The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

Research in our laboratory is focused on the following basic questions: What folding motifs underlie the large-scale chromatin folding of 10 and 30 nm chromatin fibers into interphase and mitotic chromosomes? What impact does this large-scale chromatin folding have on transcriptional regulation? How is this large-scale chromatin folding regulated? To address these questions, our laboratory is using a combination of molecular, cellular, genetic, reverse genetic, and structural experimental approaches.

During the past several years molecular and cell biology has experienced a major paradigm shift, in which regulation of local chromatin structure is now considered inseparable from regulation of transcription. Our working hypothesis is that modifications in large-scale chromatin structure will also prove to be fundamental in gene regulation. Experimental difficulties in visualizing large-scale chromatin structure currently represent a major experimental barrier to testing this hypothesis.

Our laboratory is pioneering tools for visualizing and manipulating large-scale chromatin structure. Over the past several years, we have developed a method for labeling specific chromosome sites using the lac repressor/operator interaction. We have used this method for in vivo visualization of the dynamics of individual gene loci or chromosome regions. We are working now to directly visualize the ultrastructure of specific genes.

Using this approach, our laboratory has verified the folding of 10 and 30 nm chromatin fibers into larger, ~100 nm "chromonema" fibers in mammalian cells. We have demonstrated that targeting transcriptional activators to condensed, heterochromatic chromosome regions produces a striking, large-scale chromatin decondensation involving the extension or uncoiling of these chromonema fibers. Surprisingly, targeting of transcriptional activators also can lead to a change in nuclear positioning of chromosome loci. Experiments are now in progress in to identify the molecular mechanisms underlying these activator induced changes in large-scale chromatin structure and nuclear positioning. A fundamental question is whether large-scale chromatin remodeling is carried out by novel proteins distinct from known proteins which modify local chromatin structure. Related experiments are now in progress using Drosophila melanogaster as a model organism, amendable to genetic and reverse genetic analysis.

To dissect folding of these large-scale fibers within interphase and mitotic chromosomes we have engineered chromosome regions of defined size and interphase folding patterns. Based on preliminary results, we believe these engineered chromosome regions arrest at distinct large-scale unfolding intermediates during G1 chromosome decondensation. EM reconstructions of labeled chromosome regions are in progress to reveal the underlying folding motifs underlying interphase and mitotic chromosome architecture. In vivo light microscopy has revealed a precise choreography of chromosome structural changes and nuclear positioning through the interphase cell cycle. Specific decondensation events immediately precede initiation of DNA replication and movement of chromosome arms within the nucleus. Our goal is to link these structural events with the biochemical mechanisms underlying these processes.