Sensitivity of transcription factors to DNA methylation.

Dynamic binding of transcription factors (TFs) to regulatory elements controls transcriptional states throughout organism development. Epigenetics modifications, such as DNA methylation mostly within cytosine-guanine dinucleotides (CpGs), have the potential to modulate TF binding to DNA. Although DNA methylation has long been thought to repress TF binding, a more recent model proposes that TF binding can also inhibit DNA methylation. Here, we review the possible scenarios by which DNA methylation and TF binding affect each other. Further in vivo experiments will be required to generalize these models.journal article2019 Nov 222019 11 22importe

An N-ethyl-N-Nitrosourea Mutagenesis Screen in Mice Reveals a Mutation in Nuclear Respiratory Factor 1 (Nrf1) Altering the DNA Methylation State and Correct Embryonic Development

We have established a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify novel genes playing a role in epigenetic regulation in mammals. We hypothesize that the ENU mutagenesis screen will lead to the discovery of unknown genes responsible of the maintenance of the epigenetic state as the genes found are modifiers of variegation of the transgene green fluorescent protein (GFP) expression in erythrocytes, which are named MommeD. Here we report the generation of a novel mutant mouse line, MommeD46, that carries a new missense mutation producing an amino acid transversion (L71P) in the dimerization domain of Nuclear Respiratory Factor 1 (Nrf1). The molecular characterization of the mutation reveals a decrease in the Nrf1 mRNA levels and a novel role of Nrf1 in the maintenance of the DNA hypomethylation in vivo. The heritability of the mutation is consistent with paternal imprinting and haploinsufficiency. Homozygous mutants display embryonic lethality at 14.5 days post-coitum and developmental delay. This work adds a new epi-regulatory role to Nrf1 and uncovers unknown phenotypical defects of the Nrf1 hypomorph. The generated mouse line represents a valuable resource for studying NRF1-related diseases.This research was funded by the National Health and Medical Research Council of Australia under Grant APP0552445, along with the 2020 Marie Skłodowska-Curie Individual Fellowship from the European Commission under Grant 893384, 2020 Miguel Servet fellowship from the Instituto de Salud Carlos III (Spanish National Institute of Health) under Grant CP20/00039 and European Social Fund (ESF) “Investing in your future”, and the Raine Medical Research Foundation under Grant RPG-004-19 given to A.S., and the Emergent Research Group Recognition Award from the University and Research Grants Management Agency of Catalonia (Spain) under Grant 2017SRG1620 given to M.A.S. The research was supported by CERCA Programme of Generalitat de Catalunya and the IRBLleida—Fundació Dr. Pifarré

Large-scale manipulation of promoter DNA methylation reveals context-specific transcriptional responses and stability

BACKGROUND: Cytosine DNA methylation is widely described as a transcriptional repressive mark with the capacity to silence promoters. Epigenome engineering techniques enable direct testing of the effect of induced DNA methylation on endogenous promoters; however, the downstream effects have not yet been comprehensively assessed. RESULTS: Here, we simultaneously induce methylation at thousands of promoters in human cells using an engineered zinc finger-DNMT3A fusion protein, enabling us to test the effect of forced DNA methylation upon transcription, chromatin accessibility, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that transcriptional responses to DNA methylation are highly context-specific, including lack of repression, as well as cases of increased gene expression, which appears to be driven by the eviction of methyl-sensitive transcriptional repressors. Furthermore, we find that some regulatory networks can override DNA methylation and that promoter methylation can cause alternative promoter usage. DNA methylation deposited at promoter and distal regulatory regions is rapidly erased after removal of the zinc finger-DNMT3A fusion protein, in a process combining passive and TET-mediated demethylation. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. CONCLUSIONS: These findings have important implications for epigenome engineering and demonstrate that the response of promoters to DNA methylation is more complex than previously appreciated. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-022-02728-5

Decipher Mechanisms by which Nuclear Respiratory Factor One (NRF1) Coordinates Changes in the Transcriptional and Chromatin Landscape Affecting Development and Progression of Invasive Breast Cancer

Despite tremendous progress in the understanding of breast cancer (BC), gaps remain in our knowledge of the molecular basis underlying the aggressiveness of BC and BC disparities. Nuclear respiratory factor 1 (NRF1) is a transcription factor (TF) known to control breast cancer cell cycle progression. DNA response elements bound by NRF1 positively correlate with the progression of malignant breast cancer. Mechanistic aspects by which NRF1 contributes to susceptibility to different breast tumor subtypes are still not fully understood. Therefore, the primary objective of this dissertation was to decipher mechanisms by which NRF1 coordinates changes in the transcriptional and chromatin landscape affecting development and progression of invasive breast cancer. Our hypothesis was that NRF1 reprogramming the transcription of tumor initiating gene(s) and tumor suppressor gene(s) contribute in the development and progression of invasive breast cancer. To test this hypothesis, we proposed three specific aims: (a) Decipher regulatory landscape of NRF1 networks in breast cancer. (b) Determine the role of NRF1 gene networks in different subtypes of breast cancer. (c) Determine differential NRF1 gene network sensitivity contributing to breast cancer disparities. Our approach to test these aims consisted of a systematic integration of ChIP DNA-seq, RNA-Seq, NRF1 protein-DNA motif binding, signal pathway analysis, and Bayesian machine learning. We uncovered a novel oncogenic role for NRF1. This discovery strongly supported the supposition that NRF1 overexpression is sufficient to derive breast tumorigenesis. We also observed new roles for NRF1 in the acquisition of breast tumor initiating cells, regulation of epithelial to mesenchymal transition (EMT), and invasiveness of BC stem cells. Furthermore, through the use of Bayesian network structure learning we found that the NRF1 motif was enriched in 14 associated with HER2 amplified breast cancer. Three genes—GSK3B, E2F3, and PIK3CA—were able to predict HER2 breast tumor status with 96% to100% confidence. The findings of this study also showed the roles of NRF1 sensitivity to development of lobular A, Her2+, and TNBC in different racial/ethnic groups of breast cancer patients. In summary, our study revealed for the first time the role of NRF1 in the pathogenesis of invasive BC and BC disparities

Distinct Contribution of DNA Methylation and Histone Acetylation to the Genomic Occupancy of Transcription Factors

Abstract Epigenetic modifications on chromatin play important roles in regulating gene expression. While chromatin states are often governed by multi-layered structure, how individual pathways contribute to gene expression remains poorly understood. For example, DNA methylation is known to regulate transcription factor binding but also to recruit methyl-CpG binding proteins that affect chromatin structure through the activity of histone deacetylase complexes (HDACs). Both of these mechanisms can potentially affect gene expression, but the importance of each, and whether these activities are integrated to achieve appropriate gene regulation, remains largely unknown. To address this important question, we measured gene expression, chromatin accessibility, and transcription factor occupancy in wild-type or DNA methylation-deficient mouse embryonic stem cells following HDAC inhibition. Interestingly, we observe widespread increases in chromatin accessibility at repeat elements when HDACs are inhibited, and this is magnified when cells also lack DNA methylation. A subset of these elements have elevated binding of the YY1 and GABPA transcription factors and increased expression. The pronounced additive effect of HDAC inhibition in DNA methylation deficient cells demonstrate that DNA methylation and histone deacetylation act largely independently to suppress transcription factor binding and gene expression

Influence of DNA methylation on transcription factor binding

Eukaryotic transcription factors (TFs) are key determinants of gene activity, yet they bind only a fraction of their corresponding DNA sequence motifs in any given cell type. Chromatin has the potential to restrict accessibility of binding sites; however, in which context chromatin states are instructive for TF binding remains mainly unknown. This thesis explores the contribution of DNA methylation to constrained TF binding by studying CTCF as a known methylation-sensitive TF and applying a genome-wide approach to identify further sensitive factors in mouse stem and differentiated cells. CTCF is perhaps the most prominent example for a TF that can be prevented from binding by DNA methylation in vivo. However, it is restricted by methylation only at a subset of its genomic binding sites, such as the H19/Igf2 imprinting control region (ICR). In order to understand this context dependency of CTCF methylation sensitivity, we compared CTCF binding in isogenic mouse stem cells with and without DNA methylation. Two features distinguish the fraction of sites that are bound only in the absence of DNA methylation: CpG-containing variants of the canonical CTCF motif as well as higher CpG density in the flanking regions. The H19/Igf2 ICR indeed fulfils these criteria and we show that CTCF methylation sensitivity there is independent of the complete ICR sequence, the chromosomal context and H3K9me3 marks. In order to go beyond CTCF and identify more methylation-sensitive TFs a priori, we mapped DNase I hypersensitive sites, as an indicator of TF binding, in mouse stem cells with and without DNA methylation. Methylation-restricted sites are enriched for TF motifs containing CpGs, especially for those of NRF1. In fact, NRF1 occupies several thousand additional sites in the unmethylated genome, resulting in increased genic and non-genic transcription. Restoring de novo methyltransferase activity initiates remethylation at these sites and outcompetes NRF1 binding. Even strong overexpression of NRF1 is unable to prompt binding at methylated regions. This suggests that binding of methylation-sensitive TFs relies on additional determinants to induce local hypomethylation. In support of this model, deletion of neighbouring motifs in cis or of a TF in trans causes local hypermethylation and subsequent loss of NRF1 binding. This competition between DNA methylation and TFs in vivo reveals a case of cooperativity between TFs that acts indirectly via DNA methylation. Nevertheless, the vast majority of TF binding events do not change upon removal of DNA methylation in stem cells. To investigate whether more TFs are affected in differentiated cells, for which DNA methylation is essential, we generated methylation-deficient neuronal cells that survive for several days in culture. Changes in genic transcription and chromatin accessibility are surprisingly limited in the absence of DNA methylation, although again a subset of TF motifs are enriched in methylation-restricted sites, such as NRF1 and HNF6. While this closely resembles the situation in stem cells, we observe a striking activation of specific classes of endogenous retroviruses (ERV) only in the differentiated methylation mutant. Several lines of evidence indicate that methylation-sensitive TF binding at the cAMP-responsive element (CRE motif) is responsible for ERV activation in differentiated methylation mutants including mouse cortex, which might provide a link to the ensuing cell death. Taken together, only a low percentage of TF binding events are restricted by DNA methylation in stem or differentiated cells. However, a subset of factors is methylation-sensitive at CpG-containing motifs. These factors rely on other TFs to keep their motif in an unmethylated state and their aberrant binding can have devastating consequences by repeat activation. Understanding the influence of DNA methylation on TF binding constitutes one step towards better interpretation of the rapidly growing number of epigenetic and TF binding maps. The success of the approach taken here suggests that it can be applied to other chromatin components and modifications, which should enable comprehensive prediction of TF binding and ultimately gene expression in development and disease

Prenatal methylmercury exposure and DNA methylation in seven-year-old children in the Seychelles Child Development Study

Background Methylmercury (MeHg) is present in fish and is a neurotoxicant at sufficiently high levels. One potential mechanism of MeHg toxicity early in life is epigenetic dysregulation that may affect long-term neurodevelopment. Altered DNA methylation of nervous system-related genes has been associated with adult mental health outcomes. Objective To assess associations between prenatal MeHg exposure and DNA methylation (at the cytosine of CG dinucleotides, CpGs) in three nervous system-related genes, encoding brain-derived neurotropic factor (BDNF), glutamate receptor subunit NR2B (GRIN2B), and the glucocorticoid receptor (NR3C1), in children who were exposed to MeHg in utero. Methods We tested 406 seven-year-old Seychellois children participating in the Seychelles Child Development Study (Nutrition Cohort 2), who were prenatally exposed to MeHg from maternal fish consumption. Total mercury in maternal hair (prenatal MeHg exposure measure) collected during pregnancy was measured using atomic absorption spectroscopy. Methylation in DNA from the children’s saliva was measured by pyrosequencing. To assess associations between prenatal MeHg exposure and CpG methylation at seven years of age, we used multivariable linear regression models adjusted for covariates. Results We identified associations with prenatal MeHg exposure for DNA methylation of one GRIN2B CpG and two NR3C1 CpGs out of 12 total CpG sites. Higher prenatal MeHg was associated with higher methylation for each CpG site. For example, NR3C1 CpG3 had an expected increase of 0.03-fold for each additional 1 ppm of prenatal MeHg (B = 0.030, 95% CI 0.001, 0.059; p = 0.047). Several CpG sites associated with MeHg are located in transcription factor binding sites and the observed methylation changes are predicted to lead to lower gene expression. Conclusions In a population of people who consume large amounts of fish, we showed that higher prenatal MeHg exposure was associated with differential DNA methylation at seven years of age at specific CpG sites that may influence neurodevelopment and mental health

Quantitative Analysis of the DNA Methylation Sensitivity of Transcription Factor Complexes

Although DNA modifications play an important role in gene regulation, the underlying mechanisms remain elusive. We developed EpiSELEX-seq to probe the sensitivity of transcription factor binding to DNA modification in vitro using massively parallel sequencing. Feature-based modeling quantifies the effect of cytosine methylation (5mC) on binding free energy in a position-specific manner. Application to the human bZIP proteins ATF4 and C/EBPβ and three different Pbx-Hox complexes shows that 5mCpG can both increase and decrease affinity, depending on where the modification occurs within the protein-DNA interface. The TF paralogs tested vary in their methylation sensitivity, for which we provide a structural rationale. We show that 5mCpG can also enhance in vitro p53 binding and provide evidence for increased in vivo p53 occupancy at methylated binding sites, correlating with primed enhancer histone marks. Our results establish a powerful strategy for dissecting the epigenomic modulation of protein-DNA interactions and their role in gene regulation

Discovery of non-directional and directional pioneer transcription factors by modeling DNase profile magnitude and shape

Here we describe Protein Interaction Quantitation (PIQ), a computational method that models the magnitude and shape of genome-wide DNase profiles to facilitate the identification of transcription factor (TF) binding sites. Through the use of machine learning techniques, PIQ identified binding sites for >700 TFs from one DNase-seq experiment with accuracy comparable to ChIP-seq for motif-associated TFs (median AUC=0.93 across 303 TFs). We applied PIQ to analyze DNase-seq data from mouse embryonic stem cells differentiating into pre-pancreatic and intestinal endoderm. We identified (n=120) and experimentally validated eight ‘pioneer’ TF families that dynamically open chromatin, enabling other TFs to bind to adjacent DNA. Four pioneer TF families only open chromatin in one direction from their motifs. Furthermore, we identified a class of ‘settler’ TFs whose genomic binding is principally governed by proximity to open chromatin. Our results support a model of hierarchical TF binding in which directional and non-directional pioneer activity shapes the chromatin landscape for population by settler TFs

Local regulation of DNA methylation by the transcription factor REST

Transcriptional regulation in eukaryotes is realized through intricate interactions between transcription factors and chromatin. DNA methylation constitutes a chromatin modification that is associated with transcriptional silencing (Deaton and Bird, 2011). Whole-genome methylation profiling in mammals has revealed widespread cytosine methylation with characteristic hypomethylation at cis-regulatory elements. Hypomethylation is typically present within CpG islands and distal CpG-poor regions (Stadler et al., 2011). Previous investigations have shown, that some DNA-binding factors like the RE1-silencing transcription factor (REST) directly reduce methylation at these sites. However, how DNA-binding factors mediate such local methylation changes remains largely unknown. Hence, I studied the regulation of DNA methylation by the transcription factor REST in mouse embryonic stem cells (mESCs). I ectopically expressed different REST mutants and profiled DNA methylation at distal REST binding sites. While the full-length protein is necessary and sufficient to reduce methylation at its binding sites, REST’s DNA-binding domain lacks this ability. Instead, hypomethylation at binding sites required DNA-binding factors with interaction domains. The N-terminal REST mutant for example recruits SIN3A to binding sites and shows strong DNA demethylation ability. These experiments suggest that hypomethylation is not an obligatory consequence of protein binding, but rather requires interaction domains, reflecting the potential involvement of cofactors. I inquired whether TET enzymes contribute to reduced methylation within REST binding sites. Complete Tet1/2/3 deficiency in mouse stem cells caused a strong localized hypermethylation in the immediate vicinity of the REST motif. Whether TET proteins are recruited to REST binding sites through common cofactors or indirect mechanisms remains to be determined. I also characterized chromatin accessibility and nucleosome positioning in the different REST mutant re-expression cells. Interestingly, REST mutants that were competent to decrease DNA methylation also increased chromatin accessibility and nucleosome positioning. This could potentially link the chromatin remodeling ability of transcription factors to hypomethylation around binding sites. In summary, the presented study dissected REST induced methylation patterns around binding sites and described several of its required molecular components. This presents an example for a dynamic interplay between genetic and epigenetic information