H3K36me2/3 Binding and DNA Binding of the DNA Methyltransferase DNMT3A PWWP Domain Both Contribute to its Chromatin Interaction

The PWWP domain of DNMT3 DNA methyltransferases binds to histone H3 tails containing methylated K36, and this activity is important for heterochromatic targeting. Here, we show that the PWWP domain of mouse DNMT3A binds to H3K36me2 and H3K36me3 with a slight preference for H3K36me2. PWWP domains have also been reported to bind to DNA, and the close proximity of H3K36 and nucleosomal DNA suggests a combined binding to H3K36me2/3 and DNA. We show here that the DNMT3A PWWP domain binds to DNA with a weak preference for AT-rich sequences and that the designed charge reversal R362E mutation disrupts DNA binding. The K295E mutation, as well as K295I recently identified in paraganglioma, a rare neuroendocrine neoplasm, disrupts both DNA and H3K36me2/3 binding, which is in agreement with the proximity of K295 to residues involved in K36me2/3 methyllysine binding. Nucleosome pulldown experiments show that DNA binding and H3K36me2/3 binding are important for the interaction of the DNMT3A PWWP domain with nucleosomes. Localization studies of transiently transfected fluorescently-tagged wild-type and PWWP-mutated full-length DNMT3A indicate that both interactions contribute to the subnuclear localization of DNMT3A in mouse cells. In summary, our data demonstrate that the combined binding of the DNMT3A PWWP domain to the H3 tail containing K36me2/3 and to the nucleosomal or linker DNA is important for its chromatin interaction and subnuclear targeting of DNMT3A in living cells.     

The Dnmt3a PWWP Domain Reads Histone 3 Lysine 36 Trimethylation and Guides DNA Methylation

The Dnmt3a DNA methyltransferase contains in its N-terminal part a PWWP domain that is involved in chromatin targeting. Here, we have investigated the interaction of the PWWP domain with modified histone tails using peptide arrays and show that it specifically recognizes the histone 3 lysine 36 trimethylation mark. H3K36me3 is known to be a repressive modification correlated with DNA methylation in mammals and heterochromatin in Schizosaccharomyces pombe. These results were confirmed by equilibrium peptide binding studies and pulldown experiments with native histones and purified native nucleosomes. The PWWP-H3K36me3 interaction is important for the subnuclear localization of enhanced yellow fluorescent protein-fused Dnmt3a. Furthermore, the PWWP-H3K36me3 interaction increases the activity of Dnmt3a for methylation of nucleosomal DNA as observed using native nucleosomes isolated from human cells after demethylation of the DNA with 5-aza-2′-deoxycytidine as substrate for methylation with Dnmt3a. These data suggest that the interaction of the PWWP domain with H3K36me3 is involved in targeting of Dnmt3a to chromatin carrying that mark, a model that is in agreement with several studies on the genome-wide distribution of DNA methylation and H3K36me3.     

Isw1a does not have strict limitations on the length of extranucleosomal DNAs for mobilization of nucleosomes assembled with HeLa cell histones

The Saccharomyces cerevisiae Isw1a and Isw2 ATP-dependent chromatin-remodeling complexes have important roles in vivo in the regulation of nucleosome positioning and modulation of gene activity. We studied the ability of the Isw1a- and Isw2-remodeling enzymes to reposition nucleosomes in mono- and dinucleosomes templates with variably positioned histone octamers (in the center or at the ends of the DNA fragment). To compare the Isw1a and Isw2 nucleosome-mobilizing activities, we utilized mono- and dinucleosome templates reconstituted with purified HeLa cell histones and DNA containing one or two copies of the “601” nucleosome high-affinity sequence used to specifically position nucleosomes on the DNA. The obtained data suggest that Isw1a is able to mobilize HeLa cell histone-assembled mononucleosomes with long (more than 30?bp) extranucleosomal DNAs protruding from both sides, which contrasts to the previously reported inability of Isw1 to mobilize similar nucleosomes assembled with recombinant yeast histones. The results also suggest that Isw1a and Isw2 can mobilize nucleosomes with unfavorably short linker DNA lengths, and the presence of internucleosomal interactions promotes mobilization of nucleosomes even when the linkers are short.     

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1–19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1–19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies.     

The Tudor Domain of the PHD Finger Protein 1 Is a Dual Reader of Lysine Trimethylation at Lysine 36 of Histone H3 and Lysine 27 of Histone Variant H3t

PHF1 associates with the Polycomb repressive complex 2 and it was demonstrated to stimulate its H3K27-trimethylation activity. We studied the interaction of the PHF1 Tudor domain with modified histone peptides and found that it recognizes H3K36me3 and H3tK27me3 (on the histone variant H3t) and that it uses the same trimethyllysine binding pocket for the interaction with both peptides. Since both peptide sequences are very different, this result indicates that reading domains can have dual specificities. Sub-nuclear localization studies of full-length PHF1 in human HEK293 cells revealed that it co-localizes with K27me3, but not with K36me3, and that this co-localization depends on the trimethyllysine binding pocket indicating that K27me3 is an in vivo target for the PHF1 Tudor domain. Our data suggest that PHF1 binds to H3tK27me3 in human chromatin, and H3t has a more general role in Polycomb regulation.     

Homodimeric PHD Domain-containing Rco1 Subunit Constitutes a Critical Interaction Hub within the Rpd3S Histone Deacetylase Complex

Recognition of histone post-translational modifications is pivotal for directing chromatin-modifying enzymes to specific genomic regions and regulating their activities. Emerging evidence suggests that other structural features of nucleosomes also contribute to precise targeting of downstream chromatin complexes, such as linker DNA, the histone globular domain, and nucleosome spacing. However, how chromatin complexes coordinate individual interactions to achieve high affinity and specificity remains unclear. The Rpd3S histone deacetylase utilizes the chromodomain-containing Eaf3 subunit and the PHD domain-containing Rco1 subunit to recognize nucleosomes that are methylated at lysine 36 of histone H3 (H3K36me). We showed previously that the binding of Eaf3 to H3K36me can be allosterically activated by Rco1. To investigate how this chromatin recognition module is regulated in the context of the Rpd3S complex, we first determined the subunit interaction network of Rpd3S. Interestingly, we found that Rpd3S contains two copies of the essential subunit Rco1, and both copies of Rco1 are required for full functionality of Rpd3S. Our functional dissection of Rco1 revealed that besides its known chromatin-recognition interfaces, other regions of Rco1 are also critical for Rpd3S to recognize its nucleosomal substrates and functionin vivo. This unexpected result uncovered an important and understudied aspect of chromatin recognition. It suggests that precisely reading modified chromatin may not only need the combined actions of reader domains but also require an internal signaling circuit that coordinates the individual actions in a productive way.     

Interactions of HP1 Bound to H3K9me3 Dinucleosome by Molecular Simulations and Biochemical Assays

Heterochromatin protein 1 (HP1), associated with heterochromatin formation, recognizes an epigenetically repressive marker, trimethylated lysine 9 in histone H3 (H3K9me3), and generally contributes to long-term silencing. How HP1 induces heterochromatin is not fully understood. Recent experiments suggested that not one, but two nucleosomes provide a platform for this recognition. Integrating previous and new biochemical assays with computational modeling, we provide near-atomic structural models for HP1 binding to the dinucleosomes. We found that the dimeric HP1α tends to bind two H3K9me3s that are in adjacent nucleosomes, thus bridging two nucleosomes. We identified, to our knowledge, a novel DNA binding motif in the hinge region that is specific to HP1α and is essential for recognizing the H3K9me3 sites of two nucleosomes. An HP1 isoform, HP1γ, does not easily bridge two nucleosomes in extended conformations because of the absence of the above binding motif and its shorter hinge region. We propose a molecular mechanism for chromatin structural changes caused by HP1.     

N-terminal phosphorylation of HP1α increases its nucleosome-binding specificity

Heterochromatin protein 1 (HP1) is an evolutionarily conserved chromosomal protein that binds to lysine 9-methylated histone H3 (H3K9me), a hallmark of heterochromatin. Although HP1 phosphorylation has been described in several organisms, the biological implications of this modification remain largely elusive. Here we show that HP1''s phosphorylation has a critical effect on its nucleosome binding properties. By in vitro phosphorylation assays and conventional chromatography, we demonstrated that casein kinase II (CK2) is the kinase primarily responsible for phosphorylating the N-terminus of human HP1α. Pull-down assays using in vitro-reconstituted nucleosomes showed that unmodified HP1α bound H3K9-methylated and H3K9-unmethylated nucleosomes with comparable affinity, whereas CK2-phosphorylated HP1α showed a high specificity for H3K9me3-modified nucleosomes. Electrophoretic mobility shift assays showed that CK2-mediated phosphorylation diminished HP1α''s intrinsic DNA binding, which contributed to its H3K9me-independent nucleosome binding. CK2-mediated phosphorylation had a similar effect on the nucleosome-binding specificity of fly HP1a and S. pombe Swi6. These results suggested that HP1 phosphorylation has an evolutionarily conserved role in HP1''s recognition of H3K9me-marked nucleosomes.     

Analysis of nucleosome repositioning by yeast ISWI and Chd1 chromatin remodeling complexes

ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin remodeling activities in eukaryotes from yeast to man. Many of these complexes have been found to reposition nucleosomes but with different directionalities. We find that the yeast Isw1a, Isw2, and Chd1 enzymes preferentially move nucleosomes toward more central locations on short DNA fragments whereas Isw1b does not. Importantly, the inherent positioning properties of the DNA play an important role in determining where nucleosomes are relocated to by all of these enzymes. However, a key difference is that the Isw1a, Isw2, and Chd1 enzymes are unable to move nucleosomes to positions closer than 15 bp from a DNA end, whereas Isw1b can. We also find that there is a correlation between the inability of enzymes to move nucleosomes close to DNA ends and the preferential binding to nucleosomes bearing linker DNA. These observations suggest that the accessibility of linker DNA together with the positioning properties of the underlying DNA play important roles in determining the outcome of remodeling by these enzymes.