Single-molecule footprinting identifies context-dependent regulation of enhancers by DNA methylation

Kreibich, Elisa; Kleinendorst, Rozemarijn; Barzaghi, Guido; Kaspar, Sarah; Krebs, Arnaud R

doi:10.1016/j.molcel.2023.01.017

Cited by 55 publications

(51 citation statements)

References 83 publications

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“…Several previous in vitro studies (85, 89, 90) have observed that the binding of a large percentage of TFs seem to be affected by DNA methylation. However, in studies using cellular context, this is not the case and a recent paper showed that the function of 97% of enhancers is DNA methylation insensitive (87), indicating that binding of TFs in vivo is not DNA methylation dependent. This means that DNA methylation might impact the binding of only a small number of TFs considered here in native chromatin.…”

Section: Discussionmentioning

confidence: 99%

“…In our analysis, we did not consider the role of DNA methylation on TF binding. Previous work has shown that some TFs can distinguish between methylated and unmethylated DNA (84)(85)(86)(87)(88). Several previous in vitro studies (85,89,90) have observed that the binding of a large percentage of TFs seem to be affected by DNA methylation.…”

Section: Several Transcription Factors Have No or Limited Preference ...mentioning

confidence: 98%

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Identification of mammalian transcription factors that bind to inaccessible chromatin

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Pisante

Nagy

et al. 2023

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Transcription factors (TFs) are proteins that affect gene expression by binding to regulatory regions of DNA in a sequence specific manner. The binding of TFs to DNA is controlled by many factors, including the DNA sequence, concentration of TF, chromatin accessibility and co-factors. Here, we systematically investigated the binding mechanism of hundreds of TFs by analysing ChIP-seq data with our explainable statistical model, ChIPanalyser. This tool uses as inputs the DNA sequence binding motif; the capacity to distinguish between strong and weak binding sites; the concentration of TF; and chromatin accessibility. We asked whether TFs preferred to bind to DNA in open or dense chromatin conformation and found that approximately one third of TFs are predicted to bind the genome in a DNA accessibility independent fashion. Our model predicted this to be the case when the TF binds to its strongest binding regions in the genome, and only a small number of TFs have the capacity to bind dense chromatin at their weakest binding regions, such as CTCF USF2 and CEBPB. Our study demonstrated that the binding of hundreds of human and mouse TFs is predicted by ChIPanalyser with high accuracy and showed that many TFs can bind dense chromatin.

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Section: Discussionmentioning

confidence: 99%

Section: Several Transcription Factors Have No or Limited Preference ...mentioning

confidence: 98%

Identification of mammalian transcription factors that bind to inaccessible chromatin

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Pisante

Nagy

et al. 2023

Preprint

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“…The data argued that the opening of CREs upon differentiation usually preceeds the loss of 5mC at enhancers and shows that methylation is compatible with chromatin accessibility at enhancers. We recently confirmed this observation by measuring if the presence of 5mC antagonises chromatin accessibility on the same DNA molecules at enhancers genome‐wide using single‐molecule footprinting (SMF) [43]. At single‐molecule resolution, the epigenetic heterogeneity characterising enhancers can be deconvolved to ask if methylated enhancer molecules have lower TF‐binding activity than their unmethylated counterparts at the same locus .…”

Section: Causal Evidence Linking Dna Methylation With Repression Of E...mentioning

confidence: 98%

“…For the vast majority of enhancers tested, the presence of 5mC is not associated with lower chromatin accessibility. Yet, for a narrow subset of cell‐type specific enhancers (<5%), chromatin accessibility is reduced on methylated molecules, identifying enhancers where 5mC instructs TF binding and chromatin accessibility [43]. Together, these data argue that while 5mC does not generally influence the activity of most enhancers, specific sets of enhancers are repressed by 5mC and depend on DNA demethylation for their activation.…”

Section: Causal Evidence Linking Dna Methylation With Repression Of E...mentioning

confidence: 99%

Relevance of DNA methylation at enhancers for the acquisition of cell identities

Kreibich

Krebs

2023

FEBS Letters

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DNA methylation (5mC) is an essential epigenetic mark associated with transcriptional silencing. The role of 5mC in transcriptional repression is well established for a few hundred genes through methylation of their promoters. Yet, whether 5mC contributes more broadly to gene expression is an important open question. 5mC removal has recently been associated with the activation of enhancers, opening the possibility that 5mC may globally contribute to the expression of genes defining cell identities. Here, we will review the evidence and molecular mechanisms that link 5mC with the activity of enhancers. We will discuss the spread and amplitude of the potential gene expression changes controlled by 5mC at enhancers, and how these may contribute to the determination of cell identities during development.

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“…Here, histone modifications and CpG dinucleotide methylation are added to chromatin by TFs and transcription [38][39][40][41] . These same modifications can then, in turn, modify how TFs read the DNA [42][43][44] . CpG methylation, and some repressive chromatin modifications such as H3K27 trimethylation can also persist through cell division [44][45][46][47][48] to form a memory of past TF activity.…”

Section: Box 1: Contrasting the Protein And Cis-regulatory Codesmentioning

confidence: 99%

Hold out the genome: A roadmap to solving the cis-regulatory code

Boer

Taipale

2023

Preprint

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Gene expression is regulated by transcription factors that work together to read cis-regulatory DNA sequences. The “cis-regulatory code” - the rules that cells use to determine when, where, and how much genes should be expressed - has proven to be exceedingly complex, but recent advances in the scale and resolution of functional genomics assays and Machine Learning have enabled significant progress towards deciphering this code. However, we will likely never solve the cis-regulatory code if we restrict ourselves to models trained only on genomic sequences; regions of homology can easily lead to overestimation of predictive performance, and there is insufficient sequence diversity in our genomes to learn all relevant parameters. Fortunately, randomly synthesized DNA sequences enable us to test a far larger sequence space than exists in our genomes in each experiment, and designed DNA sequences enable a targeted query of the sequence space to maximally improve the models. Since cells use the same biochemical principles to interpret DNA regardless of its source, models that are trained on these synthetic data can predict genomic activity, often better than genome-trained models. Here, we provide an outlook on the field, and propose a roadmap towards solving the cis-regulatory code by training models exclusively on non-genomic DNA sequences, and using genomic sequences solely for evaluating the resulting models.

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Single-molecule footprinting identifies context-dependent regulation of enhancers by DNA methylation

Cited by 55 publications

References 83 publications

Identification of mammalian transcription factors that bind to inaccessible chromatin

Identification of mammalian transcription factors that bind to inaccessible chromatin

Relevance of DNA methylation at enhancers for the acquisition of cell identities

Hold out the genome: A roadmap to solving the cis-regulatory code

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