H3K4me3 inversely correlates with DNA methylation at a large class of non-CpG-island-containing start sites
- Dheepa Balasubramanian†1,
- Batool Akhtar-Zaidi†1, 2,
- Lingyun Song3,
- Cynthia F Bartels1,
- Martina Veigl4,
- Lydia Beard4,
- Lois Myeroff4,
- Kishore Guda4,
- James Lutterbaugh4,
- Joseph Willis4, 5,
- Gregory E Crawford3,
- Sanford D Markowitz1, 4, 6Email author and
- Peter C Scacheri1, 2, 4Email author
© Balasubramanian et al.; licensee BioMed Central Ltd. 2012
Received: 1 December 2011
Accepted: 28 May 2012
Published: 28 May 2012
In addition to mutations, epigenetic silencing of genes has been recognized as a fundamental mechanism that promotes human carcinogenesis. To date, characterization of epigenetic gene silencing has largely focused on genes in which silencing is mediated by hypermethylation of promoter-associated CpG islands, associated with loss of the H3K4me3 chromatin mark. Far less is known about promoters lacking CpG-islands or genes that are repressed by alternative mechanisms.
We performed integrative ChIP-chip, DNase-seq, and global gene expression analyses in colon cancer cells and normal colon mucosa to characterize chromatin features of both CpG-rich and CpG-poor promoters of genes that undergo silencing in colon cancer.
Epigenetically repressed genes in colon cancer separate into two classes based on retention or loss of H3K4me3 at transcription start sites. Quantitatively, of transcriptionally repressed genes that lose H3K4me3 in colon cancer (K4-dependent genes), a large fraction actually lacks CpG islands. Nonetheless, similar to CpG-island containing genes, cytosines located near the start sites of K4-dependent genes become DNA hypermethylated, and repressed K4-dependent genes can be reactivated with 5-azacytidine. Moreover, we also show that when the H3K4me3 mark is retained, silencing of CpG island-associated genes can proceed through an alternative mechanism in which repressive chromatin marks are recruited.
H3K4me3 equally protects from DNA methylation at both CpG-island and non-CpG island start sites in colon cancer. Moreover, the results suggest that CpG-rich genes repressed by loss of H3K4me3 and DNA methylation represent special instances of a more general epigenetic mechanism of gene silencing, one in which gene silencing is mediated by loss of H3K4me3 and methylation of non-CpG island promoter-associated cytosines.
The development of cancer is closely associated with the stepwise accumulation of not only somatic mutations, but also epigenetic alterations that alter chromatin structure and lead to dysregulated gene expression. Current dogma holds that for normal somatic cells, trimethylated lysine 4 on histone H3 (H3K4me3) represents a chromatin landmark that is present at the transcription start sites (TSSs) of protein-coding genes that are either actively transcribed or that are held in a 'poised' state permissive for gene transcription . However, it is also well established that during the process of malignant transformation, loss of H3K4me3 occurs at TSSs of genes that undergo transcriptional inactivation as a result of promoter hypermethylation [2, 3]. The loss of H3K4me3 is consistent with a model whereby DNA methylation at CpG islands is initiated by removal of H3K4me by LSD1 (lysine-specific demethylase 1) and JmjC families of demethylases, followed by targeting of DNA methyltransferase (DNMT)3A/Dnmt3B-Dnmt3L complexes, which deposit methyl groups [2, 4–7]. However, de novo DNA methylation is an infrequent event occurring on only a very small fraction of CpG island-containing promoters. Far less is known about promoters lacking CpG islands or genes that are repressed by alternative mechanisms, mainly because genome-wide surveys of epigenetic modifications have only recently become technically feasible.
Here, we used ChIP-chip and expression analyses to systematically study the relationship between H3K4me3 and genes that are transcriptionally repressed in colon cancer compared to normal colon mucosa. We found that the majority of protein-coding genes contain H3K4me3, as expected. Interestingly, H3K4me3 is retained among a set of genes that undergo transcriptional repression, or 'silencing' during the process of malignant transformation. Repressed genes that retain H3K4me3 are also located in open regions of chromatin that are hypersensitive to DNaseI digestion, nearly always contain CpG islands, and frequently acquire histone modifications associated with transcriptional repression. Consistent with the established inverse correlation between DNA methylation and H3K4me3, we also detected a class of repressed genes that virtually lack detectable levels of H3K4me3 and show increased DNA methylation compared to normal colon mucosa. While the increased DNA methylation accompanying loss of H3K4me3 is easily detected at promoters containing CpG islands, we find this increase is often more prevalent at the scattered CpG sites in the promoters of genes devoid of CpG islands. We propose a model whereby H3K4me3 equally protects from DNA methylation at both CpG island and non-CpG island start sites, suggesting that the mechanisms associated with DNA methylation-associated gene silencing in colon cancer are similar for CpG and non-CpG island-containing genes.
Materials and methods
Cell lines and tissue samples
The VACO cell lines (VACO429, VACO432, VACO441 and VACO425) were cultured as previously described . SW480 was obtained from the American Type Culture Collection. Normal colon mucosa was obtained from a scraping of fresh resected colon. Cell viability was examined by staining with Trypan Blue, and estimated at 85 to 95%. Extraction of colonic crypts was performed by EDTA fractionation. Briefly, colon mucosa was first dissected from each sample taking care to maintain tissue integrity. The mucosa was then cut into thin strips, gently agitated in cell dissociation buffer (Invitrogen 13151-014 Carlsbad, CA, USA), rinsed in phosphate-buffered saline, and then agitated in fresh buffer. The tissue strips were then rapidly pipetted until the solution became cloudy due to the release of crypts from the mucosa. Mucosa strips were removed from the solution and discarded. The remaining solution was examined microscopically for the presence of viable, intact crypts free of contaminating debris.
Chromatin immunoprecipitation (ChIP)-chip experiments were performed as previously described  using the following antibodies: H3K4 trimethyl (Abcam ab8580 (Cambridge, MA, USA); Upstate 39159 (Billerica, MA, USA)), H3K9 dimethyl (Abcam, ab1220), H3K20 trimethyl (Abcam ab9053), and H3K27 trimethyl (Abcam ab6002). The following three array platforms were used: (1) NimbleGen (Madison, WI, USA) human 2.1M deluxe promoter arrays; (2) NimbleGen 385K promoter arrays; and (3) Agilent (Santa Clara, CA, USA) custom tiling arrays. The NimbleGen human 2.1M promoter array contains 50- to 70-mer probes spanning 7.2 kb upstream and 3.2kb downstream of each TSS at a resolution of 1 oligo per 100 bp. NimbleGen 385K arrays contain 50- to 70-mer probes spanning 1 kb upstream and 500 bp downstream of approximately 19,000 TSSs at a resolution of 1 oligo per 100 bp. To account for potential differences in the dyes on the array, ChIP/input ratios for each probe were scaled by subtracting the bi-weight mean for the log-ratio values from each log-ratio. ChIP-chip signals were then quantile normalized so that separate ChIP-chip experiments could be directly compared. Agilent microarrays contained 44,000 features, with oligos that spanned 10 kb downstream and 5 kb upstream of the TSS of the desired genes. Agilent array data were processed and normalized using Feature Extraction. For the comparison of multiple histone marks between normal colon mucosa and SW480, ChIP-chip data were processed with the software package ACME using a window size of 500 bp and a threshold of 95% . The minimum P-values for signals located near (±1 kb from each TSS) were then calculated and plotted.
RNA was purified by cesium chloride density gradient centrifugation from all cell lines, normal colon mucosa, and normal colon crypt preparations. RNA was then labeled and hybridized to Affymetrix (Santa Clara, CA, USA) Human Exon 1.0 ST exon arrays according to standard protocols. All microarray data were processed in a single batch using the Affymetrix Expression Console software, obtaining gene level expression data using 'core' probe sets (the highest confidence level probe sets, associated with BLAT alignments of mRNA with annotated full-length coding sequence regions) using median normalization and the PLIER (probe logarithmic intensity error estimation) algorithm. Genes located on Y, random, and the mitochondrial chromosomes were excluded. Repressed genes were considered those with an expression value of <31, while genes with expression values >75 were considered expressed or 'on'. These thresholds were determined through analysis of the distribution of expression of all genes sampled on the microarray. K4-independent genes were designated as those repressed (<31) in the colon cancer cell lines compared to normal colon crypt (>75), and containing enrichment of H3K4me3 in normal colon mucosa and colon cancer cell lines at levels corresponding to the right side of the bimodal distribution of H3K4me3 levels of all genes. K4-dependent genes were designated as repressed genes (<31) that showed levels of H3K4me3 corresponding to the left side of the bimodal distribution.
DNase-seq was performed as previously described . Sequencing was performed using an Illumina (San Diego, CA, USA) GAII, and 35-bp reads were obtained for all samples. Sequences were aligned to the human genome reference sequence (Hg18) using MAQ . The number of aligned sequence reads were as follows: SW480, 21,461,814; V432, 16,003,416; V429, 7,110,678. Aligned sequences were processed with F-seq . The maximum peak signal within 2 kb of all human TSSs was then extracted from the genome-wide DNase-seq profiles, so that each TSS could be assigned a single score. Scores were Z-score transformed so that individual samples could be directly compared.
Pyrosequence analysis of bisulfite converted and non-converted DNA was performed by EpigenDx (Worcester, MA, USA). CpG sites analyzed were located at the following positions relative to the TSSs of each indicated gene (HG19 genome assembly): MMP28, (-589, -582, -563, -536, -504,-486); PTGDR, (55, 58, 68, 86, 124, 126, 130, 148, 150, 153, 162, 166,175); HMGCS2, (59, 78, 98, 125); ACSL5, (22, 28, 55, 97, 117, 150, 154, 176, 200); BCAS1, (-262, -223, -195, -132); FRK, (-503, -493, -484, -442, -431); UBA7, (-123, -134); BCL2L14, (55, 19); PIGR, (-673, -656, -650, -597); CD177, (-150, -6, 5, 7); GUCY2C, (-124, -161, -126); TNFSF10, (-43, -98); SEMA6D, (-94, -90, -81, -76, -60, -57, -50, -42); MOBKL2B, (-77, -71, -67, -54, -50, -47, -41, -39, -30, -23, -19, -13, -11, -8, -6); SLC39A5, (-297, -218, -162, -102, -50).
SW480 cells were seeded at 105 per T75 flask on day 0. The cultures were treated for 24 h on days 2 and 5 with 5-azacytidine at 1 µg/µl. The media was changed 24 h after the addition on 5-azacytidine (on days 3 and 6). RNA harvested 8 days after the initial 5-azacytidine treatment was DNase-treated and purified using the RNeasy kit (Qiagen, Germantown, MD, USA). cDNA was prepared using the high capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA), and standard quantitative RT-PCR analyses were performed.
H3K4me3 ChIP-chip in colon cancer cell lines
Repressed genes can be distinguished into two classes, based on the presence or absence of H3K4me3
We next set out to identify genes that are repressed in each of the colon cancer samples compared to normal colon mucosa. For these experiments, we prepared RNA from each of the five colon cancer cell lines, as well as microdissected histologically normal colon mucosa, and five individual preparations of epithelial crypts purified by fractionation from normal colon mucosa. Samples were hybridized to Affymetrix Human Exon 1.0 ST Arrays, which are known to be more reliable for transcript quantification than standard 3'-UTR microarrays. Similarly to the H3K4me3 data, the genome-wide distribution of gene expression is largely bimodal (Additional file 2A). This allowed us to divide genes into two main categories: (1) abundantly expressed or 'on'; and (2) near background levels or 'off'. Among all samples analyzed, we found that, on average, 49% of genes were expressed and 33% of genes were off; 18% of genes fell in the trough of the bimodal distribution and could not be neatly classified as either silent or expressed.
Verification of K4-dependent and K4-independent classes
The levels of H3K4me3 for genes in each class were validated by standard ChIP on biological replicate samples (Additional file 3A). The loss of the H3K4me3 signal at K4-dependent genes cannot be due to homozygous deletions, as these regions could be successfully amplified by genomic PCR. For further verification, we performed hybridizations of H3K4me3 ChIPs from samples V425, V432, V441, SW480 and two independent preparations of normal colon mucosa to NimbleGen 385K promoter arrays and repeated the data analysis. Consistent with the previous results, both K4-dependent and -independent genes were evident, and the relative proportions of each class were similar within and between each cell line to the proportions found using the 2.1M feature arrays (Additional file 4A). Next, we examined the exon-tiling array data to confirm that genes designated as repressed were in fact repressed across all exons, and that when expressed in the crypt were expressed across all exons, consistent with the canonical transcript from the locus. We found that, compared to genes in crypt that were designated as 'on', and that conformed to the canonical transcript and its associated promoter, the expression levels across all exons of genes designated as repressed were at or near background levels. The exon usage across a representative example of a repressed gene is shown in Additional file 4B. Lastly, we investigated whether K4-dependent and K4-independent genes repressed in cell culture were similarly under-expressed in primary tumors. Using global expression data, we first selected K4-dependent and K4-independent genes that were repressed by at least two-fold in all five colon cancer cell lines relative to five epithelial colon crypt samples. We then determined the percentage of these genes that were also repressed in 120 primary tumors relative to 16 normal mucosa samples. Of all genes, only 7.7% of genes repressed in the cell lines are also repressed in tumors relative to mucosa, whereas 76% of K4-dependent and K4-independent genes repressed in cancer cell lines validated as repressed in primary tumors (P < 2.2 × 10-16 by exact binomial test). Collectively, these data strongly support the existence of the two classes of repressed genes in colon cancer, indicate that alternative promoter usage is unlikely to account for the difference in H3K4me3 status between the two classes, and suggest that most genes identified as repressed in the cell culture models are genuinely repressed in colon cancer.
K4-dependent and -independent genes show differences in chromatin structure
K4-dependent genes show DNA hypermethylation
We next tested whether K4-dependent genes that lack CpG islands could be reactivated upon treatment with 5-azacytidine. Three out of four K4-dependent genes tested showed a significant increase in transcript levels upon treatment with 5-azacytidine (P ≤ 0.01; Figure 4d), consistent with hypermethylation of the scattered CpG sites under the H3K4me3 mark being functionally involved in these genes' silencing. The data indicate that DNA hypermethylated K4-dependent repressed genes do not necessarily contain CpG islands, and that repressed K4-dependent genes lacking both CpG islands and H3K4me3 are very likely to be DNA methylated in regions that lose the H3K4me3 mark. The results are also consistent with previously reported reactivation of hypermethylated genes lacking CpG islands upon treatment with 5-azacytidine .
Characterization of histone marks at K4-independent and K4-dependent genes
K4-dependent and -independent genes are functionally distinct
We used Panther to determine whether specific pathways or biological processes are enriched among K4-dependent and -independent genes, and if so, whether they differ between the two classes . Intriguingly, several pathways previously linked to colorectal carcinogenesis were enriched in the K4-independent set, including transforming growth factor-beta, insulin and Wnt signaling (Additional file 6). In contrast, K4-dependent genes were enriched in axon guidance, pyrimidine metabolism and cadherin signaling. Both classes were enriched for genes involved in apoptosis and platelet-derived growth factor signaling, as well as pathways associated with B- and T-cell activation.
We next tested whether genes in each class show tissue-specific expression in colon crypts, and if so, whether the degree of crypt-specific expression differed between the two classes. To do this, we compared global gene expression levels between normal colon crypt, HepG2, K562, and NB4 cells and computed a tissue-specificity score for each gene using the method of Shannon entropy . We then plotted the distribution of colon-specificity scores for K4-dependent and -independent repressed genes, all genes, and 1,000 randomly selected genes. Both K4-dependent and -independent genes showed a high degree of crypt tissue-specific expression, with K4-dependent genes being significantly more crypt-specific than K4-independent genes (P < 0.0001) (Additional file 7A). These findings are consistent with other studies showing that genes lacking CpG islands, which comprise a large fraction of the K4-dependent class of repressed genes, are generally associated with tissue-specific expression .
Lastly, we tested whether K4-dependent genes show an increased propensity for silencing in colon cancer compared to K4-independent genes, or vice versa. To do this, the transcriptional status of genes designated as K4-dependent or K4-independent in the five colon cell lines was surveyed in an additional 35 colon cancer cell lines. We then plotted the distribution of the median expression values for each set as a histogram (Additional file 7B). Although the expression of K4-dependent genes is more variable than K4-independent genes, the overall distribution of K4-dependent genes is significantly shifted to the left of that of K4-independent genes (P = 0.015), indicating that K4-dependent genes are repressed more often in colon cancer than K4-independent genes. Although confirmatory studies are required, these findings raise the possibility that genes targeted for silencing in colon cancer are more often inactivated by mechanisms involving removal of H3K4me3 than by K4-independent mechanisms.
Two studies in which epigenetic silencing marks were profiled in prostate cancer cells (PC3) revealed that loci marked with H3K27me3 are devoid of DNA hypermethylation, raising the possibility that gene silencing by H3K27me3 occurs independently of promoter methylation [20, 21]. These findings are consistent with our results, with one notable exception. The studies by Kondo et al.  indicate that promoters marked with H3K27me3 in the absence of DNA methylation are mostly devoid of CpG islands, while the data presented here indicate that this trend is far more prevalent for CpG island-containing promoters. In fact, only a small fraction of the genes we designated as K4-dependent, which are frequently devoid of promoter CpG islands, were found to contain significant levels of H3K27me3. The discrepancy between our results and the published studies is currently not clear, but could be related to differences in cancer type.
Previous studies have shown that low CpG promoters display no significant correlation between gene activity and the abundance of methylated cytosines . The discrepancy between these findings and our results is likely due to methodological differences related to transcript quantification. Here, mRNA transcript levels were directly quantified using all exon microarrays. In contrast, in the previous studies promoter activity was 'presumed' based on occupancy of RNA polymerase II, which is now known to bind both active and 'paused' promoters that are weakly transcriptionally active [1, 22, 23]. Our findings are consistent with more recent studies showing significant positive correlations between DNA hypermethylation and low gene activity at CpG-poor promoters in multiple human tissues .
We find it particularly intriguing that K4-dependent repressed genes, which lack H3K4me3, are apparently devoid of repressive histone modifications, including H3K27me3, H3K9me2, and H4K20me3. While we cannot rule out the possibility that these genes acquire a currently unknown repressive mark, one possibility is that the loss of H3K4me3, together with methylation of residual CpGs, is sufficient for gene silencing in cancer. Further studies in which H3K4me3 is restored at these genes' promoters could help test this hypothesis.
We conclude that the presence of the H3K4me3 mark at low CpG-content TSSs protects from DNA methylation and transcriptional repression in colon cancer. Quantitatively, of transcriptionally repressed genes that lose H3K4me3 and become DNA hypermethylated in colon cancer, more typically lack CpG islands than contain CpG islands.
Based on these findings, we propose that CpG-rich genes repressed by loss of H3K4me3 and DNA methylation represent rare examples of a more general epigenetic mechanism of gene repression, one in which silencing is mediated by loss of H3K4me3 and methylation of non-CpG island promoter-associated cytosines. Lastly, we note that the identification of gene silencing in cancer in association with CpG island methylation has led to the discovery of 5-azacytidine as a drug able to reactivate expression of such genes and to the use of 5-azacytidine for cancer treatment. It will similarly be of interest to seek to identify other pharmacologic agents that induce re-expression of repressed genes that lack CpG islands.
chromatin immunoprecipitation combined with microarray technology
DNase I hypersensitive site sequencing
histone H3 trimethylated at lysine 4
histone H3 dimethylated at lysine 9
histone H4 trimethylated at lysine 20
transcription start site.
This work was supported in part by the following NIH grants: R01HD056369 and R01CA160356 to PCS, and 1P50CA150964 and NIH UO1 CA152756 to SM. BAZ is a predoctoral student in the Molecular Medicine PhD Program of Cleveland Clinic and Case Western Reserve University, funded, in part, by the Med into Grad initiative of the Howard Hughes Medical Institute.
- Guenther MG, Levine SS, Boyer LA, Jaenisch R, Young RA: A chromatin landmark and transcription initiation at most promoters in human cells. Cell. 2007, 130: 77-88. 10.1016/j.cell.2007.05.042.PubMedPubMed CentralView ArticleGoogle Scholar
- Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S, Rebhan M, Schubeler D: Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome. Nat Genet. 2007, 39: 457-466. 10.1038/ng1990.PubMedView ArticleGoogle Scholar
- Okitsu CY, Hsieh CL: DNA methylation dictates histone H3K4 methylation. Mol Cell Biol. 2007, 27: 2746-2757. 10.1128/MCB.02291-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Ciccone DN, Su H, Hevi S, Gay F, Lei H, Bajko J, Xu G, Li E, Chen T: KDM1B is a histone H3K4 demethylase required to establish maternal genomic imprints. Nature. 2009, 461: 415-418. 10.1038/nature08315.PubMedView ArticleGoogle Scholar
- Wang J, Hevi S, Kurash JK, Lei H, Gay F, Bajko J, Su H, Sun W, Chang H, Xu G, Gaudet F, Li E, Chen T: The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA methylation. Nat Genet. 2009, 41: 125-129. 10.1038/ng.268.PubMedView ArticleGoogle Scholar
- Jia D, Jurkowska RZ, Zhang X, Jeltsch A, Cheng X: Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. Nature. 2007, 449: 248-251. 10.1038/nature06146.PubMedPubMed CentralView ArticleGoogle Scholar
- Ooi SK, Qiu C, Bernstein E, Li K, Jia D, Yang Z, Erdjument-Bromage H, Tempst P, Lin SP, Allis CD, Cheng X, Bestor TH: DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature. 2007, 448: 714-717. 10.1038/nature05987.PubMedPubMed CentralView ArticleGoogle Scholar
- Willson JK, Bittner GN, Oberley TD, Meisner LF, Weese JL: Cell culture of human colon adenomas and carcinomas. Cancer Res. 1987, 47: 2704-2713.PubMedGoogle Scholar
- Scacheri PC, Davis S, Odom DT, Crawford GE, Perkins S, Halawi MJ, Agarwal SK, Marx SJ, Spiegel AM, Meltzer PS, Collins FS: Genome-wide analysis of menin binding provides insights into MEN1 tumorigenesis. PLoS Genet. 2006, 2: e51-10.1371/journal.pgen.0020051.PubMedPubMed CentralView ArticleGoogle Scholar
- Boyle AP, Davis S, Shulha HP, Meltzer P, Margulies EH, Weng Z, Furey TS, Crawford GE: High-resolution mapping and characterization of open chromatin across the genome. Cell. 2008, 132: 311-322. 10.1016/j.cell.2007.12.014.PubMedPubMed CentralView ArticleGoogle Scholar
- Li H, Ruan J, Durbin R: Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res. 2008, 18: 1851-1858. 10.1101/gr.078212.108.PubMedPubMed CentralView ArticleGoogle Scholar
- Boyle AP, Guinney J, Crawford GE, Furey TS: F-Seq: a feature density estimator for high-throughput sequence tags. Bioinformatics. 2008, 24: 2537-2538. 10.1093/bioinformatics/btn480.PubMedPubMed CentralView ArticleGoogle Scholar
- Pilozzi E, Onelli MR, Ziparo V, Mercantini P, Ruco L: CDX1 expression is reduced in colorectal carcinoma and is associated with promoter hypermethylation. J Pathol. 2004, 204: 289-295. 10.1002/path.1641.PubMedView ArticleGoogle Scholar
- Loh K, Chia JA, Greco S, Cozzi SJ, Buttenshaw RL, Bond CE, Simms LA, Pike T, Young JP, Jass JR, Spring KJ, Leggett BA, Whitehall VL: Bone morphogenic protein 3 inactivation is an early and frequent event in colorectal cancer development. Genes Chromosomes Cancer. 2008, 47: 449-460. 10.1002/gcc.20552.PubMedView ArticleGoogle Scholar
- Veigl ML, Kasturi L, Olechnowicz J, Ma AH, Lutterbaugh JD, Periyasamy S, Li GM, Drummond J, Modrich PL, Sedwick WD, Markowitz SD: Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci USA. 1998, 95: 8698-8702. 10.1073/pnas.95.15.8698.PubMedPubMed CentralView ArticleGoogle Scholar
- Han H, Cortez CC, Yang X, Nichols PW, Jones PA, Liang G: DNA methylation directly silences genes with non-CpG island promoters and establishes a nucleosome occupied promoter. Hum Mol Genet. 2011, 20: 4299-4310. 10.1093/hmg/ddr356.PubMedPubMed CentralView ArticleGoogle Scholar
- Bernstein BE, Mikkelsen TS, Xie X, Kamal M, Huebert DJ, Cuff J, Fry B, Meissner A, Wernig M, Plath K, Jaenisch R, Wagschal A, Feil R, Schreiber SL, Lander ES: A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell. 2006, 125: 315-326. 10.1016/j.cell.2006.02.041.PubMedView ArticleGoogle Scholar
- Mi H, Dong Q, Muruganujan A, Gaudet P, Lewis S, Thomas PD: PANTHER version 7: improved phylogenetic trees, orthologs and collaboration with the Gene Ontology Consortium. Nucleic Acids Res. 2010, 38: D204-210. 10.1093/nar/gkp1019.PubMedPubMed CentralView ArticleGoogle Scholar
- Schug J, Schuller WP, Kappen C, Salbaum JM, Bucan M, Stoeckert CJ: Promoter features related to tissue specificity as measured by Shannon entropy. Genome Biol. 2005, 6: R33-10.1186/gb-2005-6-4-r33.PubMedPubMed CentralView ArticleGoogle Scholar
- Kondo Y, Shen L, Cheng AS, Ahmed S, Boumber Y, Charo C, Yamochi T, Urano T, Furukawa K, Kwabi-Addo B, Gold DL, Sekido Y, Huang TH, Issa JP: Gene silencing in cancer by histone H3 lysine 27 trimethylation independent of promoter DNA methylation. Nat Genet. 2008, 40: 741-750. 10.1038/ng.159.PubMedView ArticleGoogle Scholar
- Gal-Yam EN, Egger G, Iniguez L, Holster H, Einarsson S, Zhang X, Lin JC, Liang G, Jones PA, Tanay A: Frequent switching of Polycomb repressive marks and DNA hypermethylation in the PC3 prostate cancer cell line. Proc Natl Acad Sci USA. 2008, 105: 12979-12984. 10.1073/pnas.0806437105.PubMedPubMed CentralView ArticleGoogle Scholar
- Muse GW, Gilchrist DA, Nechaev S, Shah R, Parker JS, Grissom SF, Zeitlinger J, Adelman K: RNA polymerase is poised for activation across the genome. Nat Genet. 2007, 39: 1507-1511. 10.1038/ng.2007.21.PubMedPubMed CentralView ArticleGoogle Scholar
- Core LJ, Waterfall JJ, Lis JT: Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science. 2008, 322: 1845-1848. 10.1126/science.1162228.PubMedPubMed CentralView ArticleGoogle Scholar
- Nagae G, Isagawa T, Shiraki N, Fujita T, Yamamoto S, Tsutsumi S, Nonaka A, Yoshiba S, Matsusaka K, Midorikawa Y, Ishikawa S, Soejima H, Fukayama M, Suemori H, Nakatsuji N, Kume S, Aburatani H: Tissue-specific demethylation in CpG-poor promoters during cellular differentiation. Hum Mol Genet. 2011, 20: 2710-2721. 10.1093/hmg/ddr170.PubMedView ArticleGoogle Scholar
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