By the recruitment of HATs and the SWI/SNF complex, MYC can remodel the nucleosomal topography to promote transcription. But the question remains as to whether MYC can initiate transcription. Results have shown that MYC cannot initiate de novo
transcription, but is necessary to maintain a nucleosomal landscape permissive for transcription (Knoepfler et al., 2006
; Soufi et al., 2012
). Extensive chromosome immunoprecipitation and sequencing (ChIP-Seq) analysis has revealed MYC E-box binding is dependent on chromatin context. Without specific euchromatic islands characterized by methylated H3K4 and H3K79 and acetylated H3, MYC is unable to access E-boxes on its target loci (Guccione et al., 2006
). Intriguingly, MYC also binds to euchromatic islands not on E-boxes, which may also explain MYC E-box binding degeneracy.
In NB, a genome-wide assessment of MYCN using the tetracycline (TET) suppressible MYCN SHEP NB model system and ChIP hybridizations to gene promoters (ChIP-Chip) revealed that MYCN binding associates with euchromatic histone marks H3K4me and H3K9Ac (Cotterman et al., 2008
). The correlation of MYCN binding and microarray analyses revealed that when MYCN was induced, MYCN was found bound to genes that were already transcriptionally active. MYCN was not associated with de novo
gene transcription. This is consistent with the recent models of MYC binding at or near transcription start sites (TSS) and functioning to amplify transcription of actively transcribed genes (Lin et al., 2012
; Nie et al., 2012
; Soufi et al., 2012
). An interesting aspect of the Cotterman’s study was the finding that upon loss of MYCN expression there was a global loss of the euchromatic marks even at sites that were not bound by MYCN (Cotterman et al., 2008
). This implies that MCYN plays a role in regulating euchromatic regions in a manner that is independent of its role as a classic transcription factor. One caveat raised in this study was whether the assays were sufficiently sensitive to detect low affinity MYCN binding sites. The utilization of more sensitive and quantitative technologies such as MYCN ChIP-seq should clarify this in the future.
MYC dependence on chromatin context is further supported by studies revealing the temporal basis for chromatin remodeling. During cellular reprogramming ectopic expression of transcription factors can induce global chromatin changes as a cell reverts from a differentiated state to a pluripotent state. Temporal analysis of reprogramming revealed that among the reprogramming factors Oct4, Sox2, Klf4, and MYC (OSKM), only OSK can bind to distal enhancer regions at an early time during reprogramming. Subsequently MYC is recruited to the TSS to stabilize chromatin binding and promote transcription. Furthermore, OSK proteins are all able to bind to one side of the DNA helix, suggesting they are able to bind to heterochromatic regions. MYC does not bind to heterochromatic regions. MYC is predominantly found on euchromatic regions indicating it requires a prior transcriptional stimuli or de novo
factors to bind to DNA to initiate its transcriptional function (Soufi et al., 2012
Since MYC cannot initiate de novo
transcription, the question arises as to how MYC can promote the induction of transcription initiated by reprogramming factors. Regulation of gene transcription in complex chromatin models is still evolving and occurs in at least five steps: (1) initiation, (2) promoter pausing, (3) mRNA capping, (4) promoter escape, and (5) transcript elongation. A clue to MYC function during initiation lies with MYC binding to TBP (Figure
; McEwan et al., 1996
). MYC is a direct TBP binding partner and may be able to couple the pre-initiation complex to its target promoters. Moreover, MYC has been implicated at mRNA capping, promoter escape, and elongation.
Until recently, the predominant or rate limiting mechanism regulating initiation of transcription on gene promoters was thought to be the binding or loading of Pol II onto target DNA sites, termed initiation. Pol II bound at promoters is marked by phosphorylation by TFIIH on serine 5 of its carboxy-terminal domain (CTD), p-Ser5-Pol II. Genome-wide analyses of Pol II binding demonstrated that p-Ser5-Pol II commonly resides at promoters or is paused 20–40 bases downstream of the promoters of many non-expressed genes. The capping machinery is recruited to the CTD of Pol II and results in the addition of a methylated guanine at the 5′-triphosphate of the nascent mRNA strand. MYC also binds to TFIIH and the increased p-Ser5-Pol II increases capping associated methylation. Capping is required for the recruitment of the translation machinery and mRNA binding to ribosomes. The acetylated lysine binding bromodomain containing protein BRD4, recruits P-TEFb complex which phosphorylates serine 2 on the CTD region of Pol II (p-Ser2-Pol II) causing elongation of mRNA transcripts and promoter escape (Figure
; Rahl et al., 2010
). MYC also binds to P-TEFb enhancing P-TEFb kinase activity. While MYC effects on transcriptional elongation are dependent on its DNA binding activity, evidence indicates that MYC effects on capping are independent of its DNA binding activity (Cole and Cowling, 2008
; Cowling and Cole, 2010
By studying reciprocal events in differentiation and reprogramming the progression from transcriptional initiation to elongation can be extrapolated and pathologic consequences can be evaluated. During reprograming OSK function much like pioneering factors during embryonic development and differentiation. Pioneer factors initially bind to heterochromatic regions and then recruit cofactors and other transcription factors to initiate transcription (Cirillo et al., 2002
; Zaret and Carroll, 2011
). MYC would then be recruited to promoter regions to reinforce the permissive chromatin state. In neural progenitors loss of MYC results in increased H3K9me2, a mark of repressive chromatin, decreased acetylated H3 and H4, marks of active chromatin, and overall chromatin condensation characterized by increased heterochromatin. The changes in histone architecture are also noted to be cell cycle and differentiation independent, but higher MYC expression did correlate with increased nuclear size (Knoepfler et al., 2006
). This change in nuclear size can be seen in Figure
, in which MYCN transfection into the NB cell line SK-N-AS (clone 14.2) results in cells with a relative nuclear size twice that of the parental or control-transfected (8B) cell lines which contain a single copy of MYCN and do not express MYCN mRNA (Figure
). Decreased euchromatic regions have also been identified in tumor models when MYC is knocked down (Wu et al., 2007
). The results suggest MYC is essential in euchromatin maintenance after its initial recruitment. In this context, the importance of MYC in neural progenitors may derive its importance from stimulating transcription of nascent factors necessary for neural proliferation before being down-regulated to initiate terminal differentiation.
FIGURE 3 MYCN influences global chromatin structure. (A) The SK-N-AS cell line contains a single copy of MYCN and does not express significant quantities. Upon transfection of SK-N-AS with MYCN (14-2, Vector control: 8B), one can see enlarged nuclei in high MYCN (more ...)
MYC AND TRANSCRIPTIONAL REPRESSION
As a transcriptional amplifier, transcripts targeted by MYC would be expected to be up-regulated, but there remains a sizeable cohort of down-regulated MYC targets. MYC does not bind to corepressor complexes, so it is reasonable to surmise the down-regulated genes are secondary or indirect effects of MYC function. A recent study by Valentijn et al. (2012)
identified a MYCN gene signature set in NB cell lines of 157 genes in which a subset was transcriptionally repressed genes (Valentijn et al., 2012
). ChIP analyses on promoter arrays indicated that MYCN exhibited relatively weaker binding to the TSS of repressed genes compared to binding at the TSS of up-regulated genes. This is consistent with the recent MYC ChIP-seq studies that did not find MYC binding to TSS of genes repressed by MYC expression (Lin et al., 2012
; Nie et al., 2012
Early models of transcriptional suppression associated with MYC and MYCN involve an indirect mechanism in which MYC/MAX dimers compete with other bHLH-Zip transcription factors for E-box binding to alter target gene activity. For example the MYC antagonist MAD also has chromatin modifying abilities that may explain how it can modulate MYC function. MAD is able to recruit histone deacetylases (HDACs), via SIN3 a transcriptional corepressor, to target loci to induce transcriptional silencing (Laherty et al., 1997
; Ayer, 1999
; Knoepfler and Eisenman, 1999
). Thus the model arises where MYC/MAX recruit HATs to induce chromatin acetylation making it more permissive for transcription and MAD/MAX recruit HDACs to deacetylate chromatin thereby silencing transcription (Figure
). In NB, the combined treatment of RA and Interferon-gamma (INF-γ) has been shown to dramatically decrease cell growth and induce differentiation (Lucarelli et al., 1995
). It was recently shown that the transcriptional repression of two genes associated with the growth of NB cells, ornithine decarboxylase (ODC) and human telomerase reverse transcriptase (hTERT) was associated with a shift in promoter occupancy of MYCN/MAX binding at steady-state to MAD/MAX binding after RA and INF-γ treatment. This results in a decrease in H4 acetylation at these promoters and decreased expression of ODC and hTERT mRNA (Cetinkaya et al., 2007
). The precise levels of activation and silencing probably exist in stoichiometric balance and are dependent on environmental and cellular mechanisms regulating MYC and MAD expression.
Another model of MYCN-mediated transcriptional repression invokes the association of MYC binding to the MIZ and SP1 transcriptional activators resulting in the recruitment of HDACs. This has been demonstrated for two genes associated with good prognosis and differentiation in NB, NTRK1(TrkA), and p75(NGFR). Upon induction of MYCN, there is increased HDAC1 and decreased acetylated H3 binding over the TSS of these promoters. The functional presence of MYCN was found to attenuate their promoter activity (Iraci et al., 2011
). One can see how the increased levels of MYCN that accompanies gene amplification would disrupt normal cellular transcription factor stoichiometry leading to aberrant gene regulation.
Most recently MYC has been found to transcriptionally amplify epigenetic modifiers with transcriptional repressive activities. For example, in ESCs MYC stimulates all components of the polycomb repressive complex 2 (PRC2), including embryonic ectoderm development (Eed), suppressor of zeste 12 (Suz12), and histone methyltransferase enhancer of zeste homolog 2 (EZH2), in ESCs (Zhang et al., 2005
; Neri et al., 2012
). PRC2 functions to epigenetically silence gene expression, but at a different histone residue. PRC2 catalyzes trimethylation of H3K27 (H3K27me3). It is believed that the persistent H3K27me3 at particular gene loci results in the recruitment of the PRC1 complex to induce chromatin compaction (reviewed in Bernstein et al., 2007
; Simon and Kingston, 2009
). In NB relatively high levels of EZH2 are associated with undifferentiated NB tumors (Wang et al., 2012
). The dysregulation of EZH2 may be due to a number of factors including MYCN, increased chromosome 7 copy number or loss of a regulator miR-101, which resides on 1p36. Functionally EZH2 was reported to suppress a number of genes with tumor suppressor activity in NB including the MYCN-regulated genes CLU and p75(NGFR). Enhanced EZH2 and it target H3K27me3 binding were detected at potential MYCN binding sites at steady-state conditions but the addition of an HDAC inhibitor was associated with decreased EZH2 and H3K27me3 binding and increased CLU and p75 expression (Wang et al., 2012
). It is not known whether PRC2 complex components directly bind to MYC/MYCN but the enhanced binding of EZH2 and H3K27me3 covered an E-box in the CLU promoter and was over the TSS area identified to bind MYCN in the p75(NGFR) promoter (Iraci et al., 2011
Furthermore, in NB cell lines and tumor samples, MYCN stimulates B cell-specific Moloney murine leukemia virus integration site (BMI1) expression. BMI1 is a member of the PRC1 complex whose targets are often hypermethylated suggesting epigenetic silencing (Ochiai et al., 2010
). As part of a suppressive complex, BMI1 has been shown to repress tumor suppressor genes and catalyze events leading to NB tumorigenesis. BMI1 is highly expressed in NB cell lines and NB tumor samples and was shown to have anti-apoptotic effects via stimulation of RING1A/B ubiquitin-mediated degradation of P53 (Cui et al., 2007
; Calao et al., 2012
). The pro-survival effects of BMI1 suggest MYCN induction leads to greater chances for tumor initiating events. Beyond BMI1’s transcriptional targets in NB, PRC1 has also been shown to inhibit the transcriptional pre-initiation complex thereby inhibiting Pol II-mediated transcription (Lehmann et al., 2012
). The summation of the events suggests BMI1/PRC1 suppress active transcription and catalyze epigenetic silencing, which may lead to NB tumorigenesis.
Given MYCN’s role in stimulating expansion of neural progenitors, it is not unexpected that differentiation genes like NTRK1 or p75(NGFR) are repressed by MYCN. A global survey by ChIP-Chip of ESCs for MYC and Miz-1 binding sites indicated that almost 30% of Miz-1-regulated genes could be repressed by MYC. These included homeobox genes, developmental proteins and genes involved in regulation of apoptosis. In NB tumor samples, within the MYCN gene signature set, 30% of the down-regulated genes in the set are neuronal tissue-specific genes while only 2% of such genes are up-regulated (Valentijn et al., 2012
). This is consistent with one of the first studies on MYCN regulation in NB cells which indicated that retinoid mediated down-regulation of MYCN occurred prior to evidence of differentiation (Thiele et al., 1985