Hidenori Kato

Architecture of the high mobility group nucleosomal protein 2-nucleosome complex as revealed by methyl-based NMR

Chromatin structure and function are regulated by numerous proteins through specific binding to nucleosomes. The structural basis of many of these interactions is unknown, as in the case of the high mobility group nucleosomal (HMGN) protein family that regulates various chromatin functions, including transcription. Here, we report the architecture of the HMGN2-nucleosome complex determined by a combination of methyl-transverse relaxation optimized nuclear magnetic resonance spectroscopy (methyl-TROSY) and mutational analysis. We found that HMGN2 binds to both the acidic patch in the H2A-H2B dimer and to nucleosomal DNA near the entry/exit point, “stapling” the histone core and the DNA. These results provide insight into how HMGNs regulate chromatin structure through interfering with the binding of linker histone H1 to the nucleosome as well as a structural basis of how phosphorylation induces dissociation of HMGNs from chromatin during mitosis. Importantly, our approach is generally applicable to the study of nucleosome-binding interactions in chromatin.

Structural insights into the histone H1-nucleosome complex

Significance Linker H1 histones control the accessibility of linker DNA between two neighbor nucleosomes to DNA-binding proteins and regulate chromatin folding. We investigated the structure of the H1–nucleosome complex through a combination of multidimensional nuclear magnetic resonance spectroscopy, site-directed mutagenesis-isothermal-titration calorimetry and computational design/modeling. The results lead to a unique structural model for the globular domain of H1 in complex with the nucleosome that contains residue-level information and have implications for the dynamics of chromatin in vivo. In addition, our approach will be useful for testing the hypothesis that the globular domain of H1 variants might have distinct binding geometries within the nucleosome, and thereby contribute to the heterogeneity of chromatin structure. Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1–nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo.

NMR structure of chaperone Chz1 complexed with histones H2A.Z-H2B.

The NMR structure of budding yeast chaperone Chz1 complexed with histones H2A.Z-H2B has been determined. Chz1 forms a long irregular chain capped by two short α-helices, and uses both positively and negatively charged residues to stabilize the histone dimer. A molecular model that docks Chz1 onto the nucleosome has implications for its potential functions.

Characterization of the N-terminal Tail Domain of Histone H3 in Condensed Nucleosome Arrays by Hydrogen Exchange and NMR

The N-terminal tail domains (NTDs) of histones play important roles in the formation of higher-order structures of chromatin and the regulation of gene functions. Although the structure of the nucleosome core particle has been determined by X-ray crystallography at near-atomic resolution, the histone tails are not observed in this structure. Here, we demonstrate that large quantities of nucleosome arrays with well-defined DNA positioning can be reconstituted using specific DNA sequences and recombinant isotope-labeled histones, allowing for the investigation of NTD conformations by amide hydrogen exchange and multidimensional nuclear magnetic resonance (NMR) methods. We examined the NTD of Drosophila melanogaster histones H3 in condensed nucleosome arrays. The results reveal that the majority of the amide protons in the NTD of H3 are protected from exchange, consistent with the NTDs having formed folded structures. Our study demonstrates hydrogen exchange coupled with NMR can provide residue-by-residue characterization of NTDs of histones in condensed nucleosome arrays, a technique that may be used to study NTDs of other histones and those with post-translational modifications.

Histone H4 K16Q mutation, an acetylation mimic, causes structural disorder of its N-terminal basic patch in the nucleosome

Histone tails and their posttranslational modifications play important roles in regulating the structure and dynamics of chromatin. For histone H4, the basic patch K(16)R(17)H(18)R(19) in the N-terminal tail modulates chromatin compaction and nucleosome sliding catalyzed by ATP-dependent ISWI chromatin remodeling enzymes while acetylation of H4 K16 affects both functions. The structural basis for the effects of this acetylation is unknown. Here, we investigated the conformation of histone tails in the nucleosome by solution NMR. We found that backbone amides of the N-terminal tails of histones H2A, H2B, and H3 are largely observable due to their conformational disorder. However, only residues 1-15 in H4 can be detected, indicating that residues 16-22 in the tails of both H4 histones fold onto the nucleosome core. Surprisingly, we found that K16Q mutation in H4, a mimic of K16 acetylation, leads to a structural disorder of the basic patch. Thus, our study suggests that the folded structure of the H4 basic patch in the nucleosome is important for chromatin compaction and nucleosome remodeling by ISWI enzymes while K16 acetylation affects both functions by causing structural disorder of the basic patch K(16)R(17)H(18)R(19).

Evidence for two senescence loci on human chromosome I

Microcell‐mediated introduction of a neo‐tagged human chromosome I (HC‐1‐neo) into several immortal cell lines has previously been shown to induce growth arrest and phenotypic changes indicative of replicative senescence. Somatic cell hybridization studies have localized senescence activity for immortal hamster 10W‐2 cells to a cytogenetically defined region between 1q23 and the q terminus. Previous microcell‐mediated chromosome transfer experiments showed that a chromosome 1 with an interstitial q‐arm deletion (del‐1q) lacks senescence inducing activity for several immortal human cell lines that are sensitive to an intact HC‐1‐neo. In contrast, our studies reveal that the del‐1q chromosome retains activity for 10W‐2 cells, indicating that there are at least two senescence genes on human chromosome 1. Sequence‐tagged site (STS) content analysis revealed that the q arm of the del‐1q chromosome has an interstitial deletion of approximately 63 centimorgans (cM), between the proximal STS marker D1S534 and distal marker D1S412, approximately 1q12 to 1q31. This deletion analysis provides a candidate region for one of the senescence genes on 1q. In addition, because this deletion region extends distally beyond 1q23, it localizes the region containing a second senescence gene to approximately 1q31‐qter, between D1S422 and the q terminus. STS content analysis of a panel of 11 10W‐2 microcell hybrid clones that escaped senescence identified 2 common regions of loss of 1q material below the distal breakpoint of del‐1q. One region is flanked by markers D1S459 and ACTN2, and the second lies between markers W1‐4683 and D1S1609, indicating that the distal 1q senescence gene(s) localizes within 1q42‐43. Genes Chromosom Cancer 16:55–63 (1996).

An evolving tail of centromere histone variant CENP-A

The reproduction and development of all organisms depends upon the accurate segregation of chromosomes during mitosis. Chromosome segregation is facilitated by the assembly of the kinetochore complex and its attachment to mitotic spindle microtubules at the chromosomal centromere. The centromere is specified by the nucleosome in which canonical histone H3 is replaced by its variant CENP-A. A key question in centromere biology is how the kinetochore complex recognizes the centromere nucleosome. Earlier studies have shown that human centromere proteins C (CENP-C) and N (CENP-N), components of the kinetochore complex, specifically recognize the CENP-A nucleosome,1,2 and CENP-C is found in all model systems for centromere studies.3 It is also known that the CENP-C central region (residues 426–537) and the CENP-C motif (residues 736–758) are required for CENP-C targeting to the centromere.2,4 The CENP-C central region directly binds to the CENP-A nucleosome in vitro,2 whereas the targeting mechanism for the CENP-C motif is unknown.