Structure and Dynamics in the Nucleosome Revealed by Solid-State NMR.

Eukaryotic chromatin structure and dynamics play key roles in genomic regulation. In the current study, the secondary structure and intramolecular dynamics of human histone H4 (hH4) in the nucleosome core particle (NCP) and in a nucleosome array are determined by solid-state NMR (SSNMR). Secondary structure elements are successfully localized in the hH4 in the NCP precipitated with Mg2+ . In particular, dynamics on nanosecond to microsecond and microsecond to millisecond timescales are elucidated, revealing diverse internal motions in the hH4 protein. Relatively higher flexibility is observed for residues participating in the regulation of chromatin mobility and DNA accessibility. Furthermore, our study reveals that hH4 in the nucleosome array adopts the same structure and show similar internal dynamics as that in the NCP assembly while exhibiting relatively restricted motions in several regions consisting of residues in the N-terminus, Loop 1, and the α3 helix region.

Structural and dynamical investigation of histone H2B in well-hydrated nucleosome core particles by solid-state NMR.

H2A-H2B dimer is a key component of nucleosomes and an important player in chromatin biology. Here, we characterized the structure and dynamics of H2B in precipitated nucleosome core particles (NCPs) with a physiologically relevant concentration using solid-state NMR. Our recent investigation of H3-H4 tetramer determined its unique dynamic properties and the present work provides a deeper understanding of the previously observed dynamic networks in NCP that is potentially functionally significant. Nearly complete 13C, 15N assignments were obtained for H2B R30-A121, which permit extracting unprecedented detailed structural and amino-acid site-specific dynamics. The derived structure of H2B in the well-hydrated NCP sample agrees well with that of X-ray crystals. Dynamics at different timescales were determined semi-quantitatively for H2B in a site-specific manner. Particularly, higher millisecond-microsecond dynamics are observed for H2B core regions including partial α1, L1, partial α2, and partial L3. The analysis of these regions in the context of the tertiary structure reveals the clustering of dynamical residues. Overall, this work fills a gap to a complete resonance assignment of all four histones in nucleosomes and delineates that the dynamic networks in NCP extend to H2B, which suggests a potential mechanism to couple histone core with distant DNA to modulate the DNA activities.

Histone H4 lysine 20 mono-methylation directly facilitates chromatin openness and promotes transcription of housekeeping genes.

Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction.

Solid-state NMR 13C, 15N assignments of human histone H3 in the nucleosome core particle.

Nucleosome core particle (NCP), the basic unit of chromatin in eukaryotic cells, consists of ~ 147 bp DNA wrapped around a histone octamer (HO) formed by two H2A-H2B dimers and one (H3-H4)2 tetramer. Histones undergo various post-translational modifications (PTMs), which regulates genomic activities in different cellular phases. High-resolution structures have been solved for many nucleosomes primarily including NCPs. However, the atomic-resolution structures of nucleosome arrays and chromatin fiber, as well as the dynamics of nucleosomes remain poorly understood. Solid-state NMR (SSNMR) is one of the premier techniques to answer these questions. In this study, we present the 13C and 15N chemical shifts assignments for the globular domain of human histone H3 (hH3) using multidimensional SSNMR experiments. The obtained spectra are of outstanding resolution and the assignments are nearly 100% complete for the backbone 13C and 15N spins of R42-G132 and ~ 80% when taking into account the side chains. The secondary structure derived from the chemical shifts agrees with the previously reported X-ray crystal structure. The reported chemical shifts can be carried over to future SSNMR studies of structure and dynamics of hH3 in NCPs, nucleosome array, chromatin fibers and nucleosome-protein complexes.

Dynamic networks observed in the nucleosome core particles couple the histone globular domains with DNA.

The dynamics of eukaryotic nucleosomes are essential in gene activity and well regulated by various factors. Here, we elucidated the internal dynamics at multiple timescales for the human histones hH3 and hH4 in the Widom 601 nucleosome core particles (NCP), suggesting that four dynamic networks are formed by the residues exhibiting larger-scale µs-ms motions that extend from the NCP core to the histone tails and DNA. Furthermore, despite possessing highly conserved structural features, histones in the telomeric NCP exhibit enhanced µs-ms dynamics in the globular sites residing at the identified dynamic networks and in a neighboring region. In addition, higher mobility was observed for the N-terminal tails of hH3 and hH4 in the telomeric NCP. The results demonstrate the existence of dynamic networks in nucleosomes, through which the center of the core regions could interactively communicate with histone tails and DNA to potentially propagate epigenetic changes.

Amino acid selective unlabeling in protein NMR spectroscopy.

Three-dimensional structure determination of proteins by NMR requires the acquisition of multidimensional spectra followed by assignment of chemical shifts to the respective nuclei. In order to speed up this process, resonances corresponding to individual amino acid types are often selectively identified and assigned. One of the ways of achieving this is by using the method of "selective unlabeling." In this method, resonances from one or more amino acid types are suppressed selectively in the NMR spectra, which can be achieved using both cell-based and cell-free methods. This helps not only in identifying them but also results in spectral simplification by reducing the number of peaks observed. Further, the assignments are not limited to amino acids that are specifically unlabeled. Using specially designed NMR experiments, assignments of amino acids in the neighborhood of those being selectively unlabeled can also be obtained. In this chapter, we discuss the theoretical and practical aspects of selective unlabeling focusing on how the sample is prepared, which amino acid or a combination of amino acids should be optimally chosen for unlabeling, and how this method can be used for sequential assignments of proteins.

Insulin-like growth factor system in cancer: novel targeted therapies.

Insulin-like growth factors (IGFs) are essential for growth and survival that suppress apoptosis and promote cell cycle progression, angiogenesis, and metastatic activities in various cancers. The IGFs actions are mediated through the IGF-1 receptor that is involved in cell transformation induced by tumour. These effects depend on the bioavailability of IGFs, which is regulated by IGF binding proteins (IGFBPs). We describe here the role of the IGF system in cancer, proposing new strategies targeting this system. We have attempted to expand the general viewpoint on IGF-1R, its inhibitors, potential limitations of IGF-1R, antibodies and tyrosine kinase inhibitors, and IGFBP actions. This review discusses the emerging view that blocking IGF via IGFBP is a better option than blocking IGF receptors. This can lead to the development of novel cancer therapies.