Supporting Online Material for

www.sciencemag.org/cgi/content/full/321/5892/1086/DC1

Supporting Online Material for

Epigenetic Reprogramming by Adenovirus e1a

Roberto Ferrari, Matteo Pellegrini, Gregory A. Horwitz, Wei Xie, Arnold J. Berk, Siavash K. Kurdistani*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 22 August 2008, Science 321, 1086 (2008) DOI: 10.1126/science.1155546

This PDF file includes:

Materials and Methods SOM Text

Figs. S1 to S13

Table S1

References

Figure S1. Schematic structure of small e1a and its interactions with cellular proteins required for oncogenic transformation. Small e1a has three conserved regions (CRs), the N-terminus-CR1, CR2 and CR4, and lacks CR3, which is present in large E1A. The domains required for various e1a functions are indicated as bars beneath the map. The location and consensus sequences of binding motifs necessary for interaction with p300/CBP, RB and CtBP are as indicated. CR1 and conserved residues in the N-terminus are required for interaction with p300/CBP. CR2 binds to the RB protein family, primarily through an LXCXE-motif in CR2 but residues in CR1 also contribute to the binding affinity. See Frisch and Mymryk, 2002 and Liu and Marmorstein, 2007 for discussions of the contributions of each of these regions to the binding. The region of acetylation site in e1a by CBP/p300 is also depicted. (Adapted form Berk, A.J., 2005 and Turnell, A.S. and Mymryk, J.S. 2006)

CR1 CR4 243

N CR2 C

42 72 115 139 205 242

FXD/EXXXL DLXCXE PXD/NLS

Binding motifs

Nuclear localization signal Acetylation site

Repression of Transformation with G12V-HRAS (required for transformation

with E1B) S-phase induction

Transformation with E1B/G12V-HRAS

Transcription Activation/repression

Suppression of differentiation

Functional domains E1A-binding proteins targeted

during transformation

p300/CBP

RB p107

p130 CtBP

1

Figure S2. Infected IMR90 fibroblasts enter the S-phase between 18 and 24 h post infection (p.i.). Percent of cells in S-phase before and every 6 h p.i. of contact inhibited IMR90 cells. Cells were infected with dl1500 (which expresses WT small e1a), R2G, ΔCR2, and the complete E1A deletion mutant, dl312. Mock control is also shown.

Time h p.i.

Percent nuclei with BrdUr

0 10 20 30 40 50 60

6 12 18 24 30 36

Mock dl312 dl1500 R2G CR2

2

GO class p-Value*

cytokines 5.6E-7

cell adhesion 3.8E-6

Inflammatory response 3.7E-5 response to other organism 4.2E-3

development 9.5E-31

cell-cell signaling 4.7E-18 response to wounding 1.3E-14 cell differentiation 6.1E-11

1

3

cluster

biopolymer metabolism 1.1E-65

RNA metabolism 3.6E-40

cell cycle 2.1E-28

ubl conjugation pathway 4.1E-20

2

*p-values are corrected for multiple hypothesis testing

A

INTERPRO: Term number p-value*

IPR001356:Homeobox 120 9.1E-4

IPR012287:Homeodomain-related 134 8.0E-3

Figure S3. The three cluster of genes bound by small e1a are enriched for biologically-related genes. A) GO analyses of the three clusters in Fig 1A. B) Cluster 3 genes (Fig 1A), which are enriched for small e1a binding at 24 h p.i., show significant enrichment for homeobox-domain containing genes. GO analysis was done with the

Functional Annotation Tool at David Gene Ontology NIAID website (NIH)

(http://david.abcc.ncifcrf.gov/home.jsp). Shown are the enriched domains using the INTERPRO database. The p- values are benjamini corrected for multiple-hypothesis testing.

B

*p-values are corrected for multiple hypothesis testing

3

POLE2

-5.5 TSS 2.5

-2 6

-5.5 TSS 2.5

e1a binding nln(IP/INP) ^{e1a 24h}

0

Cluster 2 nlnR(IP/IP)

-4 8

-4 8

e1a binding nln(IP/INP)

Cluster 1 p130 binding ln(IP/IP)

0 0

IL7R

e1a 6h p130 24h

-5.5 TSS 2.5

nlnR(IP/IP) 0 -5.5 TSS 2.5

H3K18ac 6h

-2

4 _{p300 6h}

p300 24h H3K18ac 24h

-2.5 6

-5.5 TSS 2.5 -2.5

0 2.5

Expression ln(dl1500/mock) -1

0 1

Expression ln(dl1500/mock)

TSS 2.5

p300 6h p300 24h

TSS 2.5

e1a6h e1a 24h

e1a binding nln(IP/INP) 0 -2

6 HELLS

-2 4

-5.5

-2 8

-5.5 TSS 2.5

H3K18ac 6h H3K18ac 24h

-2.5 0 2.5

Expression ln(dl1500/mock)

Cluster 3

HOXB6

nlnR(IP/IP) -5.5 TSS 2.5

0 -2

6

-5.5 TSS 2.5

0

e1a 6h e1a 24h

e1a binding nln(IP/INP)

-2.5 0 2.5

Expression ln(dl1500/mock) -2

6

-2

6 _{p300 6h}

p300 24h H3K18ac 6h

H3K18ac 24h -4

6

-4 6

-5.5 TSS 2.5

nlnR(IP/IP)

-5.5 -5.5 TSS 2.5

Figure S4. Representative genes from each cluster showing time-course ChIP values for selected parameters. A) Interleukine 7 Receptor (IL7R) as an example of cluster 1 genes. Shown are e1a binding at 6 h p.i., p130 at 24 h p.i., p300 binding and H3K18ac at 6 and 24h p.i. as well as expression data at 24h p.i. B) DNA polymerase epsilon subunit 2 (POLE2) and Lymphoid Specific Helicase (HELLS) as examples of cluster 2 genes. Shown are e1a and p300 bindings and H3K18ac at 6 and 24 h p.i. as well as expression data at 24 h p.i. C) Homeobox B6 (HOXB6) as an example of cluster 3 genes. Shown are e1a and p300 bindings and H3K18ac at 6 and 24 h p.i. as well as expression data at 24 h p.i.

p300 6h

p300 24h H3K18ac 6h

H3K18ac 24h

0

0 0

0 0

0

nlnR(IP/IP)nlnR(IP/IP)

nlnR(IP/IP)

A

B

C

e1a 6h

e1a 24h e1a 6h

4

Figure S5. Small e1a binding is enriched at potential E2F target genes early after e1a expression. The target genes of various E2Fs were determined based on ChIP-chip studies in other cell lines (9). A) Average WT e1a binding (WT, left panel; R2G, middle panel; ∆CR2, right panel) across the promoter regions of E2F4, B) E2F1 and C) E2F6 target genes at 6 and 24 h p.i. (blue and green lines, respectively). D) Calculated Z-scores for enrichment of each E2F target genes in each of the three e1a clusters (Fig 1A). E) log(p-values) for the Z-scores in D are indicated.

WTe1a-clusters E2F1 E2F4 E2F6 1 -20.42 -20.62 -33.03

2 27.73 35.61 24.55

3 -8.80 -14.57 0.93

WTe1a-clusters E2F1 E2F4 E2F6

1 -182.691 -186.259 <-200.000 2 <-200.000 <-200.000 -263.433

3 -34.8036 -93.5664 -0.72337

Log(p-values)

D

E

B

Average binding nln(IP/INP)

WTe1a

-0.4 -0.2 0 0.2 0.4

TSS 2.5

TSS

-5.5 2.5

24h6h

-5.5

-0.4 -0.2 0 0.2

0.4 _6h

24h

WTe1a

Average binding nln(IP/INP)

-0.4 0 0.4

0.8 R2Ge1a

TSS

-5.5 2.5

-0.4 0 0.4 0.8

TSS

-5.5 2.5

R2Ge1a

Average binding nln(IP/INP)Average binding nln(IP/INP)

-0.3 0 0.3

0.6 CR2e1a

TSS

-5.5 2.5

-0.4 0 0.4 0.8

TSS

-5.5 2.5

CR2e1a

Average binding nln(IP/INP)Average binding nln(IP/INP)

E2F1 target genesE2F6 target genes

-0.8 -0.4 0.0 0.4 0.8

TSS

-5.5 2.5

24h6h

A

-0.6 -0.3 0.0 0.3 0.6

E2F4 target genes TSS

-5.5 2.5

6h24h

Average binding nln(IP/INP) -0.4 -0.2 0 0.2

0.4 6h

24h

TSS

-5.5 2.5 Average binding nln(IP/INP) Average binding nln(IP/INP)

wt e1a R2G e1a Δ CR2 e1a

C

24h6h

24h6h 24h6h

24h6h

5

Figure S6. FIRE analysis of the three gene clusters identifies recurrent motifs within 1 Kb region upstream the TSS. Genes associated with a cluster were scanned for recurrent motifs within 1 kb region upstream of the TSS using the FIRE algorithm (http://tavazoielab.princeton.edu/FIRE/). The identified motifs are ranked by a calculated Z-score. The sequence logos represent the optimized motifs. Heat map for motif enrichment is shown on the left according to the scale.

cluster

1 2 3

6

Figure S7. H3K18ac chromatin immunoprecipitation in e1a-infected cells yields three times less DNA than from mock-infected cells. Cellular hypoacetylation in small e1a infected cells has been detected by IF, WB and mass spectrometry (see text and Horwitz et al. 2008 in the same issue).

Chromatin immunoprecipitation of H3K18ac in e1a infected IMR90 fibroblasts also gives three fold less DNA compared to mock-infected. Histone H3 ChIP does not show similar depletion from the e1a infected cells.

0 1 2 3 4 5 6 7

H3K18ac H3

DNA per IP (ng)

Mock Infected

7

PCAF CBP

TSS

-5.5 2.5 TSS

3.20.2-2.0 3.52.5-3.2

1 2

cluster

3

2 0 -2 n(lnR)

Figure S8. PCAF, and to lesser extent CBP, are also enriched in clusters 1 and 2 at 24 h p.i. Shown are the results of ChIP-chip assays for PCAF and CBP binding in dl1500- vs. mock-infected cells. The values represent relative enrichment (yellow) or depletion (blue) from dl1500- vs. mock-infected cells, respectively, and are denoted according to the bar shown on the left.

The Z-scores for enrichment of PCAF or CBP for each cluster are as indicated.

8

p107

Lane 1 2 3 4

β-tubulin

6

0 12 24

105 kDa 119 kDa 55 kDa RB

P- RB

Time p.i. (h)

e1a

-0.5 0.0 0.5 1.0 1.5 2.0 2.5

Expression relative to mock infection (log2 ratio)

6h 12h 24h

Figure S9. Small e1a increases the level of the RB-family member p107. A) Expression levels of RB, p107 and p130 mRNA at 6, 12 and 24 h p.i. in

average of three independent experiments. B) Levels of RB and p107 proteins before and after e1a expression in IMR90 fibroblasts at the indicated times were determined by western blotting. P-RB represents the phosphorylated form of RB and β-tubulin was used as a loading control.

A B

p130 p107 RB

9

p300 R2G 24h

TSS

-5.5 2.5 2.51.7-2.6

1 2

cluster

2

3

0 -2 n(lnR)

Figure S10. Expression of R2Ge1a does not result in significant p300 binding to promoters of cluster 1 and 2 genes at 24 h p.i. Shown are the results of ChIP-chip assays for p300 binding in R2Ge1a- vs. mock- infected cell at 24 h p.i. The R2G mutation in e1a disrupts its interaction with the p300/CBP HATs. The clusters are as defined in Fig 1A. The Z-scores for enrichment of p300 in each cluster are as indicated. In contrast to p300 redistribution by WTe1a (Fig 2), the R2Ge1a is unable to induce significant re-localization of p300.

10

-20 -10 0 10 20

-20 -10 0 10 20

wt e1a exp

R2G e1a exp

r = 0.12

-20 -10 0 10 20

-20 -10 0 10 20

wt e1a exp

r = 0.69

Figure S11. The R2Ge1a did not induce the gene expression changes observed with WTe1a. Shown are the correlations of the expression levels of IMR90 cells expressing the indicated e1a mutants vs. WTe1a at 24 h p.i.

11

Figure S12. Differences in gene expression profiles induced by WT and ΔCR2 small e1a for selected genes. Although the overall H3K18ac and expression patterns induced by WT and ΔCR2 small e1a expression in IMR90 primary fibroblasts were similar, differences in expression of several critical genes may explain the inability of ΔCR2 small e1a to transform cells. The level of expression at 24 h p.i. is shown for A) p53, B) telomerase, C) cyclin dependent kinase Inhibitors 1A (p21, Cip1/WAF1) and 2B (p15, INK4B), and D) CDKN2C (p18, INK4C) and CDKN3 (CDK2-associated dual specificity phosphatase). The values are log

ratio of WT or ΔCR2 small e1a vs. mock-infected cells.

-0.6 -0.3 0.0 0.3 0.6 0.9 1.2

Expression relative to mock infection (Log2 ratio)

TP53

deltaCR2 dl1500

-2.0 0.0 2.0 4.0 6.0 8.0

TERT

Expression relative to mock infection (Log2 ratio)

deltaCR2 dl1500

0.0 0.6 1.2 1.8 2.4 3.0

CDKN2C CDKN3

Expression relative to mock infection (Log2 ratio)

deltaCR2 dl1500

-10.0 -8.0 -6.0 -4.0 -2.0 0.0

CDKN1A CDKN2B

Expression relative to mock infection (Log2 ratio)

deltaCR2 dl1500

A B

C D

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e1a

Development and differentiation Anti-viral response genes

Cell Proliferation

deacetylation p300

e1a e1a p300

p300

p300

deacetylation

acetylation

p107 Rb

family E2F DP1

e1a

Rb p130

Repression

Activation

Repression

Time

Figure S13: Models for e1a-induced transcriptional activation and repression: Regulation of transcription by small e1a is discussed separately for each of the three gene clusters depicted in Fig 1.

Cluster 1 genes (cellular anti-viral responses): p300 (and possibly CBP and PCAF) associate with promoter regions of anti-viral response genes as they are induced by host cell transcription factors in response to infection. Small e1a associates with these genes through its interaction with p300, since e1a-binding is largely eliminated by the R2G mutation (Fig 3A). By 12h, e1a dissociates from cluster 2 promoters (because of acetylation by p300/CBP?), but p300, Rb, and p130 remain associated. By 24h (and possibly earlier), Rb and p130 also associate with these genes which also become densely packed with nucleosomes (increased density of H3), show H4K16 hyperacetylation, little H3K18 or H3K9 acetylation and are repressed (Figs 1, 2).

Cluster 2 genes (growth and cell cycle): Small e1a binds to the Rb-family proteins bound to E2Fs and possibly other transcription factors, causing the displacement of Rb-proteins from E2Fs as also observed in vitro (ref. 1). This results in removal of repressive chromatin modifying complexes associated with Rb-proteins and H3K9 hyperacetylation (note H3K9 hyperacetylation was induced by R2G but less so by ΔCR2 e1a mutants). Small e1a-induced binding of p300/CBP and PCAF cause hyperacetylation of H3K18 at these genes (and probably other H3 and H4 lysines), and transcription induction at 24h. Despite depletion of e1a at 24h, p300/CBP and PCAF remain associated possibly by binding acetylated lysines and/or the unmasked E2F activation domains.

Cluster 3 genes (genes involved in differentiation and development and cell-cell signaling): Small e1a and p107 and perhaps its associated repressive chromatin modifying complexes bind to these genes, causing deacetylation and repression. Depletion of p300 and H3K18ac from these genes may also contribute to their repression (this may be how ΔCR2e1a represses cluster 3 genes).

E2F DP1

TF

13

Epigenetic reprogramming by adenovirus e1a (Science 2008)

Roberto Ferrari, Matteo Pellegrini, Gregory A. Horwitz, Wei Xie, Arnold J. Berk and Siavash K.

Kurdistani

University of California, Los Angeles (UCLA), Los Angeles, CA 90095

Supplemental Experimental Procedures

Cell culture and viruses. IMR90 human primary lung embryo fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS) at 37°C in 5% CO2. Propagation of viruses was done as described(1).

Whole-genome expression profiling. Samples of 250 ng of RNA from independent infections from WTe1a-, R2Ge1a-, ΔCR2e1a- or mock-infected primary IMR90 fibroblasts were amplified and labeled with Cy5 (e1a) and Cy3 (mock) (Perkin Elmer), respectively, using the Agilent Two Colors Low RNA Input Linear Amplification Labeling Kit according to manufacturer's instructions. Labeled RNA was hybridized to the Agilent Human whole- genome array (G2534-60011) and analyzed as described below. For most analyses, we included the genes for which both ChIP and expression data were available for a total of 13792 genes.

Western Analysis. Antibodies used in this study are listed in Table S1 (see below). Cells from a 10 cm confluent dish (both dl1500- and mock-infected IMR90 fibroblasts) were harvested and whole cell extract was prepared and subjected to standard Western blotting.

Chromatin immunoprecipitation and microarray hybridization

Chromatin immunoprecipitation was performed essentially as described(2), with few modifications. Briefly, 2x10⁷ IMR90 lung fibroblasts were grown on 10 cm dishes to confluence. After 24 h, the cells were incubated with mock- or the dl1500 adenovirus for 1 h in low serum media (2%). At the indicated times post infection (p.i.), formaldehyde was added for 10 min at 37°C. After PBS washing, cross-linked cells were scraped from the plates and washed with 1 ml of PBS containing protease inhibitors (Roche). Cells were resuspended in 400 µl of Lysis buffer and incubated for 10 min on ice and immediately sonicated. 100 µl of the lysate (corresponding to 5x10⁶ cells) were used for immunoprecipitation with a given antibody (listed in table S1); 10 µl of the lysate was used as input. After overnight reversal of crosslinking at 65°C, samples were treated with RNase A for 30 min at 37°C and subsequently purified using the Qiagen Qiaquick PCR purification Kit. 10 ng of each IP and INP DNA were amplified using the WGA Kit (Sigma)(3). 2 μg of amplified material were labeled with Cy3 or Cy5 (PerkinElmer) using the Bioprime Labeling Kit (Invitrogen). DNA was mixed with 35 µl of random priming solution (Invitrogen Bioprime Kit) to a final volume of 75 µl, boiled for 5 min and quickly cooled in an ice-water bath for 5 min. The

14

labeling reaction was completed with 60U Klenow, dNTPs (0.12 mM dATP, dGTP and dTTP and 0.06 mM dCTP), 1.28 mM Cy3 and Cy5 for input and IP (or IP from mock and IP from dl1500-infected cells) respectively, and incubated for 3h at 37°C. The labeled DNA was purified using Qiagen Qiaquick PCR purification Kit and the incorporation was measured with Nanodrop. Hybridization onto the Human Promoter array (Agilent-G4489A), washing, and scanning were carried out according to the manufacturer's instructions. The arrays were scanned using an Agilent DNA Microarray scanner. Data extraction and analyses were carried out using the Agilent Feature Extraction software (version 9.1.3.1) and Chip Analytics software (version 1.2). Probe signals were extracted with the Agilent Feature extraction software, normalized with Lowess normalization using the Chip Analytics software, and statistically analyzed as described below.

BrdU incorporation assay

IMR90 cells were pulse labeled with 30 µM BrdU (Sigma) for 30 min at 37°C before fixing. Antibody access was achieved by denaturing cells in 2 N HCl for 10 min at room temperature prior to neutralizing in phosphate buffered saline and staining with anti-BrdU (Roche).

Statistical methods for the analysis of ChIP-chip data

Each ChIP profile was normalized to generate mean value of zero and variance of one, since the assay accurately captures the relative enrichment of an IP across an array. The profiles were clustered using K-means clustering with 3 groups using the Cluster software(4). Z-scores for each cluster were computed by calculating the average value of each segment in each gene of the cluster and multiplying by the square root of the number of these segments. Shades of yellow and blue represent relative enrichment and depletion respectively. Scale bars for ChIP- chip experiments vary from 2 to -2 n(ln) (normalized log natural ratio between infected and mock-infected cells) except for RB-family members ChIP-chip (from 1 to -1 n(ln)).

To compare e1a and E2F binding, we downloaded the E2f binding data published in(5). We used the average of the E2F binding values measured in GM06990 and MCF10 cells. We identified approximately 10,000 promoters for which we had both e1a and E2F binding data. The Z-score for E2F binding in each cluster was computed as above: calculate the average value of E2F binding for a cluster, subtract the mean value of E2F binding over all genes, divide by the standard deviation of E2F binding and multiply the ratio by the square root of the number of genes in the cluster. E2F target genes were selected using a cutoff of 2 fold enrichment in binding(5).

Gene expression analysis

Expression data were extracted using Agilent Feature Extraction software (version 9.1.3.1). Raw data were natural log (ln) transformed and signals from multiple probes for the same gene were averaged. Each array was normalized so that the mean was zero and standard deviation was one. Data from three independent replicate experiments were averaged.

15

Antibody Source E1A (M73) Berk, A.J.

H3K18ac Suka N, et al. 2001 H3K9ac Suka N, et al. 2001 H4K16ac Suka N, et al. 2001 H3 (ab1425) Abcam

p300 (N‐15): sc‐584 Santa Cruz p107 (C‐18): sc‐318 Santa Cruz p130 (C‐20): sc‐317 Santa Cruz RB (Ab‐1) Lab vision PCAF (ab12188) Abcam CBP (ab3652) Abcam

Table S1. Antibodies used in this study.

16

Supplemental Online Discussion

Small e1a has long been known to activate cell cycle genes by displacing the RB-family proteins and their associated repressive chromatin-modifying complexes from E2F transcription factors(6-8). Data presented here show that activation of these genes is also associated with transient binding of e1a and the re-localization of p300, CBP and PCAF HATs to their promoter regions, and the resulting acetylation of H3K18 (Fig 2). CBP and p300 are required for H3K18 acetylation since knockdown of both HATs results in global reduction of H3K18ac(9).

However, we cannot rule out that p300/CBP knockdown does not affect PCAF HAT activity as well since PCAF associates with CBP/p300(10). Consequently one or all of these HATs may be primarily responsible for acetylating H3K18 at promoter regions. The sequestration of p300, and probably CBP and PCAF, to the promoter regions of a limited set of genes could contribute to the global hypoacetylation of H3K18 induced by small e1a(9). Since small e1a also binds downstream of the TSS in cluster 2 genes (Fig 1A), it may activate their transcription by stimulating the elongation phase of transcription as well as transcription initiation. It is interesting to note that small e1a is an unstable protein, turning over with a half-time of ~80 min(11). Thus, the e1a proteins bound to cluster 3 genes at 24 h are different from e1a molecules associated with the promoter regions of cluster 1 and 2 genes at 6 h. At later times post infection, e1a binds principally to promoters of developmental, differentiation and cell-cell signaling genes (cluster 3). These genes are also bound by the p107 repressor at 24 h (Fig 2), probably contributing to their deacetylation and repression. The unexpected finding that e1a binds to repressed genes may explain why e1a makes a multitude of direct and indirect interactions with other repressors of transcription such as CtBP(12), the Polycomb proteins(13, 14) and DNA methyltransferases(15), which may also contribute to e1a-mediated repression.

In addition to changes in gene expression, the temporal order of e1a binding to its target genes may harbor important lessons for cellular transformation. The e1a interaction with p300 and the RB-family proteins are both required for the successive order of target-gene binding by e1a. But how precisely these interactions contribute to target gene selection by e1a remains to be determined. The p300 HAT acetylates e1a at lysine 239, inhibiting the binding of CtBP to conserved region 4 (fig S1), as well as at other sites not yet mapped(16). Acetylation at one of these sites could affect e1a’s affinity for other transcription factors/co-factors and, consequently, its temporal binding pattern. Considering that neither the R2Ge1a nor ΔCR2e1a is able to induce cell cycling, progression through the cell cycle, in and of itself, may also contribute to WTe1a target gene selection. Since small e1a causes de-differentiation of normal cells(17-19), the order of target-gene selection by e1a probably has important consequences for how reversal of cell differentiation can be achieved (e.g., cellular reprogramming or cancer dedifferentiation).

Small e1a has highlighted the unique function of H3K18ac in regulation of transcription(20, 21). The data from the R2G and ΔCR2 mutants (Fig 3) indicate that re-localization of H3K18ac is required for transcriptional reprogramming by e1a and depends on the e1a-p300 interaction. In contrast, re-localization of H3K9ac was insufficient to induce transcriptional changes and depended on the high-affinity e1a CR2-RB-family protein

17

interactions. The inability of the ΔCR2e1a mutant to displace the RB-proteins and their associated histone deacetylase complexes(7) may explain the defect in the stimulation of H3K9ac by this mutant (compare Fig 2 with Fig 3C) by preventing the displacement of histone deacetylases associated with the RB-family proteins(8).

Interestingly, hyperacetylation of H4K16 was observed at the repressed antiviral genes by 24 h (cluster 1).

Although H4K16ac inhibits folding of the 30 nm chromatin fiber(22), this modification was previously found to also correlate with gene repression in both S. cerevisiae (20) and S. pombe (23). Since e1a induces S-phase entry, it is important to note that cell cycle effects (independent of e1a) may also contribute to some of the changes in histone modification patterns and gene expression of e1a-infected cells. Future experiments should be able to de- convolute the e1a-specific from cell-autonomous effects.

Oncogenic RAS drives G0-arrested cells into S-phase by functioning at the cell membrane, activating several interacting signal transduction networks that ultimately regulate multiple transcription factors in the nucleus to control cell cycling(24). In contrast, our data have revealed that, far from being a simple “trigger switch” that initiates a cascade of events leading to cell cycling and cell transformation, the e1a oncoprotein directly regulates thousands of host cell genes by binding to their promoter regions in a precise, time-dependent order. Small e1a directly reprograms the cellular gene expression profile to enhance the proliferative capacity of the transformed cell with concomitant reduction in the cell’s differentiation potential and antiviral defense responses. So, both promotion of S-phase entry and repression of differentiation and antiviral responses are active processes directly orchestrated by the 243 amino acid residue protein. Considering that e1a alters the expression of a large group of histone modifiers such as the H3K27 methyltransferase Ezh2 (see gene expression data), histone modifications other than H3K18ac as well as DNA methylation may also contribute to oncogenic transformation by e1a.

References

1. K. R. Spindler, C. Y. Eng, A. J. Berk, J Virol 53, 742 (Mar, 1985).

2. G. Wang et al., Mol Cell 17, 683 (Mar 4, 2005).

3. H. O'Geen, C. M. Nicolet, K. Blahnik, R. Green, P. J. Farnham, Biotechniques 41, 577 (Nov, 2006).

4. M. J. de Hoon, S. Imoto, J. Nolan, S. Miyano, Bioinformatics 20, 1453 (Jun 12, 2004).

5. X. Xu et al., Genome Res 17, 1550 (Nov, 2007).

6. J. R. Nevins, Curr Opin Genet Dev 4, 130 (Feb, 1994).

7. X. Liu, R. Marmorstein, Genes Dev 21, 2711 (Nov 1, 2007).

8. A. J. Berk, Oncogene 24, 7673 (Nov 21, 2005).

9. G. A. Horwitz, Zhang, K., McBrian, M.A., Grunstein, M., Kurdistani, S.K. & Berk A.J., Science, (2008).

10. X. J. Yang, V. V. Ogryzko, J. Nishikawa, B. H. Howard, Y. Nakatani, Nature 382, 319 (Jul 25, 1996).

11. K. R. Spindler, A. J. Berk, J Virol 52, 706 (Nov, 1984).

12. G. Chinnadurai, Mol Cell 9, 213 (Feb, 2002).

13. M. Fuchs et al., Cell 106, 297 (Aug 10, 2001).

14. M. E. McLaughlin-Drubin, K. W. Huh, K. Munger, J Virol, (Jun 25, 2008).

15. W. A. Burgers et al., Oncogene 26, 1650 (Mar 8, 2007).

16. Q. Zhang, H. Yao, N. Vo, R. H. Goodman, Proc Natl Acad Sci U S A 97, 14323 (Dec 19, 2000).

17. K. A. Webster, G. E. Muscat, L. Kedes, Nature 332, 553 (Apr 7, 1988).

18. T. Braun, E. Bober, H. H. Arnold, Genes Dev 6, 888 (May, 1992).

19. M. Tiainen, D. Spitkovsky, P. Jansen-Durr, A. Sacchi, M. Crescenzi, Mol Cell Biol 16, 5302 (Oct, 1996).

20. S. K. Kurdistani, S. Tavazoie, M. Grunstein, Cell 117, 721 (Jun 11, 2004).

21. B. D. Strahl, C. D. Allis, Nature 403, 41 (Jan 6, 2000).

22. M. Shogren-Knaak et al., Science 311, 844 (Feb 10, 2006).

23. M. Wiren et al., EMBO J 24, 2906 (Aug 17, 2005).

24. M. Malumbres, M. Barbacid, Nat Rev Cancer 3, 459 (Jun, 2003).

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