Seminar

Spotlight on a Very Special Nucleosome

Nikolina Sekulic, PhD

The centromere is the locus that ensures proper segregation of chromosomes through generations. The histone H3 variant, centromere protein A (CENP-A) is the prime candidate for epigenetic specification of centromeres. It has been a long-standing question in the field how CENP-A distinguishes centromeric nucleosomes from canonical H3 nucleosomes. Based on our previous observation that (CENP-A/H4)2 tetramers have pronounced rigidity and exhibit rotation at the CENP-A/CENP-A interface (Sekulic et al., 2010, Nature 467:347-51) we proposed a model where CENP-A nucleosomes have an altered histone core where the H2A/H2B dimers pivot outwards to accommodate this rotation. A crystal structure of the CENP-A nucleosome (Tachiwana et al., 2011, Nature 476:232-5), however, lacked any change in histone subunit packing relative to canonical nucleosomes. To probe for alternative conformations in solution we have undertaken solution-based experiments: small-angle scattering techniques (SAXS and SANS) - to exploit larger conformational changes, fluorescence resonance energy transfer (FRET) - to probe for particular distance differences between labeled parts of the protein or DNA in two different nucleosomes and MNase digestion - to test flexibility of DNA termini. Our solution methods confirm alterations of nucleosome core in the CENP-A containing nucleosomesresulting in wider DNA gyre separation andDNA tightening in agreement with our model, while MNase digestions indicate more flexible, open DNA termini. We extend our study to test if this unique nucleosomeconformation stays preserved in the presence of specific CENP-A-nucleosome binding protein, CENP-C. Interestingly, we find that changes in the nucleosome core are reverted upon this binding while flexibility of the DNA ends persists. Together, our data, besides having profound implications for understanding the chromatin at the foundation of the kinetochore, also provide a striking example of conformational changes at the level of a nucleosome.