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- Role of epigenetics in male and female germline development
- Role of retroelements in gene regulation
- Interplay between epigenetic marks and DNA methylation in the early embryo
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Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
Classic genomic imprinted loci display parent-of-origin transcription in adult cells. Such allele-specific expression is thought to be driven by epigenetic marks, including DNA methylation (DNAme) and histone modifications, established in the gametes. However, the extent of monoallelic transcription in early mammalian development has remained relatively unexplored. As chromatin is highly dynamic before implantation, parent-of- origin directed transcription may be particularly high at this stage.To identify parent-of-origin controlled transcription, including during early embryogenesis, I helped develop MEA, a Methylomic and Epigenomic Allele-specific analysis pipeline designed to integrate whole genome bisulphite (WGBS)-, RNA- and ChIP-sequencing datasets. To demonstrate the utility and sensitivity of MEA, I processed existing mouse and human datasets and uncovered classical as well as novel candidate imprinted genes.Subsequently, I applied MEA to analyze WGBS and ChIP-seq data generated from adult gametes and preimplantation mouse embryos, which yielded female/maternal and male/paternal epigenomic profiles. Surprisingly, despite global DNAme loss following fertilization, I uncovered several dozen CpG island promoters that are de novo methylated on the paternal genome in 2-cell embryos, coincident with H3K4me3 loss on the same allele. A subset of these loci is hypermethylated in androgenetic blastocysts but hypomethylated in parthenogenetic blastocysts, confirming the paternal genome is susceptible to post-fertilization DNAme activity. Notably, this zygotic paternal DNAme gain was ablated following genetic depletion of maternal DNMT3A. Parental DNAme levels at these loci are harmonized in the post-implantation embryo and beyond: thus, unlike classic imprinted regions, these novel DMRs are established on the paternal genome by maternal stores of DNMT3A in the zygote and likely only transiently impart allelic transcription in the embryo. Indeed, expression analysis of DNMT3A maternal KO preimplantation embryos revealed that genes normally gaining paternal DNAme following fertilization are prematurely activated from the paternal allele in 4C embryos.Taken together, the results presented in my thesis illustrate the benefits of increasing the range and sensitivity of allele-specific analyses, and uncover zygotic de novo DNAme activity at CpG island promoters on the paternal genome against a backdrop of acute global demethylation.
Phosphorylation of histone H3 at serine 10 (H3S10ph) has been observed in two paradoxical contexts, depending on the stage of the cell cycle – it is a hallmark of condensed mitotic chromatin, but is also found at specific mitogen-inducible genic promoters and distal enhancers. In the work presented in this thesis, I derived a mouse embryonic stem cell (ESC) line harbouring an endogenous fluorescent cell cycle reporter (FUCCI) in combination with next-generation sequencing to comprehensively map H3S10ph at distinct stages of the mammalian cell cycle and examined the crosstalk between this mark and the repressive mark, H3K9me. I found that H3S10ph broadly demarcates gene-rich, early-replicating euchromatic regions in G1, marking up to 30% of the ESC genome. Reminiscent of H3S10ph deposited by JIL-1 kinase in Drosophila, interphase H3S10ph pervasively marks gene-dense regions to prevent the accumulation of H3K9me2 at actively transcribed genes in ESCs. In contrast to H3S10ph at euchromatin, mitotic phosphorylation mediated by Aurora kinase is also detectable in ESCs in G1 at young endogenous retroviruses (ERVs), specifically in combination with H3K9me3. Finally, to identify the kinases responsible for interphase H3S10ph, I generated knockout (KO) ESC lines of serine kinases homologous to JIL-1, including Msk1/2 and Rsk1/2/3/4. Msk2-/- ESCs showed the greatest loss of interphase H3S10ph at active promoter/enhancers. Furthermore, known H3K9me3 repressed targets, including imprinted genes, young ERVs and germline genes, were downregulated in MSK2-deficient cells. Taken together, this work revealed that H3S10ph plays a previously unappreciated role in interphase chromatin architecture and facilitates appropriate genic transcription by countering the repressive effects exerted by H3K9 methyltransferases.
Schimke immuno-osseous dysplasia (SIOD) is a rare autosomal recessive multisystemic disorder characterized by disproportionate short stature due to skeletal dysplasia, renal disease due to focal segmental glomerulosclerosis (FSGS), T-cell immunodeficiency, and vascular disease. SIOD is caused by mutations in the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin, subfamily A-like 1 (SMARCAL1) gene, which encodes for a DNA annealing helicase with roles in DNA replication, DNA repair, and gene expression. Although SMARCAL1 functions to maintain genomic integrity, it is not known how SMARCAL1 deficiency leads to the various clinical features of SIOD. My aim was therefore to characterize the molecular pathogenesis of the dental, vascular, renal, and immune features. Given that SMARCAL1 has a role in modulating gene expression and that phenotypic changes are typically preceded by changes in gene expression, I hypothesized that SMARCAL1 deficiency pathologically alters the expression of key genes that lead to the clinical features of SIOD. To test this, SIOD patient tissues were studied using molecular biological analyses. With respect to vascular disease, an SIOD aorta had decreased expression of elastin, and both transcriptional and post-transcriptional mechanisms contributed to the elastin deficiency. Elastin is critical for the structural integrity of the arteries and its deficiency is a known cause of vascular disease. With respect to renal disease, SIOD glomeruli have increased expression and activation of the Wnt and Notch signaling pathways. Wnt and Notch signaling are required for kidney development and the postnatal reactivation of these pathways is an established cause of FSGS. With respect to immune disease, SIOD T cells have decreased mRNA and protein expression of interleukin 7 receptor alpha chain (IL7R). IL7R is critical for T-cell development and its deficiency is a known cause of T-cell immunodeficiency. In conclusion, the gene expression alterations detected are known causes of disease and differ among the tissues studied. These findings suggest that SMARCAL1 deficiency may cause each disease feature by tissue-specific gene expression changes. Further studies are required to define the mechanism of how SMARCAL1 deficiency alters the expression of these genes.
Transcription of endogenous retroviruses (ERVs) is inhibited by de novo DNA methylation during gametogenesis, a process initiated after birth in oocytes and at approximately embryonic day 15.5 (E15.5) in prospermatogonia. However earlier in germline development, the genome, including most retrotransposons, is progressively demethylated. As DNA methylation reaches a low point in E13.5 primordial germ cells (PGCs) of both sexes, raising the question whether repressive histone methylations play a role in silencing of retrotransposons at this stage of development. To answer this question, I first focused on developing low input assays for profiling histone modifications, DNA methylation and transcription from rare cell populations. In close collaboration with Dr. Julie Brind’Amour, I was able to develop the “SmallCell” protocol package, which enables chromatin immunoprecipitation, bisulfite conversion of DNA, RNA isolation-reverse transcription using ~1000 cells per assay, but also construction of sequencing library from pictograms of DNA. This allows profiling of epigenetic information at both locus-specific and genome-wide scales. I then developed the “InterSeq” software (R package) to intersect and explore different types of epigenomic data. This package allows converting sequencing data into genomic interval measures in spreadsheet (SeqData), interfacing this spreadsheet into flowcytometry data (SeqFrame), and an intuitive graphical interface to gate and explore the inter-relationship between different types of epigenomic sequencing data similar to flowcytometry (SeqViz). With these tools we first determined whether retrotransposons are marked by H3K9me3 and H3K27me3. Although these repressive histone modifications are found predominantly on distinct genomic regions in E13.5 PGCs, they concurrently mark partially methylated long terminal repeats and LINE1 elements. Germline-specific conditional knockout of the H3K9 methyltransferase SETDB1 yields a decrease of both marks and DNA methylation at H3K9me3-enriched retrotransposon families. Strikingly, Setdb1 knockout E13.5 PGCs show concomitant derepression of many marked ERVs, including IAP, ETn, and ERVK10C elements, and ERV-proximal genes, a subset in a sex-dependent manner. Furthermore, Setdb1 deficiency is associated with a reduced number of male E13.5 PGCs and postnatal hypogonadism in both sexes. Taken together, these observations reveal that SETDB1 is an essential guardian against proviral expression prior to the onset of de novo DNA methylation in the germline.
Histone lysine methylation is essential for mammalian development and maintenance of somatic cell identity, as evidenced by a group of Mendelian diseases and cancers linked with mutations in lysine methyltransferases (KMTs). The transcriptional silencing of a class of retrotransposons known as endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs) provides a unique model system in which to investigate epigenetic regulation by the H3K9 family of KMTs and characterize novel molecular mechanisms of relevance to human biology and disease. In mESCs, class I and II ERVs are silenced by the SETDB1/KAP1 complex, which deposits histone H3K9 trimethylation (H3K9me3). In contrast, class III MERVL ERVs are silenced by the G9a/ GLP complex, which deposits H3K9me2. The molecular mechanisms governing the recruitment of these KMTs to their genomic ERV targets remain poorly understood. The goal of this work was to identify and characterize novel factors that regulate the functions of these KMTs in ERV silencing. In the first part of my thesis work, I identified the RNA-binding protein and transcription factor hnRNP K as a novel co-repressor for the SETDB1/KAP1 complex. HnRNP K coordinates recruitment of the KMT SETDB1 by KAP1 to its ERV targets. This function of hnRNP K involves a previously uncharacterized influence on the levels of chromatin protein SUMOylation. In the second part of my thesis work, I demonstrated that MERVL elements are also repressed by hnRNP K and can remain inactive in the absence of H3K9me2, likely due to the lack of transcriptional activators. HnRNP K forms a novel RNA-dependent complex with G9a/GLP, is required for global H3K9me2 and provides a repressive barrier to MERVL expression in the presence and absence of H3K9me2. Taken together my work has provided significant insights into the epigenetic repression of ERV transcription by KMTs and demonstrates that hnRNP K is a novel co-repressor for two different KMT complexes. As recent studies have linked mutations in HNRNPK to the novel Mendelian disorder Au-Kline syndrome and cancer, these insights should also guide future studies on the role of hnRNP K in regulation of KMT-mediated signaling pathways in human disease.
Endogenous retroviruses (ERVs) are found in genomes of all higher eukaryotes. As retrotransposition is deleterious, pathways have evolved to repress these retroelements. While DNA methylation transcriptionally represses ERVs in differentiated cells, this epigenetic mark is dispensable for maintaining proviral silencing during early stages of mouse embryogenesis and in embryonic stem cells (mESCs). Studies in diverse species have found histone H3K9 methylation and DNA methylation to function together to repress retrotransposons. However, until recently, little was known about the role of this histone modification in proviral silencing in mESCs. Interestingly, our analysis of mESCs lacking G9a, an H3K9-specific lysine methyltransferase (KMTase) revealed that although ERVs lost H3K9 di-methylation (me2) and DNA methylation, they remained silent. Strikingly, the levels of H3K9 tri-methylation (me3) remained unaltered, suggesting that this mark may instead be responsible for maintaining these parasitic elements transcriptionally inactive. The first stage of my research focused on identifying the enzyme depositing H3K9me3 at ERVs and on determining its role in proviral silencing. I discovered that Setdb1, another H3K9-specific KMTase, was indeed depositing H3K9me3 at a subset of ERVs and was required for maintaining transcriptional repression. Interestingly, this silencing pathway operated independently of DNA methylation. Through collaboration, we also discovered that this pathway played a diminished role in differentiated cells. Taken together, these findings indicate the existence of a DNA methylation-independent proviral silencing pathway in mESCs. The second stage of my research focused on the establishment of transcriptional repression of newly integrated proviruses. By employing an exogenous retroviral construct, I discovered a dramatic silencing defect in mESCs lacking G9a, which phenocopied cells depleted of the de novo DNA methyltransferases. Furthermore, efficient DNA methylation of proviruses required G9a-mediated H3K9me2. These findings reveal that histone modifications and DNA methylation function in concert to defend the genome against invading retroviral elements in mESCs. Taken together with discoveries regarding the mechanism of DNA demethylation in early embryos, I propose that cells undergoing DNA methylation reprogramming predominantly employ histone modification-based pathways to maintain these parasitic elements in a silent state; however, the establishment of transcriptional repression for newly integrated elements also requires de novo DNA methylation.
Master's Student Supervision (2010 - 2018)
Chromatin replication during cell division must be accurately orchestrated to ensure genetic and epigenetic information is transmitted to cell progeny. Upon cell division, newly synthesized histones assemble onto the newly formed chromatin to replace the disassociated parental histones. As these newly synthesized histones do not contain the same post-translational modifications as their adjacent parental histones, these modifications must be recapitulated after each cell division. The trimethylation of lysine 27 (K27me3) on histone H3 is associated with transcriptional repression, and is deposited by EZH2, a member of the PRC2 complex. Using a Gal4 DNA binding domain (Gal4DBD) fused to EZH2 coupled with FLP/FRT-based deletion of a gal4 binding site cassette, I provide evidence that, once established, the maintenance of H3K27me3 does not require the presence of the DNA binding sites necessary for the initial deposition of this mark. These results suggest that the presence of specific histone marks may be sufficient to promote reiterative deposition of the same mark on nascent histones in association with DNA replication.
- Maternal DNMT3A-dependent de novo methylation of the zygotic paternal genome inhibits gene expression in the early embryo (2020)
- Setting the chromatin stage in oocytes (2020)
Nature Cell Biology,
- Epigenetic regulation of unique genes and repetitive elements by the KRAB zinc finger protein ZFP57 (2019)
- Evolution of imprinting via lineage-specific insertion of retroviral promoters (2019)
- Evolution of imprinting via lineage-specific insertion of retroviral promoters (2019)
- Histone H3K9 Methyltransferase G9a in Oocytes Is Essential for Preimplantation Development but Dispensable for CG Methylation Protection. (2019)
- SETD2 regulates the maternal epigenome, genomic imprinting and embryonic development (2019)
- Development and application of an integrated allele-specific pipeline for methylomic and epigenomic analysis (MEA) (2018)
BMC Genomics, 19 (1)
- H3S10ph broadly marks early-replicating domains in interphase ESCs and shows reciprocal antagonism with H3K9me2 (2018)
Genome Research, 28 (1), 37-51
- LTR retrotransposons transcribed in oocytes drive species-specific and heritable changes in DNA methylation (2018)
- Epigenetic modifier drugs trigger widespread transcription of endogenous retroviruses. (2017)
- Evidence for Converging DNA Methylation Pathways in Placenta and Cancer (2017)
Developmental Cell, 43 (3), 257-258
- On the role of H3.3 in retroviral silencing. (2017)
- Activation of Endogenous Retroviruses in Dnmt1(-/-) ESCs Involves Disruption of SETDB1-Mediated Repression by NP95 Binding to Hemimethylated DNA. (2016)
- Activation of Endogenous Retroviruses in Dnmt1−/− ESCs Involves Disruption of SETDB1-Mediated Repression by NP95 Binding to Hemimethylated DNA (2016)
Cell Stem Cell, 19 (1), 81-94
- ChAsE: Chromatin analysis and exploration tool. (2016)
Bioinformatics, 32 (21), 3324-3326
- Dynamic and flexible H3K9me3 bridging via HP1β dimerization establishes a plastic state of condensed chromatin (2016)
Nature Communications, 7
- Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire. (2016)
Molecular Cell, 62 (5), 766-776
- An ultra-low-input native ChIP-seq protocol for genome-wide profiling of rare cell populations (2015)
Nature Communications, 6
- hnRNP K coordinates transcriptional silencing by SETDB1 in embryonic stem cells. (2015)
PLoS Genetics, 11 (1)
- Setdb1 is required for germline development and silencing of H3K9me3-marked endogenous retroviruses in primordial germ cells. (2015)
Genes and Development, 28 (18), 2041-2055
- Systematic identification of factors for provirus silencing in embryonic stem cells. (2015)
Cell, 163 (1), 230-245
- VisRseq: R-based visual framework for analysis of sequencing data (2015)
BMC Bioinformatics, 16 (11)
- ALEA: a toolbox for allele-specific epigenomics analysis. (2014)
Bioinformatics, 30 (8), 1172-1174
- Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells. (2014)
Nature, 516 (7531), 405-409
- Regulation of DNA methylation turnover at LTR retrotransposons and imprinted loci by the histone methyltransferase Setdb1. (2014)
Proceedings of the National Academy of Sciences of the United States of America, 111 (18), 6690-6695
- An interactive analysis and exploration tool for epigenomic data (2013)
Computer Graphics Forum, 32 (3 PAR), 91-100
- Distinct roles of KAP1, HP1 and G9a/GLP in silencing of the two-cell-specific retrotransposon MERVL in mouse ES cells (2013)
Epigenetics and Chromatin, 6 (1)
- Genome-wide mapping of chromatin marks from 1,000 cells to study epigenetic reprogramming in primordial germ cells. (2013)
- Kinetics and epigenetics of retroviral silencing in mouse embryonic stem cells defined by deletion of the D4Z4 element. (2013)
Molecular Therapy, 21 (8), 1536-1550
- Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells. (2013)
Nature, 500 (7461), 222-226
- Histone H3K4 demethylation is negatively regulated by histone H3 acetylation in Saccharomyces cerevisiae. (2012)
Proceedings of the National Academy of Sciences of the United States of America, 109 (45), 18505-18510
- Rare treatable neurologic diseases. (2012)
- Silencing of endogenous retroviruses: when and why do histone marks predominate? (2012)
Trends in Biochemical Sciences, 37 (4), 127-133
- DNA methylation and SETDB1/H3K9me3 regulate predominantly distinct sets of genes, retroelements, and chimeric transcripts in mESCs. (2011)
Cell Stem Cell, 8 (6), 676-687
- H3K9me3-binding proteins are dispensable for SETDB1/H3K9me3-dependent retroviral silencing (2011)
Epigenetics and Chromatin, 4 (1)
- Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing. (2011)
Proceedings of the National Academy of Sciences of the United States of America, 108 (14), 5718-5723
- Retrotransposon-induced heterochromatin spreading in the mouse revealed by insertional polymorphisms. (2011)
PLoS Genetics, 7 (9)
- Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. (2010)
Nature, 464 (7290), 927-931
- H2A.Z and DNA methylation: irreconcilable differences. (2009)
Trends in Biochemical Sciences, 34 (4), 158-161
- Targeting of EZH2 to a defined genomic site is sufficient for recruitment of Dnmt3a but not de novo DNA methylation. (2009)
Epigenetics, 4 (6), 404-414
- DNA methylation in ES cells requires the lysine methyltransferase G9a but not its catalytic activity. (2008)
EMBO Journal, 27 (20), 2691-2701
- An unmethylated 3' promoter-proximal region is required for efficient transcription initiation. (2007)
- RNA polymerase II: just stopping by. (2007)
Cell, 130 (1), 16-18
- Dynamics, stability and inheritance of somatic DNA methylation imprints. (2006)
Journal of Theoretical Biology, 242 (4), 890-899
- Intragenic DNA methylation alters chromatin structure and elongation efficiency in mammalian cells. (2004)
Nature Structural and Molecular Biology, 11 (11), 1068-1075
- DNA methylation density influences the stability of an epigenetic imprint and Dnmt3a/b-independent de novo methylation. (2002)
Molecular and Cellular Biology, 22 (21), 7572-7580
- C(m)C(a/t)GG methylation: a new epigenetic mark in mammalian DNA? (2001)
- CmC(a/t)GG methylation: A new epigenetic mark in mammalian DNA? (2001)
Proceedings of the National Academy of Sciences of the United States of America, 98 (18), 10034-10036
- Methylation-mediated proviral silencing is associated with MeCP2 recruitment and localized histone H3 deacetylation. (2001)
Molecular and Cellular Biology, 21 (23), 7913-7922
- Position effects are influenced by the orientation of a transgene with respect to flanking chromatin. (2001)
Molecular and Cellular Biology, 21 (1), 298-309
- Reporters of gene expression: enzymatic assays. (2001)
Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.], Chapt
- Targeting silence: the use of site-specific recombination to introduce in vitro methylated DNA into the genome. (2001)
Science"s STKE [electronic resource] : signal transduction knowledge environment, 2001 (83)
- Dynamic analysis of proviral induction and De Novo methylation: implications for a histone deacetylase-independent, methylation density-dependent mechanism of transcriptional repression. (2000)
Molecular and Cellular Biology, 20 (3), 842-850
- Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation. (2000)
Molecular and Cellular Biology, 20 (24), 9103-9112
- Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting- based assay for mammalian β-glucuronidase enzymatic activity (1999)
Journal of Biological Chemistry, 274 (2), 657-665
- Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity. (1999)
- Detection and isolation of gene-corrected cells in Gaucher disease via a fluorescence-activated cell sorter assay for lysosomal glucocerebrosidase activity. (1997)
Blood, 89 (9), 3412-3420
- Enzyme-generated intracellular fluorescence for single-cell reporter gene analysis utilizing Escherichia coli beta-glucuronidase. (1996)
- Enzyme-generated intracellular fluorescence for single-cell resporter gene analysis utilizing Escherichia coli β-glucuronidase (1996)
Cytometry, 24 (4), 321-329
- Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins. (1996)
Proceedings of the National Academy of Sciences of the United States of America, 93 (16), 8508-8511