The hierarchical packaging of eukaryotic chromatin plays a central role in transcriptional regulation and other DNA-related biological processes. Here, we report the 11-angstrom-resolution cryogenic electron microscopy (cryo-EM) structures of 30-nanometer chromatin fibers reconstituted in the presence of linker histone H1 and with different nucleosome repeat lengths. The structures show a histone H1-dependent left-handed twist of the repeating tetranucleosomal structural units, within which the four nucleosomes zigzag back and forth with a straight linker DNA. The asymmetric binding and the location of histone H1 in chromatin play a role in the formation of the 30-nanometer fiber. Our results provide mechanistic insights into how nucleosomes compact into higher-order chromatin fibers.

Dot1 is an evolutionarily conserved histone methyltransferase that methylates lysine-79 of histone H3 in the core domain. Unlike other histone methyltransferases, Dot1 does not contain a SET domain, and it specifically methylates nucleosomal histone H3. We have solved a 2.5 A resolution structure of the catalytic domain of human Dot1, hDOT1L, in complex with S-adenosyl-L-methionine (SAM). The structure reveals a unique organization of a mainly alpha-helical N-terminal domain and a central open alpha/beta structure, an active site consisting of a SAM binding pocket, and a potential lysine binding channel. We also show that a flexible, positively charged region at the C terminus of the catalytic domain is critical for nucleosome binding and enzymatic activity. These structural and biochemical analyses, combined with molecular modeling, provide mechanistic insights into the catalytic mechanism and nucleosomal specificity of Dot1 proteins.

Dot1 is a non-SET domain protein that methylates histone H3 at lysine 79, a surface-exposed residue that lies within the globular domain. In the context of a nucleosome, H3 lysine 79 is located in close proximity with lysine 123 of histone H2B, a major site for ubiquitination by Rad6. Here we show that Rad6-mediated ubiquitination of H2B lysine 123 is important for efficient methylation of lysine 79, but not lysine 36, of histone H3. In contrast, lysine 79 methylation of H3 is not required for ubiquitination of H2B. Our study provides a new example of trans-histone regulation between modifications on different histones. In addition, it suggests that Rad6 affects telomeric silencing, at least in part, by influencing methylation of histone H3.Histones are subjected to post-translational modifications such as acetylation, phosphorylation, ubiquitination, and methylation. Methylation on specific lysine or arginine residues is carried out by distinct classes of enzymes. The CARM1/ PRMT1 class of enzymes mediates arginine methylation, while the SET domain-containing enzymes mediate lysine methylation (1, 2). In Saccharomyces cerevisiae, arginine methylation of histones has not been described, but the N-terminal tail of histone H3 is methylated at lysines 4 and 36. Set1 is the lysine 4 methylase because lysine 4 methylation is abolished in a set1 deletion strain and the Set1 complex methylates lysine 4 in vitro (3-6). Set1 methylation of H3 lysine 4 is important for rDNA silencing (3, 7). Lysine 36 methylation of H3 is mediated by Set2, and artificial recruitment of Set2 to a promoter results in transcriptional repression (8). Unlike the case in higher eukaryotes, methylation of histone H3 at lysines 9 and 27 and methylation of histone H4 at lysine 20 are not observed in S. cerevisiae (1,9,10).Recently S. cerevisiae Dot1 and the related human protein have been identified as histone methylases that specifically methylate lysine 79 within the globular domain of histone H3 (11-14). These studies demonstrate that histone H3 can be methylated outside the tail region, and they provide the first example of a non-SET domain protein that mediates lysine methylase activity. Based on the crystal structure of a nucleosome, lysine 79 is a surface-exposed residue that is located within loop 1 between helix 1 and helix 2 of histone H3 (15). Dot1 methylates lysine 79 only in the context of nucleosomes, indicating that a certain structural feature of the nucleosome is required for enzymatic activity (11,12). A similar requirement for the nucleosomal configuration has been reported for Set2 and SET8/PR-SET7 (8 -10).In yeast cells, either loss or overexpression of Dot1 results in impaired telomeric silencing (16). Telomeric silencing is also disrupted by mutations of lysine 79 of histone H3 or by mutations that abolish the catalytic activity of Dot1, suggesting that Dot1 influences telomeric silencing largely through methylation of lysine 79 (11, 12). This defect in telomeric silencing might reflect an interaction between Sir proteins a...

The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target.