Cell-cycle-dependent structural transitions in the human CENP-A nucleosome in vivo

In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in?vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.     

Chromosomal histone modification patterns--from conservation to diversity

The organization of DNA into chromatin regulates expression and maintenance (replication, repair, recombination, segregation) of genetic information in a dynamic manner. The N-terminal tails of the nucleosomal core histones are subjected to post-translational modifications such as acetylation, methylation, phosphorylation, ubiquitination, glycosylation, ADP-ribosylation, carbonylation and sumoylation. These modifications, together with DNA methylation, control the folding of the nucleosomal array into higher order structures and mediate signalling for cellular processes. Although histones and their modifications are highly conserved, recent data show that chromosomal distribution of individual modifications (acetylation, methylation, phosphorylation) can differ along the cell cycle as well as among and between groups of eukaryotes. This implies the possibility of evolutionary divergence in reading the "histone code".     

Histone tail network and modulation in a nucleosome

The structural unit of eukaryotic chromatin is a nucleosome, comprising two histone H2A/H2B heterodimers and one histone (H3/H4)₂ tetramer, wrapped around by ∼146-bp core DNA and linker DNA. Flexible histone tails sticking out from the core undergo posttranslational modifications that are responsible for various epigenetic functions. Recently, the functional dynamics of histone tails and their modulation within the nucleosome and nucleosomal complexes have been investigated by integrating NMR, molecular dynamics simulations, and cryo-electron microscopy approaches. In particular, recent NMR studies have revealed correlations in the structures of histone N-terminal tails between H2A and H2B, as well as between H3 and H4 depending on linker DNA, suggesting that histone tail networks exist even within the nucleosome.     

Genome-wide patterns of histone modifications in yeast

Post-translational histone modifications and histone variants generate complexity in chromatin to enable the many functions of the chromosome. Recent studies have mapped histone modifications across the Saccharomyces cerevisiae genome. These experiments describe how combinations of modified and unmodified states relate to each other and particularly to chromosomal landmarks that include heterochromatin, subtelomeric chromatin, centromeres, origins of replication, promoters and coding regions. Such patterns might be important for the regulation of heterochromatin-mediated silencing, chromosome segregation, DNA replication and gene expression.     

Computational analysis suggests a highly bendable, fragile structure for nucleosomal DNA

Eukaryotic chromosomal DNA coils around histones to form nucleosomes. Although histone affinity for DNA depends on DNA sequence patterns, how nucleosome positioning is determined by them remains unknown. Here, we show relationships between nucleosome positioning and two structural characteristics of DNA conferred by DNA sequence. Analysis of bendability and hydroxyl radical cleavage intensity of nucleosomal DNA sequences indicated that nucleosomal DNA is bendable and fragile and that nucleosome positional stability was correlated with characteristics of DNA. This result explains how histone positioning is partially determined by nucleosomal DNA structure, illuminating the optimization of chromosomal DNA packaging that controls cellular dynamics.     

Histone modifications dictate specific biological readouts

The basic unit of chromatin is the nucleosomal core particle, containing 147 bp of DNA that wraps twice around an octamer of core histones. The core histones bear a highly dynamic N-terminal amino acid tail around 20-35 residues in length and rich in basic amino acids. These tails extending from the surface of nucleosome play an important role in folding of nucleosomal arrays into higher order chromatin structure, which plays an important role in eukaryotic gene regulation. The amino terminal tails protruding from the nuclesomes get modified by the addition of small groups such as methyl, acetyl and phosphoryl groups. In this review, we focus on these complex modi- fication patterns and their biological functions. Moreover, these modifications seem to be part of a complex scheme where distinct histone modifications act in a sequential manner or in combination to form a ＂histone code＂ read by other proteins to control the structure and/or function of the chromatin fiber. Errors in this histone code may be involved in many human diseases especially cancer, the nature of which could be therapeutically exploited. Increasing evidence suggests that many proteins bear multiple, distinct modifications, and the ability of one modification to antagonize or synergize the deposition of another can have significant biological consequences.     

Nucleosome depletion alters the chromatin structure of Saccharomyces cerevisiae centromeres.

Saccharomyces cerevisiae centromeric DNA is packaged into a highly nuclease-resistant chromatin core of approximately 200 base pairs of DNA. The structure of the centromere in chromosome III is somewhat larger than a 160-base-pair nucleosomal core and encompasses the conserved centromere DNA elements (CDE I, II, and III). Extensive mutational analysis has revealed the sequence requirements for centromere function. Mutations affecting the segregation properties of centromeres also exhibit altered chromatin structures in vivo. Thus the structure, as delineated by nuclease digestion, correlated with functional centromeres. We have determined the contribution of histone proteins to this unique structural organization. Nucleosome depletion by repression of either histone H2B or H4 rendered the cell incapable of chromosome segregation. Histone repression resulted in increased nuclease sensitivity of centromere DNA, with up to 40% of CEN3 DNA molecules becoming accessible to nucleolytic attack. Nucleosome depletion also resulted in an alteration in the distribution of nuclease cutting sites in the DNA surrounding CEN3. These data provide the first indication that authentic nucleosomal subunits flank the centromere and suggest that nucleosomes may be the central core of the centromere itself.     

A native peptide ligation strategy for deciphering nucleosomal histone modifications

Post-translational modifications of histones influence both chromatin structure and the binding and function of chromatin-associated proteins. A major limitation to understanding these effects has been the inability to construct nucleosomes in vitro that harbor homogeneous and site-specific histone modifications. Here, we describe a native peptide ligation strategy for generating nucleosomal arrays that can harbor a wide range of desired histone modifications. As a first test of this method, we engineered model nucleosomal arrays in which each histone H3 contains a phosphorylated serine at position 10 and performed kinetic analyses of Gcn5-dependent histone acetyltransferase activities. Recombinant Gcn5 shows increased histone acetyltransferase activity on nucleosomal arrays harboring phosphorylated H3 serine 10 and is consistent with peptide studies. However, in contrast to analyses using peptide substrates, we find that the histone acetyltransferase activity of the Gcn5-containing SAGA complex is not stimulated by H3 phosphorylation in the context of nucleosomal arrays. This difference between peptide and array substrates suggests that the ability to generate specifically modified nucleosomal arrays should provide a powerful tool for understanding the effects of post-translational histone modifications.     

Plant chromatin -- epigenetics linked to ATP-dependent remodeling and architectural proteins

Studies in organisms belonging to different eukaryotic kingdoms have revealed that the structural state of chromatin is controlled by interactions of DNA, small RNAs and specific proteins, linked to a self-reinforcing complex network of biochemical activities involving histone and DNA modifications and ATP-dependent nucleosome remodeling. However, these findings must now be reinterpreted in light of the recent discovery of the highly dynamic character of interphase chromosomes exemplified by the constant flux of enzymatic and structural proteins through both eu- and heterochromatin and by short- and long-range chromosome movements in the nucleus. The available data on chromosome organization in Arabidopsis thaliana and links between proteins influencing chromatin structure and DNA and histone modifications documented in this model plant provide strong supportive evidence for the dynamic nature of chromosomes.