Background: The clinical utility of DNA methylation as a predictive or prognostic biomarker requi... more Background: The clinical utility of DNA methylation as a predictive or prognostic biomarker requires scalable resequencing protocols for bisulfite-converted DNA. Key features of any validation method should be adaptability for low-or high-throughput needs and high reproducibility, and should only require minimal amounts of precious clinical sample as input material. Critically, this method should also deliver robust results when working with bisulfite-converted DNA extracted from formalin-fixed, paraffin-embedded (FFPE) blocks. Results: We report here for the first time on comparison studies between the Fluidigm Access Array system and multiplex assays for multiplex bisulfite PCR resequencing. The requirement of the Fluidigm Access Array system for high template amounts and its sensitivity to variations in template quality rendered it unsuitable for bisulfite PCR applications utilizing FFPE DNA. In response to this limitation, we established a multiplex bisulfite PCR assay capable of delivering robust methylation data using minimal amounts of FFPE clinical DNA. To evaluate the parameters and reproducibility of this assay, 57 amplicons were used to prepare sequencing libraries in triplicate for 13 FFPE tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%). Analysis of this data demonstrated that this multiplex assay had high reproducibility (mean standard deviation of 1.4% for methylation values), was low cost, required low sample input (50 ng of DNA or less), and could be scaled for both low-and high-throughput needs. Notably, ExoSAP-IT (exonuclease I) treatment to remove residual primers in bisulfite resequencing libraries appeared to degrade the library and generate a high-molecular weight smear which may impact on the degree of methylation assessed. Conclusions: Multiplex bisulfite PCR assays represent a convenient and scalable method for validation and screening of methylated DNA regions from archival FFPE DNA. Moreover, the library construction process detailed here can be rapidly optimized and implemented with a minimal amount of work, can be performed using the standard equipment found in any molecular biology laboratory, and can be easily adapted for use on both genomic DNA and bisulfite DNA applications. However, in preparing bisulfite libraries for sequencing, the use of ExoSAP-IT is not recommended due to potential off-target nuclease effects which may impact downstream methylation analysis.
Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel opti... more Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel options for tumour stratification. Here, we perform whole-genome methylation capture sequencing on small amounts of DNA isolated from formalin-fixed, paraffin-embedded tissue from triple-negative breast cancer (TNBC) and matched normal samples. We identify differentially methylated regions (DMRs) enriched with promoters associated with transcription factor binding sites and DNA hypersensitive sites. Importantly, we stratify TNBCs into three distinct methylation clusters associated with better or worse prognosis and identify 17 DMRs that show a strong association with overall survival, including DMRs located in the Wilms tumour 1 (WT1) gene, bi-directional-promoter and antisense WT1-AS. Our data reveal that coordinated hypermethylation can occur in oestrogen receptor-negative disease, and that characterizing the epigenetic framework provides a potential signature to stratify TNBCs. Together, ...
Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the... more Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer Mapping the sequences controlling 93 breast cancer-associated miRNA genes EJH Wee et al
Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we s... more Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we show in prostate cancer that miRNA-205 (miR-205) transcription is commonly repressed and the MIR-205 locus is hypermethylated. LOC642587, the MIR-205 host gene of unknown function, is also concordantly inactivated. We show that miR-205 targets mediator 1 (MED1, also called TRAP220 and PPARBP) for transcriptional silencing in normal prostate cells, leading to reduction in MED1 mRNA levels, and in total and active phospho-MED1 protein. Overexpression of miR-205 in prostate cancer cells negatively affects cell viability, consistent with a tumor suppressor function. We found that hypermethylation of the MIR-205 locus was strongly related with a decrease in miR-205 expression and an increase in MED1 expression in primary tumor samples (n ¼ 14), when compared with matched normal prostate (n ¼ 7). An expanded patient cohort (tumor n ¼ 149, matched normal n ¼ 30) also showed significant MIR-205 DNA methylation in tumors compared with normal, and MIR-205 hypermethylation is significantly associated with biochemical recurrence (hazard ratio ¼ 2.005, 95% confidence interval (1.109, 3.625), P ¼ 0.02), in patients with low preoperative prostate specific antigen. In summary, these results suggest that miR-205 is an epigenetically regulated tumor suppressor that targets MED1 and may provide a potential biomarker in prostate cancer management.
Mutants in the Drosophila InsP3R gene (itpr) are flight defective. Expression of the wild-type In... more Mutants in the Drosophila InsP3R gene (itpr) are flight defective. Expression of the wild-type InsP3R in aminergic interneurons rescues flight. However, molecular and cellular changes in the central nervous system of InsP3R mutants that lead to flightless behavior remain unknown. To understand the molecular basis of flight phenotypes in Drosophila InsP3R mutants a microarray screen was done with RNA isolated from adult heads and thoraces. We found down-regulation of several genes that affect the excitability of neurons and muscles. Among these the role of glutamine synthetase 2 was investigated further. This enzyme reduces glutamate levels at the synapse. Our results show that Gs2 (glutamine synthetase 2) transcripts and glutamate levels correspond precisely but inversely, with InsP3R phenotypes, which can be rescued by a mutant allele for Gs2, namely, Gs2(m13). From measuring axonal branches and synapse number at a glutamatergic synapse--the adult flight neuromuscular junction--of InsP3R mutants, we conclude that glutamate homeostasis is altered through a cell non-autonomous mechanism, and is likely to be an important basis for the observed flight defects.
DNA methylation is an essential epigenetic modification that plays a key role associated with the... more DNA methylation is an essential epigenetic modification that plays a key role associated with the regulation of gene expression during differentiation, but in disease states such as cancer, the DNA methylation landscape is often deregulated. There are now numerous technologies available to interrogate the DNA methylation status of CpG sites in a targeted or genome-wide fashion, but each method, due to intrinsic biases, potentially interrogates different fractions of the genome. In this study, we compare the affinity-purification of methylated DNA between two popular genome-wide techniques, methylated DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain-based capture (MBDCap), and show that each technique operates in a different domain of the CpG density landscape. We explored the effect of whole-genome amplification and illustrate that it can reduce sensitivity for detecting DNA methylation in GC-rich regions of the genome. By using MBDCap, we compare and contrast microarray-and sequencing-based readouts and highlight the impact that copy number variation (CNV) can make in differential comparisons of methylomes. These studies reveal that the analysis of DNA methylation data and genome coverage is highly dependent on the method employed, and consideration must be made in light of the GC content, the extent of DNA amplification, and the copy number.
Signaling by the second messenger inositol 1,4,5-trisphosphate is thought to affect several devel... more Signaling by the second messenger inositol 1,4,5-trisphosphate is thought to affect several developmental and physiological processes. Mutants in the inositol 1,4,5-trisphosphate receptor (itpr) gene of Drosophila exhibit delays in molting while stronger alleles are also larval lethal. In a freshly generated set of EMS alleles for the itpr locus we have sequenced and identified single point mutations in seven mutant chromosomes. The predicted allelic strength of these mutants matches the observed levels of lethality. They range from weak hypomorphs to complete nulls. Interestingly, lethality in three heteroallelic combinations has a component of cold sensitivity. The temporal focus of cold sensitivity lies in the larval stages, predominantly at second instar. Coupled with our earlier observation that an itpr homozygous null allele dies at the second instar stage, it appears that there is a critical period for itpr gene function in second instar larvae.
DNA methylation primarily occurs at CpG dinucleotides in mammals and is a common epigenetic mark ... more DNA methylation primarily occurs at CpG dinucleotides in mammals and is a common epigenetic mark that plays a critical role in the regulation of gene expression. Profiling DNA methylation patterns across the genome is vital to understand DNA methylation changes that occur during development and in disease phenotype. In this study, we compared two commonly used approaches to enrich for methylated DNA regions of the genome, namely methyl-DNA immunoprecipitation (MeDIP) that is based on enrichment with antibodies specific for 5'-methylcytosine (5MeC), and capture of methylated DNA using a methyl-CpG binding domain-based (MBD) protein to discover differentially methylated regions (DMRs) in cancer. The enriched methylated DNA fractions were interrogated on Affymetrix promoter tiling arrays and differentially methylated regions were identified. A detailed validation study of 42 regions was performed using Sequenom MassCLEAVE technique. This detailed analysis revealed that both enrichment techniques are sensitive for detecting DMRs and preferentially identified different CpG rich regions of the prostate cancer genome, with MeDIP commonly enriching for methylated regions with a low CpG density, while MBD capture favors regions of higher CpG density and identifies the greatest proportion of CpG islands. This is the first detailed validation report comparing different methylated DNA enrichment techniques for identifying regions of differential DNA methylation. Our study highlights the importance of understanding the nuances of the methods used for DNA genome-wide methylation analyses so that accurate interpretation of the biology is not overlooked.
The inositol (1,4,5)-trisphosphate receptor (InsP 3 R) is an intracellular calcium (Ca 21 ) relea... more The inositol (1,4,5)-trisphosphate receptor (InsP 3 R) is an intracellular calcium (Ca 21 ) release channel that plays a crucial role in cell signaling. In Drosophila melanogaster a single InsP 3 R gene (itpr) encodes a protein (DmInsP 3 R) that is ;60% conserved with mammalian InsP 3 Rs. A number of itpr mutant alleles have been identified in genetic screens and studied for their effect on development and physiology. However, the functional properties of wild-type or mutant DmInsP 3 Rs have never been described. Here we use the planar lipid bilayer reconstitution technique to describe single-channel properties of embryonic and adult head DmInsP 3 R splice variants. The three mutants chosen in this study reside in each of the three structural domains of the DmInsP 3 R-the amino-terminal ligand binding domain (ug3), the middle-coupling domain (wc703), and the channel-forming region (ka901). We discovered that 1), the major functional properties of DmInsP 3 R (conductance, gating, and sensitivity to InsP 3 and Ca 21 ) are remarkably conserved with the mammalian InsP 3 R1; 2), single-channel conductance of the adult head DmInsP 3 R isoform is 89 pS and the embryonic DmInsP 3 R isoform is 70 pS; 3), ug3 mutation affects sensitivity of the DmInsP 3 Rs to activation by InsP 3 , but not their InsP 3 -binding properties; 4), wc703 channels have increased sensitivity to modulation by Ca 21 ; and 5), homomeric ka901 channels are not functional. We correlated the results obtained in planar lipid bilayer experiments with measurements of InsP 3 -induced Ca 21 fluxes in microsomes isolated from wildtype and heterozygous itpr mutants. Our study validates the use of D. melanogaster as an appropriate model for InsP 3 R structure-function studies and provides novel insights into the fundamental mechanisms of the InsP 3 R function.
MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA... more MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.
MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA... more MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.
Background: The clinical utility of DNA methylation as a predictive or prognostic biomarker requi... more Background: The clinical utility of DNA methylation as a predictive or prognostic biomarker requires scalable resequencing protocols for bisulfite-converted DNA. Key features of any validation method should be adaptability for low-or high-throughput needs and high reproducibility, and should only require minimal amounts of precious clinical sample as input material. Critically, this method should also deliver robust results when working with bisulfite-converted DNA extracted from formalin-fixed, paraffin-embedded (FFPE) blocks. Results: We report here for the first time on comparison studies between the Fluidigm Access Array system and multiplex assays for multiplex bisulfite PCR resequencing. The requirement of the Fluidigm Access Array system for high template amounts and its sensitivity to variations in template quality rendered it unsuitable for bisulfite PCR applications utilizing FFPE DNA. In response to this limitation, we established a multiplex bisulfite PCR assay capable of delivering robust methylation data using minimal amounts of FFPE clinical DNA. To evaluate the parameters and reproducibility of this assay, 57 amplicons were used to prepare sequencing libraries in triplicate for 13 FFPE tumour samples, as well as a series of 5 methylated controls (0%, 25%, 50%, 75%, and 100%). Analysis of this data demonstrated that this multiplex assay had high reproducibility (mean standard deviation of 1.4% for methylation values), was low cost, required low sample input (50 ng of DNA or less), and could be scaled for both low-and high-throughput needs. Notably, ExoSAP-IT (exonuclease I) treatment to remove residual primers in bisulfite resequencing libraries appeared to degrade the library and generate a high-molecular weight smear which may impact on the degree of methylation assessed. Conclusions: Multiplex bisulfite PCR assays represent a convenient and scalable method for validation and screening of methylated DNA regions from archival FFPE DNA. Moreover, the library construction process detailed here can be rapidly optimized and implemented with a minimal amount of work, can be performed using the standard equipment found in any molecular biology laboratory, and can be easily adapted for use on both genomic DNA and bisulfite DNA applications. However, in preparing bisulfite libraries for sequencing, the use of ExoSAP-IT is not recommended due to potential off-target nuclease effects which may impact downstream methylation analysis.
Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel opti... more Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel options for tumour stratification. Here, we perform whole-genome methylation capture sequencing on small amounts of DNA isolated from formalin-fixed, paraffin-embedded tissue from triple-negative breast cancer (TNBC) and matched normal samples. We identify differentially methylated regions (DMRs) enriched with promoters associated with transcription factor binding sites and DNA hypersensitive sites. Importantly, we stratify TNBCs into three distinct methylation clusters associated with better or worse prognosis and identify 17 DMRs that show a strong association with overall survival, including DMRs located in the Wilms tumour 1 (WT1) gene, bi-directional-promoter and antisense WT1-AS. Our data reveal that coordinated hypermethylation can occur in oestrogen receptor-negative disease, and that characterizing the epigenetic framework provides a potential signature to stratify TNBCs. Together, ...
Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the... more Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer Mapping the sequences controlling 93 breast cancer-associated miRNA genes EJH Wee et al
Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we s... more Deregulation of microRNA (miRNA) expression can have a critical role in carcinogenesis. Here we show in prostate cancer that miRNA-205 (miR-205) transcription is commonly repressed and the MIR-205 locus is hypermethylated. LOC642587, the MIR-205 host gene of unknown function, is also concordantly inactivated. We show that miR-205 targets mediator 1 (MED1, also called TRAP220 and PPARBP) for transcriptional silencing in normal prostate cells, leading to reduction in MED1 mRNA levels, and in total and active phospho-MED1 protein. Overexpression of miR-205 in prostate cancer cells negatively affects cell viability, consistent with a tumor suppressor function. We found that hypermethylation of the MIR-205 locus was strongly related with a decrease in miR-205 expression and an increase in MED1 expression in primary tumor samples (n ¼ 14), when compared with matched normal prostate (n ¼ 7). An expanded patient cohort (tumor n ¼ 149, matched normal n ¼ 30) also showed significant MIR-205 DNA methylation in tumors compared with normal, and MIR-205 hypermethylation is significantly associated with biochemical recurrence (hazard ratio ¼ 2.005, 95% confidence interval (1.109, 3.625), P ¼ 0.02), in patients with low preoperative prostate specific antigen. In summary, these results suggest that miR-205 is an epigenetically regulated tumor suppressor that targets MED1 and may provide a potential biomarker in prostate cancer management.
Mutants in the Drosophila InsP3R gene (itpr) are flight defective. Expression of the wild-type In... more Mutants in the Drosophila InsP3R gene (itpr) are flight defective. Expression of the wild-type InsP3R in aminergic interneurons rescues flight. However, molecular and cellular changes in the central nervous system of InsP3R mutants that lead to flightless behavior remain unknown. To understand the molecular basis of flight phenotypes in Drosophila InsP3R mutants a microarray screen was done with RNA isolated from adult heads and thoraces. We found down-regulation of several genes that affect the excitability of neurons and muscles. Among these the role of glutamine synthetase 2 was investigated further. This enzyme reduces glutamate levels at the synapse. Our results show that Gs2 (glutamine synthetase 2) transcripts and glutamate levels correspond precisely but inversely, with InsP3R phenotypes, which can be rescued by a mutant allele for Gs2, namely, Gs2(m13). From measuring axonal branches and synapse number at a glutamatergic synapse--the adult flight neuromuscular junction--of InsP3R mutants, we conclude that glutamate homeostasis is altered through a cell non-autonomous mechanism, and is likely to be an important basis for the observed flight defects.
DNA methylation is an essential epigenetic modification that plays a key role associated with the... more DNA methylation is an essential epigenetic modification that plays a key role associated with the regulation of gene expression during differentiation, but in disease states such as cancer, the DNA methylation landscape is often deregulated. There are now numerous technologies available to interrogate the DNA methylation status of CpG sites in a targeted or genome-wide fashion, but each method, due to intrinsic biases, potentially interrogates different fractions of the genome. In this study, we compare the affinity-purification of methylated DNA between two popular genome-wide techniques, methylated DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain-based capture (MBDCap), and show that each technique operates in a different domain of the CpG density landscape. We explored the effect of whole-genome amplification and illustrate that it can reduce sensitivity for detecting DNA methylation in GC-rich regions of the genome. By using MBDCap, we compare and contrast microarray-and sequencing-based readouts and highlight the impact that copy number variation (CNV) can make in differential comparisons of methylomes. These studies reveal that the analysis of DNA methylation data and genome coverage is highly dependent on the method employed, and consideration must be made in light of the GC content, the extent of DNA amplification, and the copy number.
Signaling by the second messenger inositol 1,4,5-trisphosphate is thought to affect several devel... more Signaling by the second messenger inositol 1,4,5-trisphosphate is thought to affect several developmental and physiological processes. Mutants in the inositol 1,4,5-trisphosphate receptor (itpr) gene of Drosophila exhibit delays in molting while stronger alleles are also larval lethal. In a freshly generated set of EMS alleles for the itpr locus we have sequenced and identified single point mutations in seven mutant chromosomes. The predicted allelic strength of these mutants matches the observed levels of lethality. They range from weak hypomorphs to complete nulls. Interestingly, lethality in three heteroallelic combinations has a component of cold sensitivity. The temporal focus of cold sensitivity lies in the larval stages, predominantly at second instar. Coupled with our earlier observation that an itpr homozygous null allele dies at the second instar stage, it appears that there is a critical period for itpr gene function in second instar larvae.
DNA methylation primarily occurs at CpG dinucleotides in mammals and is a common epigenetic mark ... more DNA methylation primarily occurs at CpG dinucleotides in mammals and is a common epigenetic mark that plays a critical role in the regulation of gene expression. Profiling DNA methylation patterns across the genome is vital to understand DNA methylation changes that occur during development and in disease phenotype. In this study, we compared two commonly used approaches to enrich for methylated DNA regions of the genome, namely methyl-DNA immunoprecipitation (MeDIP) that is based on enrichment with antibodies specific for 5'-methylcytosine (5MeC), and capture of methylated DNA using a methyl-CpG binding domain-based (MBD) protein to discover differentially methylated regions (DMRs) in cancer. The enriched methylated DNA fractions were interrogated on Affymetrix promoter tiling arrays and differentially methylated regions were identified. A detailed validation study of 42 regions was performed using Sequenom MassCLEAVE technique. This detailed analysis revealed that both enrichment techniques are sensitive for detecting DMRs and preferentially identified different CpG rich regions of the prostate cancer genome, with MeDIP commonly enriching for methylated regions with a low CpG density, while MBD capture favors regions of higher CpG density and identifies the greatest proportion of CpG islands. This is the first detailed validation report comparing different methylated DNA enrichment techniques for identifying regions of differential DNA methylation. Our study highlights the importance of understanding the nuances of the methods used for DNA genome-wide methylation analyses so that accurate interpretation of the biology is not overlooked.
The inositol (1,4,5)-trisphosphate receptor (InsP 3 R) is an intracellular calcium (Ca 21 ) relea... more The inositol (1,4,5)-trisphosphate receptor (InsP 3 R) is an intracellular calcium (Ca 21 ) release channel that plays a crucial role in cell signaling. In Drosophila melanogaster a single InsP 3 R gene (itpr) encodes a protein (DmInsP 3 R) that is ;60% conserved with mammalian InsP 3 Rs. A number of itpr mutant alleles have been identified in genetic screens and studied for their effect on development and physiology. However, the functional properties of wild-type or mutant DmInsP 3 Rs have never been described. Here we use the planar lipid bilayer reconstitution technique to describe single-channel properties of embryonic and adult head DmInsP 3 R splice variants. The three mutants chosen in this study reside in each of the three structural domains of the DmInsP 3 R-the amino-terminal ligand binding domain (ug3), the middle-coupling domain (wc703), and the channel-forming region (ka901). We discovered that 1), the major functional properties of DmInsP 3 R (conductance, gating, and sensitivity to InsP 3 and Ca 21 ) are remarkably conserved with the mammalian InsP 3 R1; 2), single-channel conductance of the adult head DmInsP 3 R isoform is 89 pS and the embryonic DmInsP 3 R isoform is 70 pS; 3), ug3 mutation affects sensitivity of the DmInsP 3 Rs to activation by InsP 3 , but not their InsP 3 -binding properties; 4), wc703 channels have increased sensitivity to modulation by Ca 21 ; and 5), homomeric ka901 channels are not functional. We correlated the results obtained in planar lipid bilayer experiments with measurements of InsP 3 -induced Ca 21 fluxes in microsomes isolated from wildtype and heterozygous itpr mutants. Our study validates the use of D. melanogaster as an appropriate model for InsP 3 R structure-function studies and provides novel insights into the fundamental mechanisms of the InsP 3 R function.
MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA... more MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.
MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA... more MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.
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Papers by Shalima Nair