Evgenia Nikolaevna Nikolova

The zinc finger transcription factor Kaiso (ZBTB33) recognizes methylated CpG dinucleotides at silenced promoters and imprinted loci, but how it engages methylated DNA within the nucleosome remains unclear. To address this, we developed a DNMT1-based strategy for preparing site-specifically methylated nucleosomes containing the Kaiso recognition motif. Kaiso binds methylated nucleosomes with strong positional preference, with high-affinity engagement at the entry/exit site (SHL 6.5; Kd ≈ 100 nM), reduced affinity at SHL 5.5 (Kd ≈ 170 nM), and no methylation-dependent enhancement at dyad-proximal positions. Hemi- and fully methylated substrates bind Kaiso comparably at SHL 6.5, and the E535A mutation, which disrupts a key methyl-CpG contact, abolishes methylation-dependent discrimination. Solution NMR titrations of 15N-labeled H3 nucleosomes reveal that Kaiso binding perturbs a discrete set of H3 N-terminal tail residues consistent with partial release of the tail from its nucleosomal DNA contacts. This pattern closely resembles that produced by the pioneer factor Sox2 at the same nucleosomal region, suggesting H3 tail displacement is a general consequence of factor engagement at the nucleosome edge, independent of DNA-recognition mode. These results establish Kaiso as an active reader of methylated nucleosomal DNA that may prime local chromatin by exposing the H3 tail.

The zinc finger transcription factor Kaiso recognizes methylated CpG dinucleotides at silenced promoters and imprinted loci, but how it engages methylated DNA within the nucleosome remains unclear. To address this, we developed a DNMT1-based strategy for preparing site-specifically methylated nucleosomes with defined position and methylation state of the Kaiso recognition motif. Electrophoretic mobility shift assays show that Kaiso binds methylated nucleosomes with strong positional preference, with high-affinity engagement at the entry/exit site (SHL 6.5; Kd ≈ 100 nM), reduced affinity at SHL 5.5 (Kd ≈ 170 nM), and no methylation-dependent enhancement at dyad-proximal positions. Hemi- and fully methylated substrates bind Kaiso comparably at SHL 6.5, and the E535A mutation, which disrupts a key methyl-CpG contact, reduces binding in a methylation- and position-dependent manner. Solution NMR titrations of 15N-labeled H3 nucleosomes reveal that Kaiso binding perturbs a discrete set of H3 N-terminal tail residues, with chemical shifts trending toward free-peptide values, indicating release of the tail from its nucleosomal DNA contacts. This pattern closely resembles that produced by the pioneer factor Sox2 at the same nucleosomal region, suggesting H3 tail displacement is a general consequence of factor engagement at the nucleosome edge, independent of DNA-recognition mode. These results establish Kaiso as an active reader of methylated nucleosomal DNA that may prime local chromatin by exposing the H3 tail.

Epigenetic regulation of chromatin structure is strongly influenced by histone variants and post-translational modifications. The conserved histone variant H2A.Z has been functionally linked to the pioneer factors Sox2 and Oct4, which open chromatin and activate cell fate-specific transcriptional programs. However, the molecular basis of this interaction is not well understood. Here, we combine biochemistry, NMR spectroscopy, and molecular dynamics (MD) simulations to investigate how H2A.Z nucleosome dynamics influence pioneer factor binding. We find that H2A.Z enhances Sox2 and Oct4 association at distinct positions within 601 nucleosomes, correlating with increased DNA accessibility and altered H3 N-terminal tail dynamics. We show that the H3 tail competes with Sox2 for DNA binding and is more efficiently displaced with H2A.Z, while also allowing for unique Sox2-H3 tail interactions. MD simulations reveal that H2A.Z promotes DNA unwrapping, increases inter-gyre spacing, and enhances H3 tail flexibility, while simultaneously reducing contacts with DNA and with the H2A.Z C-terminal tail. This destabilizing effect is DNA-sequence dependent and prominent in the less stable Lin28B nucleosome, which Sox2 appears to substantially reshape. Together, our results suggest that H2A.Z promotes pioneer factor binding by increasing DNA accessibility and reducing histone tail competition, with broad implications for epigenetic regulation and chromatin recognition.

Epigenetic regulation of chromatin structure is strongly influenced by histone variants and post-translational modifications. The conserved histone variant H2A.Z has been functionally linked to the pioneer factors Sox2 and Oct4, which open chromatin and activate cell fate-specific transcriptional programs. However, the molecular basis of this interaction is not well understood. Here, we combine biochemistry, NMR spectroscopy, and molecular dynamics (MD) simulations to investigate how H2A.Z nucleosome dynamics influence pioneer factor binding. We find that H2A.Z enhances Sox2 and Oct4 association at distinct positions within 601 nucleosomes, correlating with increased DNA accessibility and altered H3 N-terminal tail dynamics. We show that the H3 tail competes with Sox2 for DNA binding and is more efficiently displaced with H2A.Z, while also allowing for unique Sox2-H3 tail interactions. MD simulations reveal that H2A.Z promotes DNA unwrapping, increases inter-gyre spacing, and enhances H3 tail flexibility, while simultaneously reducing contacts with DNA and with the H2A.Z C-terminal tail. This destabilizing effect is DNA-sequence dependent and prominent in the less stable Lin28B nucleosome, which Sox2 appears to substantially reshape. Together, our results suggest that H2A.Z promotes pioneer factor binding by increasing DNA accessibility and reducing histone tail competition, with broad implications for epigenetic regulation and chromatin recognition.

Nucleosomes in eukaryotic genomes present a barrier to the competent binding of many regulatory proteins. Pioneer transcription factors (pTFs) can bind their target sites on nucleosomal DNA and collaborate with other factors to locally open chromatin and promote transcription. While the interaction of pluripotency pioneer factors and functional partners Sox2 and Oct4 with nucleosomes has been widely studied, molecular details about their engagement in different nucleosome contexts remain elusive. Here, using high-resolution nuclear magnetic resonance (NMR) spectroscopy and biochemical studies, we reveal site-specific structural and dynamic information about pTF interaction with nucleosomes. We find that the affinity of Sox2 and Oct4 to the nucleosome and their synergistic binding correlates with solvent-exposed sites but is highly position and DNA sequence dependent and linked to distinct pTF conformation and dynamics. Sox2 alone forms a super-stable complex near superhelical location 5 (SHL5) with similar affinity and conformation to that of naked DNA but shows elevated dynamics at suboptimal positions. Oct4 strongly favors positions near SHL5.5 and SHL6.5 and both of its DNA binding modules, POU _S or POU _HD , are required for stable complex formation. A ternary complex forms efficiently on canonical Sox2-Oct4 composite motifs (no spacing) near nucleosome ends but is sparse at spaced motifs and absent at internal sites. Moreover, the ability of Sox2 to fold and bend DNA plays a key role in the formation of a stable nucleosome complex and cooperative Oct4 binding. Collectively, our findings describe diverse binding modes of Sox2 and Oct4 on nucleosomes that could guide their site selection and potential interaction with other chromatin factors in vivo .

Many eukaryotic transcription factors recognize the epigenetic marker 5-methylcytosine (mC) at CpG sites in DNA. Despite their structural diversity, methyl-CpG-binding proteins (MBPs) share a common mode of recognition of mC methyl groups that involves hydrophobic pockets and weak hydrogen bonds of the CH···O type. The zinc finger protein Kaiso possesses a remarkably high specificity for methylated over unmethylated CpG sites. A key contribution to this specificity is provided by glutamate 535 (E535), which is optimally positioned to form multiple interactions with mCpG, including direct CH···O hydrogen bonds. To examine the role of E535 and CH···O hydrogen bonding in the preferential recognition of mCpG sites, we determined the structures of wild type Kaiso (WT) and E535 mutants and characterized their interactions with methylated DNA by nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography, and in vitro protein–DNA binding assays. Our data show that Kaiso favors an mCpG over a CpG site by 2 orders of magnitude in affinity and that an important component of this effect is the presence of hydrophobic and CH···O contacts involving E535. Moreover, we present the first direct evidence for formation of a CH···O hydrogen bond between an MBP and 5-methylcytosine by using experimental (NMR) and quantum mechanical chemical shift analysis of the mC methyl protons. Together, our findings uncover a critical function of methyl-specific interactions, including CH···O hydrogen bonds, that optimize the specificity and affinity of MBPs for methylated DNA and contribute to the precise control of gene expression.

The inability of A-form RNA to form Hoogsteen base pairs provides a mechanism for how post-transcriptional modifications can disrupt RNA structure and might help explain why DNA is the molecular choice for storing genetic information. The B-DNA double helix can dynamically accommodate G-C and A-T base pairs in either Watson–Crick or Hoogsteen configurations. Here, we show that G-C+ (in which + indicates protonation) and A-U Hoogsteen base pairs are strongly disfavored in A-RNA. As a result,N1-methyladenosine and N1-methylguanosine, which occur in DNA as a form of alkylation damage and in RNA as post-transcriptional modifications, have dramatically different consequences. Whereas they create G-C+ and A-T Hoogsteen base pairs in duplex DNA, thereby maintaining the structural integrity of the double helix, they block base-pairing and induce local duplex melting in RNA. These observations provide a mechanism for disrupting RNA structure through post-transcriptional modifications. The different propensities to form Hoogsteen base pairs in B-DNA and A-RNA may help cells meet the opposing requirements of maintaining genome stability, on the one hand, and of dynamically modulating the structure of the epitranscriptome, on the other.

Approaches that combine experimental data and computational molecular dynamics (MD) to determine atomic resolution ensembles of biomolecules require the measurement of abundant experimental data. NMR residual dipolar couplings (RDCs) carry rich dynamics information, however, difficulties in modulating overall alignment of nucleic acids have limited the ability to fully extract this information. We present a strategy for modulating RNA alignment that is based on introducing variable dynamic kinks in terminal helices. With this strategy, we measured seven sets of RDCs in a cUUCGg apical loop and used this rich data set to test the accuracy of an 0.8 μs MD simulation computed using the Amber ff10 force field as well as to determine an atomic resolution ensemble. The MD-generated ensemble quantitatively reproduces the measured RDCs, but selection of a sub-ensemble was required to satisfy the RDCs within error. The largest discrepancies between the RDC-selected and MD-generated ensembles are observed for the most flexible loop residues and backbone angles connecting the loop to the helix, with the RDC-selected ensemble resulting in more uniform dynamics. Comparison of the RDC-selected ensemble with NMR spin relaxation data suggests that the dynamics occurs on the ps–ns time scales as verified by measurements of R_1ρ relaxation–dispersion data. The RDC-satisfying ensemble samples many conformations adopted by the hairpin in crystal structures indicating that intrinsic plasticity may play important roles in conformational adaptation. The approach presented here can be applied to test nucleic acid force fields and to characterize dynamics in diverse RNA motifs at atomic resolution.

We recently showed that Watson–Crick base pairs in canonical duplex DNA exist in dynamic equilibrium with G(syn)·C⁺ and A(syn)·T Hoogsteen base pairs that have minute populations of ∼1%. Here, using nuclear magnetic resonance R_1ρ relaxation dispersion, we show that substitution of guanine with the naturally occurring base inosine results in an ∼17-fold increase in the population of transient Hoogsteen base pairs, which can be rationalized by the loss of a Watson–Crick hydrogen bond. These results provide further support for transient Hoogsteen base pairs and demonstrate that their population can increase significantly upon damage or chemical modification of the base.