Let me begin by saying that I think I could have a satisfying career studying the histone proteins simply due to the number of great (/terrible) puns to which they lend themselves. In all fairness, my title was inspired by a 2010 Nature Reviews Cell Biology by Talbert and Henikoff (1); therefore, my sense of humor is vindicated by the editors of a high-impact journal.
I had the distinct honor of giving the first student presentation in our series concerning protein dynamics studied via backbone amide hydrogen–deuterium exchange monitored by either mass spectrometry (H/DX-MS) or NMR spectroscopy (H/DX-NMR). The BCH 761 course covering topics in epigenetics prompted me to consider protein dynamics within chromatin, the protein–DNA complex responsible for packaging eukaryotic genomes into nuclei. Chromatin is assembled by the basic unit of the nucleosome which is a disc-like structure composed of an octamer of histone proteins with approximately 150 base-pairs of DNA sequence wrapped around the outside of the disc (hence the wrap/rap pun). A set of four canonical histone proteins–H2A, H2B, H3 and H4– that are conserved throughout eukaryotic organisms comprise the most abundant “variety” of nucleosomes. However, regions of chromatin acquire distinguishing characteristics through various modifications including the incorporation of sequence-variant histone proteins into the nucleosome core. Centromeres, the location on each chromosome where the kinetochore assembles and the spindle apparatus attaches to mediate chromosomal segregation during mitosis, are defined epigenetically (without DNA sequence specificity, per se) by the nucleosomal replacement of histone H3 with the variant protein named centromeric histone protein A (CENP-A) in humans. Comparisons between H3 and CENP-A will be our center of “a tension.” (Get it? The centromeres are under tension during chromosome segregation… oh, forget it.)
In a 2011 Proceedings of the National Academy of the Sciences article (2), the Englander and Black Groups at the University of Pennsylvania School of Medicine used H/DX-MS to compare the dynamics of human histone H3 to human CENP-A when each are assembled into 12-mer nucleosome arrays. Examples of these nucleosome arrays in the de-condensed “beads on a string” conformation are shown in the electron micrograph below.
Differences in histone dynamics for the H3 and CENP-A arrays were determined by comparing the slowing of H/D exchange rates, what the authors refer to as “exchange protection”, when the arrays were condensed into a tightly packed state by the addition of a magnesium salt. Surprisingly, across the entire ~3 megadalton protein–DNA complexes the authors found substantial differences in H/D exchange protection only in a single alpha helix (helix αN) of the H3 and CENP-A proteins. (The αN helix is labeled in the crystal structure drawings below.)
For the CENP-A nucleosome array cation-induced condensation confers much less exchange protection to this helix than does condensation of the H3-containing arrays. The authors attribute significance to this difference in protection since the αN helices (two copies of H3/CENP-A per nucleosome) are the sites where DNA contacts the histone core as it enters and exits the nucleosome particle. A decrease in exchange protection at this site in CENP-A nucleosomes suggests that DNA interacts with the histone core less at these superhelix termini. This hypothesis is further supported by the crystal structure of the CENP-A nucleosome particle published by Tachiwana et al (3) in which the DNA at the termini is not sufficiently ordered to be observed in the structure. (The equivalent crystal structure for the H3 nucleosome particle shows structured DNA at the termini.)
What significance does decreased interactions between DNA and the histone core have for CENP-A nucleosomes? Panchenko et al. (2) hypothesize that the decrease in DNA-histone interactions reflects a different wrapping pattern for DNA occupying CENP-A nucleosomes as illustrated schematically in their figure below, and this conclusion is supported by other studies such as AFM measurement of CENP-A nucleosome dimensions.
Perhaps the closer packing of CENP-A nucleosomes confers additional physical resilience to centromeric chromatin so that it can better function during the pulling apart of sister chromatids at mitosis? Perhaps the different wrapping pattern allows CENP-A nucleosomes easier occupancy of the chromosome surface for more efficient kinetochore assembly? Perhaps something else is in play? Panchenko et al. (2) really could only speculate as to the biological significance of the biophysical differences in DNA–histone association they described. At this point, there still seems to be more to learn from studying the moves of this “wrap artist”, centromeric histone protein A.
1. Talbert P.B. & Henikoff S. (2010). Histone variants — ancient wrap artists of the epigenome, Nature Reviews Molecular Cell Biology, 11 (4) 264-275. DOI: 10.1038/nrm2861
2. Panchenko T., Sorensen T.C., Woodcock C.L., Kan Z.Y., Wood S., Resch M.G., Luger K., Englander S.W., Hansen J.C. & Black B.E. & (2011). Replacement of histone H3 with CENP-A directs global nucleosome array condensation and loosening of nucleosome superhelical termini, Proceedings of the National Academy of Sciences, 108 (40) 16588-16593. DOI: 10.1073/pnas.1113621108
3. Tachiwana H., Kagawa W., Shiga T., Osakabe A., Miya Y., Saito K., Hayashi-Takanaka Y., Oda T., Sato M. & Park S.Y. & (2011). Crystal structure of the human centromeric nucleosome containing CENP-A, Nature, 476 (7359) 232-235. DOI: 10.1038/nature10258