DNA (de)methylation in embryonic stem cells controls CTCF-dependent chromatin boundaries

Area of Science:

• Epigenetics and Gene Regulation
• Chromatin Dynamics
• Cellular Differentiation

Background:

• Cell-type-specific chromatin states are established during differentiation through coordinated DNA (de)methylation, nucleosome positioning, and CTCF binding.
• The TET dioxygenases (TET1, TET2) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), playing a key role in DNA demethylation.
• Understanding the interplay between these epigenetic marks and architectural proteins like CTCF is crucial for deciphering gene regulation during development.

Purpose of the Study:

• To elucidate the molecular mechanisms linking DNA (de)methylation, nucleosome positioning, and CTCF binding during cell differentiation.
• To investigate the impact of TET1 and TET2 loss on the DNA modification landscape and its consequences for chromatin structure and gene expression in mouse embryonic stem cells (ESCs).

Main Methods:

• Generation and analysis of mouse ESCs with a double knockout (DKO) of Tet1 and Tet2.
• Determination of nucleosome positioning, CTCF binding, DNA methylation (5mC, 5hmC, 5fC, 5caC), and gene expression profiles in DKO ESCs.
• Development of biophysical models to predict differential CTCF binding based on epigenetic and sequence features.

Main Results:

• Loss of TET1/TET2 in DKO ESCs led to significant changes in DNA methylation and nucleosome positioning.
• Methylation-sensitive nucleosome repositioning was a major driver of CTCF binding loss in DKO ESCs.
• CTCF binding was sensitive to 5hmC levels, with preferential loss from 5hmC-marked sites, and CTCF loss at specific sites correlated with DNA methylation spreading and gene downregulation.

Conclusions:

• A hierarchical interplay exists between cytosine modifications, nucleosome positioning, and DNA sequence in determining differential CTCF binding.
• CTCF binding dynamics are modulated by DNA methylation states and nucleosome occupancy, impacting chromatin loop and TAD organization.
• These findings reveal how perturbations in DNA demethylation pathways can lead to altered gene expression programs during cellular differentiation.