Internucleosomal Interactions in Chromatin Fibers and Metaphase Chromosomes

Intellectual Merit: The DNA of eukaryotic cells, including human cells, is found in the nucleus tightly packed in complex, hierarchically organized structures called chromosomes. At the first level of organization, the DNA is coiled around histone protein cores like string on a spool to form structures called nucleosomes. The nucleosomes interact with each other to form arrays and higher-order structures, tightly compacting the DNA to fit it in the nucleus. For proper cell functioning, the higher-order chromatin structures must unfold to make the DNA accessible to enzymes that carry out transcription, replication, recombination, and repair of DNA. Alternatively, chromatin can adopt a condensed state known as heterochromatin that is transcriptionally inactive (repressed). The objective of this project is to deduce the basic principles of interaction between nucleosomes that leads to reversible formation of different higher-order chromatin structures that underlie the condensed or decondensed states. This project will experimentally test the hypothesis that nucleosome array compaction in condensed heterochromatin is determined by a) inter-nucleosomal interactions within and between nucleosomal arrays and b) linker DNA flexibility that promotes either intra-array interactions (chromatin 'secondary structure') or inter-array interactions (chromatin 'tertiary structure').

The experimental design includes two specific aims: 1) To identify linker DNA sequence motifs modulating linker DNA conformation and inter-nucleosomal interactions in reconstituted nucleosome arrays. 2) To determine patterns of inter-nucleosomal interactions in native interphase chromatin and condensed metaphase chromosomes in situ. In the second aim, the nucleosome interactions will be fixed in living cells and then chromatin isolated, unfolded, and analyzed by Electron Microscopy. These experiments should elucidate global chromatin higher-order organization and its transitions in vivo. This work employs established biochemical and electron microscopic experimental techniques and novel biochemical approaches to capture in vivo chromatin structure ('in-situ electron microscopy-assisted nucleosome interaction capture').

Broader Impacts: It is anticipated that this project may substantially change the current models featured in modern molecular biology and genetics textbooks in which the 30 nm chromatin fiber is depicted as a universal intermediate in chromosome folding. These studies are expected to reveal alternative type(s) of higher order structures underlying dynamic chromatin compaction in interphase cells and in metaphase chromosomes. The new information and methodology that is being developed under this project is crucial for understanding spatial organization of DNA in chromatin and its relationship to fundamental mechanisms of heterochromatin formation, gene silencing, and cell differentiation. In addition to the general scientific knowledge, this project will provide new research training and education opportunities to undergraduate students participating in the Penn State Summer Undergraduate Research program, many of whom attend small rural colleges in central Pennsylvania. The students come from under-represented minority groups as well as rural environments which provide little previous exposure to experimental science. The project will play a crucial role in supporting the educational infrastructure for research and training in molecular imaging and electron microscopy. This project also includes a number of tasks especially suitable for undergraduate trainees that will allow them to relate biochemical experiments to visual changes in chromatin structure as observed by electron microscopy and to develop the initial confidence necessary for them to promote their interest and motivation for scientific research.