//tags: GpC methylation-assisted tracing, G-MAT, chromatin, epigenetics, NuRD, CTCF, MBD3, TET1, chromatin remodeling
Binding sites for the CCCTC-binding factor (CTCF) - an evolutionarily conserved transcription factor with regulatory roles that include 3D genome organization, enhancer blocking, and transcriptional regulation - exhibit a specific epigenetic pattern: highly ordered nucleosomes and DNA hypomethylation. Importantly, we currently lack a deeper understanding of the mechanisms that establish this particular epigenetic landscape: does DNA demethylation precede and enable CTCF binding, or does CTCF binding recruit additional factors to alter local DNA methylation levels?
In an effort to answer this chicken-and-egg question, researchers led by Liang Zhang, Miao Yu, Xi Wang, and Jian Yan developed a novel approach to capture dynamic transcription factor-DNA interactions and proximal proteomes at high resolution. Their new Molecular Cell study now reports on G-MAT or "GpC methylation-assisted tracing," which they employed to simultaneously map CTCF binding and endogenous DNA methylation levels on the same DNA fragment at base-pair resolution (Sun, Wu, and Xia et al.). Overall, this fascinating new study now reveals that CTCF-chromatin interactions tend to coincide with regions marked by elevated levels of DNA methylation, with the nucleosome remodeling and deacetylase (NuRD) complex acting as the central mediating factor that integrates chromatin remodeling, architectural protein binding, and active DNA demethylation to establish/maintain the epigenetic and chromatin architectural landscape required for genome regulation and cellular differentiation. Overall, this application of the newly developed G-MAT technique suggests that additional factors recruited by CTCF play an active role in determining the specific epigenetic landscape of CTCF-binding sites.
Paired-Tag technology from Epigenome Technologies generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Could an in-depth analysis of histone modifications and transcriptomic profiles of the same single cells afforded by the integration of Paired-Tag have provided complementary data and revealed more regarding how NuRD helps to establish the epigenetic landscape of CTCF binding sites?
By applying their novel approach to HEK293T human kidney cells and mouse embryonic stem cells, the authors discovered that CTCF-chromatin interactions frequently coincided with regions of elevated DNA methylation, suggesting that CTCF first binds and then alters the local chromatin environment. They then set out to identify the factors that supported CTCF-chromatin binding through the development and application of an enhanced version of the proximity-dependent biotin identification (eBioID) method.
While they identified components of the NuRD, SWI/SNF, and ISWI chromatin remodeler families as potential mediators of CTCF-chromatin interactions, previous studies had already provided evidence that SWI/SNF (Barisic et al. and Ren et al.) and ISWI (Iurlaro et al. and Barisic et al.) activity had no significant impact on CTCF binding/occupancy. Therefore, these data suggested the necessary role of the NuRD complex in CTCF chromatin occupancy. In addition, the team discovered that the NuRD component MBD3 (Methyl-CpG Binding Domain Protein 3) facilitated TET1 (ten-eleven translocation 1) methylcytosine dioxygenase-mediated DNA demethylation to support the activation/expression of nearby genes. The primary components of the NuRD complex include the ATP-dependent remodeling enzymes CHD3/4/5, the histone deacetylases HDAC1/2, the scaffold proteins MTA1/2/3, the histone chaperones RBBP4/7, the CpG-binding proteins MBD2/3, p66α and/or p66β (Boulasiki et al.). MBD2 and MBD3 possess different affinities for methylated DNA and are mutually exclusive within the NuRD complex (Liu et al.).
Finally, the authors revealed that NuRD loss and CTCF loss led to the same outcomes: the disruption of topologically associating domain insulation, loop anchors, and long-range interactions; furthermore, embryonic stem cells lacking NuRD exhibited impaired differentiation/lineage commitment by blocking epigenetic remodeling. Overall, this application of G-MAT suggests that CTCF first binds regions of elevated DNA methylation and recruits the NuRD complex to facilitate the development of the characteristic epigenetic landscape of CTCF-binding sites and the expression of nearby genes.
The canonical role of the NuRD complex involves nucleosome remodeling; here, the newly developed G-MAT approach demonstrated that CTCF assembles with the NuRD complex to establish the epigenetic landscape of CTCF sites and the high-order architecture of the mammalian genome. In addition, the NuRD complex recruits TET1 to maintain local DNA hypomethylation and to allow the binding of additional proteins required for the transcriptional activation of nearby genes. The implementation of Paired-Tag technology from Epigenome Technologies, which generates joint epigenetic and transcriptomic profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays, has the potential to provide deeper insight into such research aims. Could the simultaneous single-cell analysis of histone modification and transcriptomic profiles have provided complementary data that would have revealed more regarding how the NuRD chromatin remodeling complex helps to establish the epigenetic landscape of CTCF binding sites?
By Stuart P. Atkinson
Page Title
G-MAT Defines the Epigenetic Interplay of CTCF and NuRD
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A study employs GpC methylation-assisted tracing (G-MAT) to define how the NuRD complex establishes the epigenetic landscape of CTCF-binding sites.
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