constant expression level irrespective of gene dose. However, a simpler buffering mechanism involves either expressing a gene at a very low level where it has no effect, or at a high level where it can contribute its functions even if its expression level is decreased due to transcriptional noise or loss-of-function of one allele. A similar two state mechanism where physiological transcription factor (TF) activity levels in the relevant cell types are either too low to drive any target genes (off state), or high enough to activate all important targets (on state) could also mechanistically explain why most heterozygous null mutations of TF genes have no apparent phenotype. Our analysis of the role of MYC in normal colon is consistent with such a simple buffering model (Figure 7). However, it should be noted that this buffering mechanism does not operate in all tissues and under all conditions. For example, Myc gene dose has effects on mouse size, longevity and hematopoiesis (Davis et al., 1993;Trumpp et al., 2001;Dubois et al., 2008;Hofmann et al., 2015). In addition, the level of expression of the Myc gene has quantitative effects on cell proliferation under pathological conditions such as activation of T-cells (Heinzel et al., 2017). These results indicate that in some situations, MYC is expressed at a level where cell growth responds linearly to small changes in MYC levels ( Figure 7, middle panel). However, the lack of an overt phenotype in our model under normal physiological conditions in the absence of infection or tissue damage suggests that growth during normal tissue homeostasis in at least some adult tissues does not linearly respond to changes in MYC levels. The lack of an overt phenotype should not, however, be taken to mean that the mice have no phenotype at all. As the super-enhancer region contains several highly conserved DNA segments, and affects cell growth in culture, we expect that it will also affect responses to tissue damage or some other perturbation that we have not investigated here. Therefore, further studies are needed to determine the role of the super-enhancer region in various chronic and acute models of injury and infection. Based on our data and the earlier literature we propose that under normal physiological conditions in the intestine, the Myc gene regulatory system is in the off state, and a basal level of expression of the MYC target genes is maintained by a MYC-independent mechanism. The target genes are thus only sensitive to an increase in MYC levels. Consistently, an 80% decrease of Myc mRNA expression does not lead to a proliferation defect, or major changes in expression of known MYC target genes. In contrast, in tumors the system is locked to an on state, where MYC targets are driven to a maximal level by MYC, and the targets are now only sensitive to a decrease in MYC activity (Figure 7).

Normal adult tissue homeostasis Signal independent rapid proliferation in tumors
The requirement of MYC in tumor cells appears absolute. In transgenic animal models, overexpression of MYC leads to deregulated proliferation and tumor development in multiple tissues (Felsher and Bishop, 1999;Pelengaris et al., 1999;D'Cruz et al., 2001;Jain et al., 2002;Shachaf et al., 2004). Furthermore, inhibition of MYC almost invariably causes growth arrest of cancer cells both in culture and in vivo (Soucek et al., 2002(Soucek et al., , 2004Hart et al., 2014). Despite the importance of MYC for cancer growth, it appears that the role of MYC in controlling growth during adult tissue homeostasis is limited. In the adult tissues, MYC is expressed in rapidly proliferating compartments of the body like the intestinal crypts and skin. Deletion of Myc in these compartments does not result in prominent proliferation defects (Wilson et al., 2004;Baena et al., 2005;Bettess et al., 2005;Muncan et al., 2006). Although there is still controversy regarding MYC requirement for the intestinal homeostasis, in the skin MYC is dispensable under normal adult proliferation and homeostasis in vivo (Oskarsson et al., 2006). It is however required for Ras mediated tumorigenesis and growth of fibroblasts and keratinocytes in vitro (Mateyak et al., 1997;Oskarsson et al., 2006). Taken together, these results suggest that MYC is required for pathological proliferation, but is less important and in many cases dispensable for normal homeostasis of tissues in the adult. Our results are consistent with these observations. Prior to our study it was not clear whether the MYC dependence of cancer cells in vivo and normal cells in culture is due to shared regulatory mechanisms. Our results have uncovered striking mechanistic similarities between growth of normal cells in culture, and growth of cancer cells in vivo by showing that MYC expression depend on the same genetic elements in cultured normal cells and in cancer cells. The similarity between tumor cells and cultured normal cells also suggest that many Figure 7 continued adult, MYC is expressed at intermediate levels to elicit response from targets with high affinity binding sites (red). In cancer cells or cells grown in culture (right), upstream regulators such as Tcf7l2 and b-catenin activate the Myc super-enhancers, driving high levels of MYC expression. This leads to the formation of MYC/MAX heterodimers that strongly activate transcription of MYC target genes driving cancer cell growth. The high levels of MYC are also sufficient to induce target genes that harbor low affinity MYC binding sites (grey). The model is consistent with the model of Lorenzin et al (Lorenzin et al., 2016) who showed genes differ in their response to MYC levels due to differences in the MYC affinity of their promoters. Given that Myc super-enhancer region is tumor-specific, and induction of the MYC target genes are not required for normal homeostasis, it provides a promising target for antineoplastic therapies. DOI: 10.7554/eLife.23382.017 potential drugs that block cancer cell growth may have been inadvertently discarded due to their negative effects on growth of normal cells in culture, even when they might not have affected normal tissue homeostasis in vivo.
Our results show that the MYC super-enhancer region that carries multi-cancer susceptibility in humans contributes to the formation of multiple tumor types also in mice. Despite its role in tumor formation, it is dispensable for normal development and homeostasis. Loss of the super-enhancer region leads to low MYC expression, but the lowered expression does not translate to changes in expression of MYC target genes in the intestine. Thus, the MYC/MAX/MNT system (Grandori et al., 2000) that drives cell growth and proliferation is robustly set to an off state during normal homeostasis, whereas in cancer, the system is locked to a pathological on state. This also explains how physiological growth control can be robust to small perturbations and transcriptional noise. Taken together, our results reveal an important difference between the transcriptional states of normal and cancer cells, and suggest that therapeutic interventions that decrease the activity of the Myc superenhancer region would be well tolerated.

Mouse strains
We generated cKO Myc-196 and cKO Myc-540 strains with loxP sites flanking the regions chr15:61445326-61447611 and chr15:61789274-61791107, respectively (Taconic). These mice were crossed to EIIa-cre mouse strain (Jackson Laboratory) to generate mice with enhancer deletions. Myc-CTCF mut mouse strain was generated by mutating the CTCF-binding site at chr15:61983375-61983647 TGGCCAGTAGAGGGCAC to TGGAACGTCTTGAATGC. In order to generate large deletions at the Myc locus (Myc 42-367 and Myc 42-540 ) Myc-367 À and Myc-540 À were crossed to Myc-CTCF mut that were also heterozygous for the Rosa26-Cre (Taconic). The Myc-540 À , Myc-196 À and Myc-CTCF mut carry one lox P site at the respective loci (chr15:61445326, chr15:61618287 and chr15:61983375). The loxP site on chr15:61983375 is located immediately 5' of the mutant CTCF binding site. We obtained compound heterozygotes carrying the chr15:61445326 or the chr15:61618287 loxP site together with the loxP site on chr15:61983375 and the Rosa26-Cre. The compound heterozygotes were screened by PCR for the interallelic recombination and the resultant deletion and duplication of the intervening sequence. Mice mosaic for the deletion and duplication were backcrossed to the C57Bl/6 mice in order to segregate the chromosomes carrying the deletion. The F1 heterozygotes were intercrossed to generate mice with homozygous large deletions. Myc-335 strain has been previously described (Sur et al., 2012b). All mice used in the study were on a C57Bl/6 genetic background. All mouse experiments were conducted in accordance with the local ethical guidelines, after approval of the protocols by the ethics committee of the Board of Agriculture, Experimental Animal Authority, Stockholm South, Sweden (Dnr S50/13, S11/15 and S16/15). The sequences of the different primer pairs used for genotypings are given in Supplementary file 2.

Mammary gland whole mount analysis
Inguinal mammary glands were removed from 8 week old virgin females and spread on glass slides. These were fixed for 4 hr in Carnoy's fixative and subsequently stained O/N with Carmine Alum. The whole mounts were rinsed and dehydrated through increasing series of ethanol and cleared in xylene before mounting with the pertex mounting medium.
Quantitative PCR analysis qPCR was performed as described previously (Sur et al., 2012b). Essentially, total RNA was isolated from whole tissue by homogenizing in RNA Bee reagent (ambios AMS Biotechnology) followed by RNA isolation using Qiagen's RNA MinElute kit according to manufacturers' protocols. 0.5-1 mg of total RNA was reverse transcribed using high capacity reverse transcription kit in a 20 ml reaction (Applied Biosystems). Quantitative PCR in triplicates was performed using the SYBR select master mix (Applied Biosystems) on the LightCycler 480 instrument (Roche). For normalization, mouse bactin transcripts were used as internal controls. Following primer pairs were used for quantitative PCR analysis.

RNA-sequencing
NEBNext Ultra Directional RNA library Prep kit (NEB) was used for preparing the samples for RNAseq together with the NEBNext Poly(A) mRNA magnetic isolation module (NEB) according to manufacturers protocol. In the case of tissues 1-2 mg and for cultured fibroblasts 200 ng of total RNA was used as starting material. For library preparation, adapters and index primers from NEBNext Multiplex Oligos for Illumina kit were used. The RNA-seq library was sequenced on a HiSeq2000 (Illumina). Sequencing reads were mapped to the mouse reference genome (NCBI37/mm9) using Tophat2 (version 2.0.13; RRID:SCR_013035) (Kim et al., 2013). Cuffdiff (version 2.2.1; RRID:SCR_ 001647) was used for differential gene expression analysis and for graphical representation, Cum-meRbund package (version 2.8.2; RRID:SCR_014568) (Trapnell et al., 2012) was used. The upstream regulator analysis was performed on all the significant differentially expressed genes (Cuffdiff q-value <0.05) using QIAGEN's Ingenuity Pathway Analysis (IPA, QIAGEN Redwood city, www.qiagen.com/ingenuity;version 24718999, updated 2015-09-14; RRID:SCR_008653).

Bisulfite sequencing
Genomic DNA was isolated using Qiagen's Blood & Tissue Genomic DNA extraction kit. Around 1 mg of wild-type and 250 ng of Myc 42-540/42-540 null fibroblast genomic DNA was sonicated to 300 bp fragments using Covaris S220 sonicator. Subsequent to end polishing and A base addition, cytosine methylated paired end adapters (Integrated DNA technologies) were ligated to the DNA fragments. The adapter sequences are as follows 5'-P-GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT After adapter ligation 300-600 bp fragments were size-selected on a 2% agarose gel. Bisulfiteconversion was carried out using ZYMO EZ DNA Methylation-Gold kit (cat. no. D5005). PCR amplification with 12 and 18 cycles was carried out to prepare libraries from the wild-type and Myc 42-540/ 42-540 null mouse fibroblasts, respectively. The primer pair used for PCR amplification was as follows PE PCR Primer P1: 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTC TTCCGATCT PE PCR Primer P2: 5'-CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACC GCTCTTCCGATCT The final library was size-selected for 250-300 bp fragments on a 2% agarose gel and 150 bp sequenced from both ends on two lanes of a HiSeq 4000 (Illumina). Raw sequencing reads were quality and adapter trimmed with cutadapt version 1.8.1 (RRID:SCR_011841) in Trim Galore version 0.4.0 (RRID:SCR_011847). Trimming of low-quality ends was done using Phred score cutoff 30. In addition, all reads were trimmed by 2 bp from their 3' end. Adapter trimming was performed using the first 13 bp of the standard Illumina paired-end adapters with stringency overlap two and error rate 0.1. Read alignment was performed against mouse genome mm9 with Bismark (version v0.14.3; RRID:SCR_005604) (Krueger and Andrews, 2011) and Bowtie 2 (version 2.2.4; RRID:SCR_005476) (Langmead and Salzberg, 2012). Duplicates were removed using the Bismark deduplicate function. Extraction of methylation calls was done with Bismark methylation extractor discarding first 10 bp of both reads and reading methylation calls of overlapping parts of the paired reads from the first read (-no_overlap parameter). Genomic sites with the coverage of at least 10 reads were considered and methylation ratios smoothed with loess method across 49 bp windows.

Isolation and culture of mouse primary fibroblasts
Fibroblasts were isolated from adult mice by dissecting the skin to~1 mm 3 pieces, and allowing the pieces to adhere to cell culture plates, followed by addition of DMEM medium supplemented with 10% FCS and antibiotics. The fibroblasts were allowed to migrate out from the explants, after which the cells were collected by trypsinization and passaged in the same media for 1-3 passages. For growth assays, 2 Â 10 3 cells were plated per well in 96 well plates. Cells were trypsinized and counted using hemocytometer at respective time points.

Tumor induction Mammary tumors
Six week-old female mice were implanted s.c. with medroxypregesterone acetate (MPA) pellets (50 mg with a 90 days release period from Innovative Research of America). Subsequently 100 ml of 10 mg/ml dimethylbenz[a]anthracene (DMBA)/oil solution (Sigma) was administered via gavage at 7, 8, 10, 11, 13 and 14 weeks of age. Mice were checked twice a week for development of palpable tumors. Detection of palpable mass in the mammary gland was taken as the end point for tumorfree survival analysis.

Bladder tumors
Ten week-old male mice were administered 0.1% N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) (Sigma) in drinking water for five months. At the end of the treatment the mice were sacrificed and the bladders scored for tumor development.

Intestinal tumors
Apc min mouse strain (Jackson Laboratory RRID:MGI:5438590) was used as a model for spontaneous development of intestinal tumors.
CRISPR-Cas9 mediated deletion of super-enhancer region in GP5d cell line CRISPR-Cas9 mediated deletion of MYC super-enhancer region on chromosome 8q24 (GRCh37/ hg19 chr8: 128226403-128746490) and Immunoglobulin Heavy (IGH) gene locus on chromosome 14q32.33 (GRCh37/hg19 chr14: 106527004-107035452) were carried out in GP5d (Sigma, 95090715; RRID:CVCL_1235, confirmed by STR profiling at ECACC) colon cancer cell line stably expressing Cas9 protein. A lentiviral plasmid containing Cas9 fused via a self-cleaving 2A peptide to a blasticidin resistance gene, was packaged into lentiviral particles using the packaging plasmids psPAX2 (a gift from Didier Trono, Addgene plasmid # 12260, RRID:SCR_002037) and pCMV-VSV-G (a gift from Robert Weinberg (Addgene plasmid # 8454, RRID:SCR_002037). The virus was used to transduce GP5d colon cancer cells. 48 hr after transduction, GP5d cells expressing Cas9 (GP5d-Cas9) were selected in 5 mg/ml Blasticidin (Thermo Fisher Scientific Inc., Cat. no. A1113903). The single guide RNA (sgRNA's) were designed (http://www.broadinstitute. org/rnai/public/analysis-tools/sgrna-design) to span the entire MYC super-enhancer region and IGH locus (Figure 6), respectively (Eurofins MWG Operon). sgRNAs were cloned into an sgRNA Cloning Vector (Addgene Plasmid #41824, RRID:SCR_002037) using Gibson assembly master mix (NEBuilder HiFi DNA assembly Master Mix, Cat no. E2621S). GP5d-Cas9 (2 Â 10 6 ) cells were transfected (using FuGENE HD Transfection Reagent, Cat.no E2312) with 10 mg of eight pooled equimolar sgRNA constructs. Post transfection half of the cultured cells were collected for PCR genotyping, while the other half was re-plated for culturing. Cells were collected at day 2, 4 and subsequently every fourth day till day 32. DNA from cells was extracted (using DNeasy Blood & Tissue Kit, Qiagen Cat. no. 69506) and genotyped with 300 ng of DNA at following conditions -Initial denaturation of 95˚C for 5 min; denaturation of 98˚C for 15 s, annealing at 60˚C for 30 s, extension at 72˚C for 30 s (30 cycles for MYC super-enhancer region and 35 cycles for IGH gene locus deletion genotyping); final extension at 72˚C, 5 min. Each experiment was done in triplicate. The sequences of the different guide RNAs and primer pairs used for PCR genotyping of the deletions are given in Supplementary file 2. GP5d cells were cultured in DMEM supplemented with 10% FBS and antibiotics. The cell line was mycoplasma free. The following previously published datasets were used: