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. 2006 Jun 21;25(12):2723-34.
doi: 10.1038/sj.emboj.7601152. Epub 2006 May 25.

Myc influences global chromatin structure

Paul S Knoepfler  1 , Xiao-yong Zhang, Pei Feng Cheng, Philip R Gafken, Steven B McMahon, Robert N Eisenman
Affiliations

Myc influences global chromatin structure

Paul S Knoepfler et al. EMBO J. .

Abstract

The family of myc proto-oncogenes encodes transcription factors (c-, N-, and L-Myc) that regulate cell growth and proliferation and are involved in the etiology of diverse cancers. Myc proteins are thought to function by binding and regulating specific _target genes. Here we report that Myc proteins are required for the widespread maintenance of active chromatin. Disruption of N-myc in neuronal progenitors and other cell types leads to nuclear condensation accompanied by large-scale changes in histone modifications associated with chromatin inactivation, including hypoacetylation and altered methylation. These effects are largely reversed by exogenous Myc as well as by differentiation and are mimicked by the Myc antagonist Mad1. The first chromatin changes are evident within 6 h of Myc loss and lead to changes in chromatin structure. Myc widely influences chromatin in part through upregulation of the histone acetyltransferase GCN5. This study provides the first evidence for regulation of global chromatin structure by an oncoprotein and may explain the broad effects of Myc on cell behavior and tumorigenesis.

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Figures

Figure 1
Figure 1
Analysis of N-Myc levels and histone acetylation. (A) E12.5 Sagittal sections from control (panels i and v), N-myc null (panels ii and vi), and N-myc transgenic embryos (Tg) expressing moderately (iii and vii) and highly elevated levels of N-Myc (iv and viii) under control of the nestin promoter/enhancer. Embryos were stained for N-Myc (brown stain, panels i–iv) and AcH3 (brown stain; panels v–viii) and counterstained with methyl green (bluish). Nuclear and cytoplasmic N-Myc proteins are evident, as has been previously reported (Wakamatsu et al, 1993) (× 100 magnification). Boxed regions are shown at higher magnification in Supplementary Figure S1A. (B) CGNPs derived from N-mycflox/flox (left three columns) or c-mycflox/flox (right column) embryos were grown in medium containing Shh (3 ng/ml), infected with GFP or Cre-IRES-GFP retroviruses, and stained with DAPI and anti-AcH3. White arrows denote infected cells. (C–F) Control and nestin-cre-derived N-myc null CGNPs stained for DAPI (blue) and histone H3 diMeK9 (C), triMeK4 (D), and triMeK9 (E) (red). (F) Control and N-myc null CGNPs stained for DAPI (blue) and HP1α (monochrome). (G) Cultured E12.5-derived control and N-myc null neurospheres stained for DNA (DAPI), N-Myc (green), and AcH3 (red). Columns 3 and 4 represent null cells transfected with N-Myc. White arrow indicates untransfected cell. Yellow arrows indicate NPCs with supraphysiological levels of N-Myc. Control (flox/flox) and null (flox/flox nestin-cre) cultures were grown for at least 1 month before these analyses and exhibited a high degree of stability in culture composition and properties (extremely low, but similar rates of spontaneous differentiation, stable proliferation rates, stable neurosphere morphology and size, and stable cellular morphology upon growth as a monolayer). We consistently observed that 80–90% of the N-mycflox/flox nestin-cre+ neurosphere cells exhibited a complete loss of detectable N-Myc protein.
Figure 2
Figure 2
Histone modifications in c-myc null, mad1–transfected, and WT fibroblasts. (A) Staining for AcH4 (red) in parental (TGR), c-myc null (HO15.19), and c-myc null rat fibroblasts stably transfected with c-myc. (B) Mad1 overexpression in WT murine fibroblasts partially phenocopies loss of Myc. (C) Immunoblotting for the indicated acetylated and methylated histones H3 and H4 comparing levels in TGR, c-myc null, and c-myc null rat fibroblasts transfected with c-myc. Values in the graphs represent the ratio of arbitrary values measured by Odyssey system for bands representing modified/total histones from three independent biological repeats. (D, E) Mass spectrometric data on total and K-specific histone H4 N-terminal acetylation from three independent experiments. The relative fraction of N-terminal peptides containing 0–4 acetyl groups was determined as well as the fraction of specific K residues that were acetylated. All error bars in this figure are s.e.m.
Figure 3
Figure 3
Histone modifications, heterochromatin, and DNA accessibility in control and null N-myc CGNPs. (A) N-mycflox/flox nestin-cre CGNPs were visually sorted by nuclear size following staining with the indicated antibodies. Each color intensity in column 6 was uniformly increased in each panel to maximize detection of potential colocalization of signal. (B) Control (top) and N-myc null (bottom) CGNPs were analyzed by EM following uranyl acetate staining (panels i and iii–vi) or by DAPI (panel ii). Arrows indicate heterochromatic regions. Note: Although the scale bar in panel (i) is 2 μm and the bars in the other panels are 1 μm, the bar in (i) is also twice as long as the bars in the other panels, indicating that all images are exactly of the same magnification. (C) MNase assay on Tet-off Myc B cells with and without 72 h of tetracycline treatment.
Figure 4
Figure 4
Differentiation of control and N-myc null neurospheres influences histone modifications. IF staining of control and N-myc null neurospheres induced to differentiate for 7 days by growth factor withdrawal and retinoic acid treatment.
Figure 5
Figure 5
GCN5 is a direct Myc _target gene and can reverse histone hypoacetylation in N-myc null cells. (A, B) Shutting off Myc in the Tet-repressor Myc B cells leads to a reduction in GCN5 levels, but has no effect on the levels of other HATs, HDACs, and HMTs as indicated by IF (monochrome) and immunoblot with antisera against the indicated protein. (C, D) shRNA-mediated KD of endogenous Myc results in decreased hGCN5 mRNA levels. Treatment of the human lung cancer cell line H1299 for 48 h with shRNA directed against Myc (shMyc) results in decreased levels of Myc mRNA and protein, whereas a scrambled shRNA (shScr) had no effect. Concomitant with the loss of Myc expression is the loss of hGCN5 at both the protein and mRNA level. (E) Myc activates transcription of the hGCN5 gene in primary human cells. The human diploid fibroblast strain IMR90 was stably transduced with a retrovirus directing expression of the Myc-ER protein. Myc-ER-expressing cells were treated with 4-OHT (or EtOH as a negative control) to activate c-Myc for the times indicated. mRNA was harvested and analyzed by quantitative RT–PCR. Actin mRNA levels were determined simultaneously and used to normalize mRNA levels for the other genes. hGCN5 levels are increased by Myc activation even more dramatically than those of the known Myc _targets CAD and cyclin D2. Furthermore, mRNA for the other TRRAP-associated HATs, PCAF and TIP60, were not responsive to Myc activation. The non-Myc responsive gene ELF1a was used as a negative control. Values are expressed as fold induction. (F) Direct binding of endogenous Myc to the hGCN5 locus in vivo. The human GCN5 locus contains two matches to the CACGTG consensus Myc binding site as indicated. In addition, several matches to the non-canonical site bound by Myc in the cytochrome c gene (CATGCG) are present. To examine the binding of Myc to these sites, we utilized human diploid fibroblasts that had been either serum deprived or re-fed with 10% FCS for 2 h. Binding of endogenous Myc to three sites within the hGCN5 locus was then assessed. Inducible binding of Myc to site 2, which is adjacent to the transcriptional start site, was evident. (G) Overexpression of hGCN5 in N-myc null neurospheres reverses histone hypoacetylation as effectively as reintroduction of N-myc. Null neurospheres were transfected with N-myc or hGCN5 (red), then IF stained for acetylation of histone H3 (green).
Figure 6
Figure 6
A critical role for GCN5 in Myc's regulation of chromatin. (A) RNAi-mediated KD of endogenous GCN5 in N-myc null neurospheres. IF analysis with GCN5 antisera (red) indicating variably reduced GCN5 protein levels with five RNAi constructs _targeted against GCN5. Column 1, no RNAi treatment; column 2, control RNAi treatment; columns 3–7, treatment with indicated GCN5 RNAi. Columns 1–7 are N-myc null neurospheres, whereas column 8 is control neurospheres with no RNAi treatment. (B) RNAi KD of endogenous GCN5 blocks the ability of exogenous N-Myc to rescue histone hypoacetylation in N-myc null neurospheres. Representative IF images are shown. Columns 1–7 are cells treated with the same RNAi constructs as in (A) but also followed 48 h later by transfection with exogenous N-Myc (red) and stained for acetylated histone H3 (green) as well as DAPI (blue). Cells expressing exogenous N-Myc are indicated by white arrows. The N-Myc IF panel in column 8 for the Fl/Fl cre− control is from a separate experiment but is included to show representative control levels of endogenous N-Myc protein for comparison. (C) Quantitation of the ability of GCN5 RNAi to block rescue. Mean values from four independent groups of 10 N-Myc-transfected cells that were also previously transfected with each type of RNAi are shown with error bars of s.e.m. The differences between the values for the control RNAi and the three strongest RNAi (RNAi #2, #3, and #4) exhibit P-values <0.0002, 0.00005, and 0.0009, respectively.
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