Rochelle L. Tiedemann

Linking DNA Methyltransferases to Epigenetic Marks and Nucleosome Structure Genome-wide in Human Tumor Cells

DNA methylation, mediated by the combined action of three DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), is essential for mammalian development and is a major contributor to cellular transformation. To elucidate how DNA methylation is targeted, we mapped the genome-wide localization of all DNMTs and methylation, and examined the relationships among these markers, histone modifications, and nucleosome structure in a pluripotent human tumor cell line in its undifferentiated and differentiated states. Our findings reveal a strong link between DNMTs and transcribed loci, and that DNA methylation is not a simple sum of DNMT localization patterns. A comparison of the epigenomes of normal and cancerous stem cells, and pluripotent and differentiated states shows that the presence of at least two DNMTs is strongly associated with loci targeted for DNA hypermethylation. Taken together, these results shed important light on the determinants of DNA methylation and how it may become disrupted in cancer cells.

Distinct and overlapping control of 5-methylcytosine and 5-hydroxymethylcytosine by the TET proteins in human cancer cells

BackgroundThe TET family of dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), but their involvement in establishing normal 5mC patterns during mammalian development and their contributions to aberrant control of 5mC during cellular transformation remain largely unknown. We depleted TET1, TET2, and TET3 in a pluripotent embryonic carcinoma cell model and examined the impact on genome-wide 5mC, 5hmC, and transcriptional patterns.ResultsTET1 depletion yields widespread reduction of 5hmC, while depletion of TET2 and TET3 reduces 5hmC at a subset of TET1 targets suggesting functional co-dependence. TET2 or TET3 depletion also causes increased 5hmC, suggesting these proteins play a major role in 5hmC removal. All TETs prevent hypermethylation throughout the genome, a finding dramatically illustrated in CpG island shores, where TET depletion results in prolific hypermethylation. Surprisingly, TETs also promote methylation, as hypomethylation was associated with 5hmC reduction. TET function is highly specific to chromatin environment: 5hmC maintenance by all TETs occurs at polycomb-marked chromatin and genes expressed at moderate levels; 5hmC removal by TET2 is associated with highly transcribed genes enriched for H3K4me3 and H3K36me3. Importantly, genes prone to hypermethylation in cancer become depleted of 5hmC with TET deficiency, suggesting that TETs normally promote 5hmC at these loci. Finally, all three TETs, but especially TET2, are required for 5hmC enrichment at enhancers, a condition necessary for expression of adjacent genes.ConclusionsThese results provide novel insight into the division of labor among TET proteins and reveal important connections between TET activity, the chromatin landscape, and gene expression.

Acute Depletion Redefines the Division of Labor among DNA Methyltransferases in Methylating the Human Genome

Global patterns of DNA methylation, mediated by the DNA methyltransferases (DNMTs), are disrupted in all cancers by mechanisms that remain largely unknown, hampering their development as therapeutic targets. Combinatorial acute depletion of all DNMTs in a pluripotent human tumor cell line, followed by epigenome and transcriptome analysis, revealed DNMT functions in fine detail. DNMT3B occupancy regulates methylation during differentiation, whereas an unexpected interplay was discovered in which DNMT1 and DNMT3B antithetically regulate methylation and hydroxymethylation in gene bodies, a finding confirmed in other cell types. DNMT3B mediated non-CpG methylation, whereas DNMT3L influenced the activity of DNMT3B toward non-CpG versus CpG site methylation. Altogether, these data reveal functional targets of each DNMT, suggesting that isoform selective inhibition would be therapeutically advantageous.

Dynamic reprogramming of DNA methylation in SETD2-deregulated renal cell carcinoma

Clear cell renal cell carcinomas (ccRCCs) harbor frequent mutations in epigenetic modifiers including SETD2, the H3K36me3 writer. We profiled DNA methylation (5mC) across the genome in cell line-based models of SETD2 inactivation and SETD2 mutant primary tumors because 5mC has been linked to H3K36me3 and is therapeutically targetable. SETD2 depleted cell line models (long-term and acute) exhibited a DNA hypermethylation phenotype coinciding with ectopic gains in H3K36me3 centered across intergenic regions adjacent to low expressing genes, which became upregulated upon dysregulation of the epigenome. Poised enhancers of developmental genes were prominent hypermethylation targets. SETD2 mutant primary ccRCCs, papillary renal cell carcinomas, and lung adenocarcinomas all demonstrated a DNA hypermethylation phenotype that segregated tumors by SETD2 genotype and advanced grade. These findings collectively demonstrate that SETD2 mutations drive tumorigenesis by coordinated disruption of the epigenome and transcriptome,and they have important implications for future therapeutic strategies targeting chromatin regulator mutant tumors.

Nucleosome positioning changes during human embryonic stem cell differentiation

ABSTRACT Nucleosomes are the basic unit of chromatin. Nucleosome positioning (NP) plays a key role in transcriptional regulation and other biological processes. To better understand NP we used MNase-seq to investigate changes that occur as human embryonic stem cells (hESCs) transition to nascent mesoderm and then to smooth muscle cells (SMCs). Compared to differentiated cell derivatives, nucleosome occupancy at promoters and other notable genic sites, such as exon/intron junctions and adjacent regions, in hESCs shows a stronger correlation with transcript abundance and is less influenced by sequence content. Upon hESC differentiation, genes being silenced, but not genes being activated, display a substantial change in nucleosome occupancy at their promoters. Genome-wide, we detected a shift of NP to regions of higher G+C content as hESCs differentiate to SMCs. Notably, genomic regions with higher nucleosome occupancy harbor twice as many G↔C changes but fewer than half A↔T changes, compared to regions with lower nucleosome occupancy. Finally, our analysis indicates that the hESC genome is not rearranged and has a sequence mutation rate resembling normal human genomes. Our study reveals another unique feature of hESC chromatin, and sheds light on the relationship between nucleosome occupancy and sequence G+C content.

Abstract 2319: Dynamics of TET methylcytosine dioxygenases in 5-methylcytosine and 5-hydroxymethylcytosine patterning in human cancer cells

The Ten-eleven translocation (TET) family of dioxygenases hydroxylate 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. 5mC provides important epigenetic instructions during development, and its aberrant control is a major contributor to cellular transformation; however TET functions in regulating the epigenome, particularly in cancer, remain largely unknown. We targeted TET1, TET2, and TET3 for siRNA-mediated depletion in pluripotent human embryonic carcinoma cells and examined the impact on 5mC and 5hmC genome-wide localization. TET1, TET2, and TET3 co-regulate 5hmC at many sites, and depletion of only one of the TETs is sufficient to reduce 5hmC at these co-regulated sites, suggesting a functional co-dependence for TETs. Depletion of TET1 and TET2 had the greatest impact on 5hmC levels at high and low CpG density promoters, respectively, indicating that TETs exhibit DNA sequence-based functional specificity. All TETs prevent hypermethylation throughout the genome, especially in CpG island shores, where TET depletion resulted in prolific hypermethylation. Promoter hypermethylation resulting from TET depletion was associated with histone H2AK119 monoubiquitination, DNMT1, and DNMT3B occupancy. Surprisingly, TETs also promote cytosine methylation, as many loci became hypomethylated following TET depletion. Induction of differentiation generally caused 5hmC reduction, except at transcriptionally activated genes, which become enriched for 5hmC. Importantly, genes prone to promoter hypermethylation in cancer become depleted of intragenic 5hmC and 5mC with TET deficiency. This study highlights the multi-dimensional functions of TETs in mediating DNA methylation, hydroxymethylation, and gene expression patterns, and the results reveal that chromatin landscape and DNA sequence composition are regulators of TET function. Citation Format: Emily L. Putiri, Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Dynamics of TET methylcytosine dioxygenases in 5-methylcytosine and 5-hydroxymethylcytosine patterning in human cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2319. doi:10.1158/1538-7445.AM2014-2319

Abstract 2309: Identification of common and unique epigenetic signatures of chronic hepatitis infection and alcohol abuse in human liver disease

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Dysregulation of the intrinsic DNA methylation landscape is a ubiquitous feature of human carcinogenesis, manifested by global hypomethylation and promoter-specific hypermethylation, ultimately resulting in genome instability and tumor suppressor gene silencing, respectively. Alterations in DNA methylation are particularly apparent in hepatocellular carcinoma (HCC) which afflicts roughly 750,000 new patients each year [1]. Indeed, it has been demonstrated that a variety of tumor suppressor genes (e.g. p53, E-cadherin) are hypermethylated and silenced in HCC [2-4]. Importantly, HCC is accompanied by the premalignant stage of cirrhosis in 80% of cases. One major roadblock to understanding the methylome in HCC is the presence of multiple etiologies such as Hepatitis C virus (HCV), Hepatitis B virus (HBV), and chronic alcoholism. Therefore, we performed genome-wide methylation profiling to dissect the methylation patterns of more than 170 primary liver samples to stratify etiologic and stage-specific changes in the DNA methylation landscape. Our results profile the DNA methylation landscape across normal, cirrhotic, and HCC livers in the largest study of its kind to date. We unveil distinct locus-specific and large-scale effects of HCV, HBV, and chronic alcoholism in hepatocarcinogenesis. Furthermore, analysis indicates a specific methylation profile for individual etiologies as well as conserved patterns throughout cirrhosis and hepatocellular carcinoma. Our study demonstrates that each etiology contains potential biomarkers and targets for downstream clinical therapeutics. This study is our first step toward defining the epigenome in cirrhosis and hepatocellular carcinoma and will be combined with future genomic and epimutational data (e.g. transcription, histone modifications, miRNA) to determine the true extent and interplay between epigenetic marks across different stages of liver cancer. Overall, this research has the potential to improve our understanding of epigenetics and result in diagnostic, prognostic, and therapeutic epigenetic signatures in cirrhosis and hepatocellular carcinoma, which are expected to allow for more timely and efficient detection of disease. 1. Jemal, A., et al., Global cancer statistics. CA: a cancer journal for clinicians, 2011. 61(2): p. 69-90. 2. Tischoff, I. and A. Tannapfe, DNA methylation in hepatocellular carcinoma. World journal of gastroenterology : WJG, 2008. 14(11): p. 1741-8. 3. Calvisi, D.F., et al., Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. The Journal of clinical investigation, 2007. 117(9): p. 2713-22. 4. Lambert, M.P., et al., Aberrant DNA methylation distinguishes hepatocellular carcinoma associated with HBV and HCV infection and alcohol intake. Journal of hepatology, 2011. 54(4): p. 705-15. Citation Format: Ryan A. Hlady, Rochelle Tiedemann, William Puszyk, Chen Liu, Jeong-Hyeon Choi, Keith D. Robertson. Identification of common and unique epigenetic signatures of chronic hepatitis infection and alcohol abuse in human liver disease. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2309. doi:10.1158/1538-7445.AM2014-2309

Abstract 2305: Acute depletion reveals novel co-regulation of DNA methylation at conserved loci by DNMT1 and DNMT3B

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, 3B, 3L) and maintaining (DNMT1) DNA methylation genome-wide. Aberrant DNA methylation is frequently observed in cancer; however, little is known about how regulation of this modification goes awry. In this study, we aim to understand how DNA methylation is regulated by the DNMTs throughout the genome by identifying specific and broad changes in methylation patterning upon depletion of the DNMTs. We utilize siRNA technology to acutely deplete NCCIT embryonal carcinoma cells of DNMT mRNA (individually and in combination), and then assay the impact on genome-wide DNA methylation patterns using the HumanMethylation450 Bead Chip (450K array). Depletion of DNMT1 (individual/combination) resulted in widespread hypomethylation, most notably in gene bodies, 3′UTRs, and intergenic sequences. DNMT3 knockdown resulted in more specific changes in DNA methylation, but surprisingly, more hypermethylation (predominately in gene bodies) than hypomethylation events occurred. These specific hypermethylation events, particularly in samples with DNMT3B KD, significantly overlapped with sites hypomethylated in DNMT1 KD conditions, indicating a potential cross-regulatory role for DNMT1 and DNMT3B in regulating DNA methylation across gene bodies. To gain a more comprehensive genome-wide view of DNA methylation in the absence of DNMT3B, we performed Methyl-CpG-Binding-Domain(MBD)-seq on DNMT3B KD cells. MBD-seq revealed dynamic changes in methylation with minor hypermethylation in promoters and subtle hypomethylation across gene bodies; however, analysis of only the most significant methylation changes (> 4-fold) revealed that more hypermethylation events occur in intronic sequences, consistent with results obtained using the 450K array. To further investigate the overlap between DNMT1 hypomethylated and DNMT3B hypermethylated sites, we examined DNA methylation in HCT116 colorectal carcinoma cells lacking (KO) or over-expressing (KI) DNMT1/DNMT3B. Interestingly, a marked number of CpG sites that gained methylation in the DNMT3B KO overlapped significantly with sites that became hypermethylated in DNMT1 and DNMT3B KI, and hypomethylated in DNMT1 KO. Additionally, these HCT116 hypermethylated CpG sites gained methylation in NCCIT DNMT3B KD (individual/combination) and lost methylation in DNMT1 KD. Taken together, these results suggest that DNMT1 and DNMT3B co-regulate DNA methylation at conserved loci across cell types in an opposing fashion, providing novel insight into a potential regulatory mechanism for DNA methylation patterning. Further elucidation of this DNMT1 and DNMT3B co-regulation holds the potential to yield novel therapeutic strategies for correcting aberrant methylation events in cancer. Citation Format: Rochelle Tiedemann, Jeong-Hyeon Choi, Keith Robertson. Acute depletion reveals novel co-regulation of DNA methylation at conserved loci by DNMT1 and DNMT3B. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2305. doi:10.1158/1538-7445.AM2014-2305

Abstract 2970: Acute depletion of DNA methyltransferases reveals unique and overlapping target sites in cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) DNA methylation to regulate gene transcription and promote overall genome stability. Global changes in DNA methylation, such as hypomethylation of repetitive sequences and hypermethylation of tumor supressor gene promoters, are commonly observed in various types of cancer; however, the DNMTs’ contribution to this aberrant methylation remains largely unknown. In this study, we aim to identify unique and overlapping target sites for each of the DNMTs in order to better understand aberrant DNA methylation in cancer that will ultimately permit development of new therapeutic strategies. We utilize siRNA-mediated knockdown technology to acutely deplete the mRNA for each of the DNMTs in both individual and combinatorial fashion in NCCIT embryonal carcinoma cells. Subsequently, DNA methylation is observed genome-wide in each DNA sample using two different methodologies: (1) Methyl-CpG Binding Domain (MBD)-seq, which identifies regions of the genome that are enriched for DNA methylation, and (2) Illumina Infinium HumanMethylation450 BeadChip (450K array) allowing for specific CpG site methylation status determination. Aligned MBD-seq sequences for individual DNMT knockdowns are analyzed by read coverage nomalization within 100Kbp windows and using various peak-calling algorithms (e.g. MACS, BALM). For the 450K array, DNA samples for both individual and combination knockdowns undergo bisulfite conversion and array processing; β-values for each CpG site are derived from the array signal intensities using GenomeStudio and R/Bioconductor (minfi). Genome-wide DNA methylation analysis reveals that DNMT1 depletion, both individual and in combination with knockdown of other DNMTs, results in global demethylation among all genomic features. Interestingly, a small population of genes exhibit hypermethylation in gene-promoter CpG islands for DNMT1 (individual only) depletion. In contrast, de novo methyltransferase depletion (individual and combination) shows more specific demethylation effects. In particular, hypomethylation events resulting from DNMT3B depletion (individual and combination (not 3B+3L)) occur within gene bodies and largely outside of CpG islands and flanking regions (shores, shelves). Additionally, a number of hypermethylation events occurring within the 3’UTR region of genes are observed in de novo methyltransferase depleted samples. We anticipate further analysis will reveal unique and overlapping target sites for each of the DNMTs that will lay the ground-work necessary to characterize and understand DNMT recruitment both in normal and cancerous tissues. Citation Format: Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Acute depletion of DNA methyltransferases reveals unique and overlapping target sites in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2970. doi:10.1158/1538-7445.AM2013-2970

Abstract B34: Acute depletion reveals novel divisions of labor among human DNA methyltransferases in cancer

DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) DNA methylation genome-wide. Hypomethylation of repetitive sequences and transposable elements coupled with gene-specific promoter hypermethylation events contribute to the genomic instability and loss of tumor suppressor gene transcription observed in cancer. Regulation of aberrant methylation in cancer remains poorly understood. The aim of our study was to identify unique and overlapping target sites for each of the DNMTs to better understand regulation of normal and aberrant DNA methylation. We hypothesized that acute depletion of the DNMTs (individual and combination) mediated by siRNA technology in NCCIT human embryonic carcinoma cells would result in both distinct and broad changes in DNA methylation patterning. Genome-wide methylation was assayed using the HumanMethylation450 Bead Chip (450K array) allowing for specific CpG site methylation status determination. Select target sites were verified by bisulfite genomic sequencing. DNMT1 knockdown samples (individual/combination) revealed genome-wide hypomethylation, with the strongest demethylation occurring in gene bodies, 3′UTR, and intergenic sequences. Interestingly, only the DNMT1 individual knockdown showed significant hypermethylation in gene promoters. DNMT3 knockdowns showed more specific changes in DNA methylation, but surprisingly, more hypermethylation events occurred than hypomethylation at CpG dinucleotides. Hypermethylation observed in DNMT3 knockdown occurred primarily in gene bodies and 3′UTR, and overlapped significantly with those genes that become hypomethylated in DNMT1 knockdown, indicating a potential cross-regulatory role for the DNMTs to maintain proper regulation of DNA methylation at specific gene termini. Conversely, gene promoters were targeted for hypomethylation in DNMT3 knockdown, and did not significantly overlap with genes that become hypermethylated in DNMT1 knockdown. Of particular interest was that DNMT3B knockdown resulted in widespread non-CpG hypomethylation. In contrast, DNMT3L knockdown showed non-CpG hypermethylation, indicating a potential mechanism for regulation of non-CpG methylation where DNMT3B is responsible for non-CpG methylation, and DNMT3L acts to restrict DNMT3B9s activity at non-CpG dinucleotides. Our results reveal a complex view of DNA methylation regulation, in which DNMTs not only target specific sites for methylation, but also cooperate to establish and maintain proper levels of DNA methylation at CpG and non-CpG dinucleotides. Moving forward, we believe our results will provide the framework needed to define the regulatory mechanisms by which DNA methylation is conferred and ultimately develop therapeutic strategies to correct aberrant methylation events that occur in cancer. Citation Format: Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Acute depletion reveals novel divisions of labor among human DNA methyltransferases in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B34.