Single molecule multi-omics reveals context-dependent regulation of enhancers by DNA methylation

Abstract

Enhancers are cis-regulatory elements that control the establishment of cell identities during development. In mammals, enhancer activation is tightly coupled with local DNA de-methylation. Yet, whether this epigenetic remodelling is necessary for enhancer activation is unknown. Here, we developed a single molecule multi-omics approach to measure chromatin accessibility and transcription factor binding as a function of the presence of methylation on the same DNA molecules. We leveraged natural epigenetic heterogeneity at active enhancers to test the impact of DNA methylation on their activity in multiple cell lineages. While reduction of DNA methylation appears dispensable for the activity of most enhancers, we identify a class of cell-type-specific enhancers where presence of DNA methylation antagonises the binding of transcription factors. Genetic perturbations reveal that chromatin accessibility and transcription factor binding are a direct function of active demethylation at these loci. Thus, in addition to safeguarding the genome from spurious activation, DNA methylation directly controls transcription factor occupancy at cell-type-specific active enhancers.

Genetic risk of osteoarthritis operates during human skeletogenesis

“…The question of DNAm as a cause vs consequence has long been debated, and the answer has been hindered because of the complex involvement of multiple cis- regulatory elements (CREs) in the fine-tuning of gene expression. A recent study used single-cell technologies in mouse embryonic stem cells to investigate the co-occurrence of DNAm with chromatin accessibility, and TF occupancy ( 37 ). The authors demonstrated that in most enhancers, DNAm neither antagonizes chromatin accessibility nor the binding of TFs.…”

DNA methylation restricts coordinated germline and neural fates in embryonic stem cell differentiation

“…Several evidence suggest that cell fate choices could be epigenetically primed in the enhancer landscape of progenitors cells 55,56 . In naive and primed ESCs, somatic lineage-associated enhancers harbor heterogenous levels of DNA methylation and around 3% of them were estimated to be regulated by methyl-sensitive TFs 20 . Variegating DNA methylation reflects the competing activity of DNA methylation writers and erasers—namely DNMTs and ten-eleven translocation (TET) enzymes—during priming 15,17,57 , which seems to be particularly pronounced at enhancers of the ectoderm lineage during epiblast development 45 Accordingly, recent single cell analyses reported that embryos lacking DNMT1 have over-represented neuro-ectoderm-associated cell types 37,58 .…”

“…Moreover, lineage-specific enhancers display heterogeneous DNA methylation levels in populations of naive and primed ESCs in culture [15][16][17] , which may reflect requisite intercellular heterogeneity as cells prepare to engage towards specific lineages. Mechanistically, DNA methylation anti-correlates with chromatin features that reflect enhancer activity, such as lower nucleosome occupancy and histone 3 lysine 27 acetylation (H3K27ac) 18 , possibly by restraining the binding of DNA methylation-sensitive transcription factors (TFs) 19,20 .…”

“…Moreover, lineage-specific enhancers display heterogeneous DNA methylation levels in populations of naive and primed ESCs in culture 15–17 , which may reflect requisite intercellular heterogeneity as cells prepare to engage towards specific lineages. Mechanistically, DNA methylation anti-correlates with chromatin features that reflect enhancer activity, such as lower nucleosome occupancy and histone 3 lysine 27 acetylation (H3K27ac) 18 , possibly by restraining the binding of DNA methylation-sensitive transcription factors (TFs) 19,20 .…”

Shared retinoic acid responsive enhancers coordinately regulate nascent transcription of Hoxb coding and non-coding RNAs in the developing mouse neural tube

“…Regulation of clustered Hox genes in vertebrate systems has been linked to diverse types of cis -regulatory elements (CREs) and differences in chromatin states, epigenetic modifications, and chromosome topology/3D genome conformations (Sharpe et al, 1998; Chambeyron and Bickmore, 2004; Tarchini and Duboule, 2006; Gonzalez et al, 2007; Soshnikova and Duboule, 2009; Noordermeer et al, 2011; Mazzoni et al, 2013; Ahn et al, 2014; Noordermeer et al, 2014; De Kumar et al, 2015; Narendra et al, 2015; Narendra et al, 2016; Neijts et al, 2016; Parker and Krumlauf, 2017; Qian et al, 2018; Rodríguez-Carballo et al, 2019). Among the CREs, enhancers play an important role in modulating the activation and/or maintenance of transcription of Hox genes through their ability to interpret graded cues from signaling pathways and to integrate dynamic combinations of TFs to control gene expression patterns in a spatio-temporal and tissue-specific manner (Marshall et al, 1994; Studer et al, 1994; Tümpel et al, 2002; Berlivet et al, 2013; Delpretti et al, 2013; Paris et al, 2013; Crocker et al, 2015; Heinz et al, 2015; Parker and Krumlauf, 2017; Henriques et al, 2018; Nolte et al, 2019; Parker et al, 2019; Choi et al, 2021; Kreibich et al, 2022). Studies in mice, in a variety of different tissues, have demonstrated that there are multiple enhancers embedded within and flanking the Hox clusters which can exhibit overlapping activities (shadow enhancers), selective and competitive preferences for target genes, and they can regulate both near adjacent genes or act more globally on multiple genes in a cluster (shared enhancers) (Oosterveen et al, 2003a; Scotti and Kmita, 2012; Tschopp et al, 2012; Andrey et al, 2013; Berlivet et al, 2013; Nolte et al, 2013; Ahn et al, 2014; Qian et al, 2018).…”