ORCID Profile
0000-0001-8045-718X
Current Organisation
Garvan Institute of Medical Research
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Publisher: Springer Science and Business Media LLC
Date: 17-02-2021
DOI: 10.1186/S13148-021-01023-7
Abstract: BRG1 (encoded by SMARCA4 ) is a catalytic component of the SWI/SNF chromatin remodelling complex, with key roles in modulating DNA accessibility. Dysregulation of BRG1 is observed, but functionally uncharacterised, in a wide range of malignancies. We have probed the functions of BRG1 on a background of prostate cancer to investigate how BRG1 controls gene expression programmes and cancer cell behaviour. Our investigation of SMARCA4 revealed that BRG1 is over-expressed in the majority of the 486 tumours from The Cancer Genome Atlas prostate cohort, as well as in a complementary panel of 21 prostate cell lines. Next, we utilised a temporal model of BRG1 depletion to investigate the molecular effects on global transcription programmes. Depleting BRG1 had no impact on alternative splicing and conferred only modest effect on global expression. However, of the transcriptional changes that occurred, most manifested as down-regulated expression. Deeper examination found the common thread linking down-regulated genes was involvement in proliferation, including several known to increase prostate cancer proliferation ( KLK2 , PCAT1 and VAV3 ). Interestingly, the promoters of genes driving proliferation were bound by BRG1 as well as the transcription factors, AR and FOXA1. We also noted that BRG1 depletion repressed genes involved in cell cycle progression and DNA replication, but intriguingly, these pathways operated independently of AR and FOXA1. In agreement with transcriptional changes, depleting BRG1 conferred G1 arrest. Our data have revealed that BRG1 promotes cell cycle progression and DNA replication, consistent with the increased cell proliferation associated with oncogenesis.
Publisher: Oxford University Press (OUP)
Date: 06-08-2020
Abstract: As reference genome assemblies are updated there is a need to convert epigenome sequence data from older genome assemblies to newer versions, to facilitate data integration and visualization on the same coordinate system. Conversion can be done by re-alignment of the original sequence data to the new assembly or by converting the coordinates of the data between assemblies using a mapping file, an approach referred to as ‘liftover’. Compared to re-alignment approaches, liftover is a more rapid and cost-effective solution. Here, we benchmark six liftover tools commonly used for conversion between genome assemblies by coordinates, including UCSC liftOver, rtracklayer::liftOver, CrossMap, NCBI Remap, flo and segment_liftover to determine how they performed for whole genome bisulphite sequencing (WGBS) and ChIP-seq data. Our results show high correlation between the six tools for conversion of 43 WGBS paired s les. For the chromatin sequencing data we found from interval conversion of 366 ChIP-Seq datasets, segment_liftover generates more reliable results than USCS liftOver. However, we found some regions do not always remain the same after liftover. To further increase the accuracy of liftover and avoid misleading results, we developed a three-step guideline that removes aberrant regions to ensure more robust genome conversion between reference assemblies.
Publisher: Cold Spring Harbor Laboratory
Date: 10-02-2020
DOI: 10.1101/2020.02.09.941013
Abstract: Multiplex bisulphite PCR sequencing is a convenient and scalable method to comprehensively profile DNA methylation at selected loci. The method is useful for validation of methylation biomarker panels on large clinical cohorts, as it can be applied to DNA isolated from fresh tissue, archival formalin fixed paraffin embedded tissue (FFPET) or circulating cell free DNA in plasma. However, successful clinical implementation of DNA methylation biomarkers for disease detection using multiplex bisulphite PCR sequencing, requires user-friendly s le analysis methods and a ersity of visualisation options, which are not met by current tools. We have developed a computational pipeline with an interactive graphical interface, called MethPanel , in order to rapidly analyse multiplex bisulphite PCR sequencing data. MethPanel comprises a complete analysis workflow from genomic alignment to DNA methylation calling and supports an unlimited number of PCR licons and input s les. Moreover, MethPanel offers important and unique features, such as calculation of a polymorphism score and bisulphite PCR bias correction capabilities. MethPanel is designed so that the methylation data from all s les can be run in parallel on either a personal computer or a high performance computer. The outputs are also automatically forwarded to a shinyApp for convenient display, visualisation and sharing data with collaborators and clinicians. Importantly the data is centralised in one location, which aids storage management. MethPanel is freely available at hinhong/MethPanel MethPanel provides a novel parallel pipeline and interactive analysis tool for multiplex bisulphite PCR sequencing to assess DNA methylation marker panels for disease detection.
Publisher: Cold Spring Harbor Laboratory
Date: 04-07-2020
DOI: 10.1101/2020.07.03.187385
Abstract: BRG1 (encoded by SMARCA4 ) is a catalytic component of the SWI/SNF chromatin remodelling complex, with key roles in modulating DNA accessibility. Dysregulation of BRG1 is observed, but functionally uncharacterised, in a wide range of malignancies. We have probed the functions of BRG1 on a background of prostate cancer to investigate how BRG1 controls gene expression programs and cancer cell behaviour. Our investigation of SMARCA4 revealed that BRG1 is universally overexpressed in 486 tumours from The Cancer Genome Atlas prostate cohort, as well as in a complementary panel of 21 prostate cell lines. Next, we utilised a temporal model of BRG1 depletion to investigate the molecular effects on global transcription programs. Unexpectedly, depleting BRG1 had no impact on alternative splicing and conferred only modest effect on global expression. However, of the transcriptional changes that occurred, most manifested as down-regulated expression. Deeper examination found the common thread linking down-regulated genes was involvement in proliferation, including several known to increase prostate cancer proliferation ( KLK2 , PCAT1 and VAV3 ). Interestingly, the promoters of genes driving proliferation were bound by BRG1 as well as the oncogenic transcription factors, AR and FOXA1. We also noted that BRG1 depletion repressed genes involved in cell cycle progression and DNA replication but intriguingly, these pathways operated independently of AR and FOXA1. In agreement with transcriptional changes, depleting BRG1 conferred G1 arrest. Our data have revealed that BRG1 has capacity to drive oncogenesis by coordinating oncogenic pathways dependent on BRG1 for proliferation, cell cycle progression and DNA replication.
Publisher: MDPI AG
Date: 11-12-2020
Abstract: Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy in iduals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled s les from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission s les from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis s les. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease.
Publisher: Oxford University Press (OUP)
Date: 26-12-2021
DOI: 10.1093/BIOINFORMATICS/BTAA1060
Abstract: DNA methylation patterns in a cell are associated with gene expression and the phenotype of a cell, including disease states. Bisulphite PCR sequencing is commonly used to assess the methylation profile of genomic regions between different cells. Here we have developed MethPanel, a computational pipeline with an interactive graphical interface to rapidly analyse multiplex bisulphite PCR sequencing data. MethPanel comprises a complete analysis workflow from genomic alignment to DNA methylation calling and supports an unlimited number of PCR licons and input s les. MethPanel offers important and unique features, such as calculation of an epipolymorphism score and bisulphite PCR bias correction capabilities, and is designed so that the methylation data from all s les can be processed in parallel. The outputs are automatically forwarded to a shinyApp for convenient display, visualization and remotely sharing data with collaborators and clinicians. MethPanel is freely available at hinhong/MethPanel. Supplementary data are available at Bioinformatics online.
Publisher: Elsevier BV
Date: 09-2021
DOI: 10.1016/J.CELREP.2021.109722
Abstract: DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation.
Publisher: Springer Science and Business Media LLC
Date: 22-06-2020
DOI: 10.1186/S13148-020-00880-Y
Abstract: DNA methylation is a well-studied epigenetic mark that is frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of the disease. Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material. Here, we provide an updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis. We describe additional steps to improve performance and reliability: (1) pre-sequencing PCR optimisation which includes assessing the optimal PCR cycling temperature and primer concentration and (2) post-sequencing PCR optimisation to achieve uniform coverage of each licon. We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each licon. Here, we show that the MBPS assay can lify as little as 0.625 ng starting DNA and can detect methylation differences of 1% with a sequencing coverage of 1000 reads. Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on 1–5 ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA. The MBPS assay is a valuable approach for assessing methylated DNA regions in clinical s les with limited material. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies.
No related grants have been discovered for Phuc Loi Luu.