Come join us on Wednesday, May 18th, 2022 at 4:00 PM in the Pancoe Auditorium to see Alexander Ruthenburg discuss Subjecting the Dogma of Chromatin Marks to Analytical Chemical Biology!
Abstract
Nucleosomes, composed of DNA and histone proteins, represent the fundamental repeating unit of the eukaryotic genome; posttranslational modifications of these histone proteins are thought to influence the activity of the associated genomic regions to regulate cell identity. Yet the quantification of marks and patterns of modifications at any site in the genome remains a significant challenge. We have developed a method to quantify histone marks in the genome, using reconstituted barcoded nucleosomes bearing defined histone marks, and have now extended it subject the perhaps the most well-studied histone mark pattern to quantitative scrutiny. Traditionally, trimethylation of histone H3K4 (H3K4me3) is associated with transcriptional initiation, whereas trimethylation of H3K27 (H3K27me3) is considered transcriptionally repressive. The apparent juxtaposition of these opposing marks, termed “bivalent domains”, was proposed to specifically demarcate of small set transcriptionally-poised lineage-commitment genes that resolve to one constituent modification through differentiation, thereby determining transcriptional status. Since then, many thousands of studies have canonized the bivalency model as a chromatin hallmark of development in many cell types. However, these conclusions are largely based on chromatin immunoprecipitations (ChIP) with significant methodological problems hampering their interpretation. Absent direct quantitative measurements, it has been difficult to evaluate the strength of the bivalency model. Here, we present reICeChIP, a calibrated sequential ChIP method to quantitatively measure nucleosomes with H3K4me3 and H3K27me3 genome-wide, addressing the limitations of prior measurements. With reICeChIP, we profile bivalency through the differentiation paradigm that first established this model: from naïve mouse embryonic stem cells (mESCs) into neuronal progenitor cells (NPCs). Our results cast doubt on every aspect of the bivalency model; in this context, we find that bivalency is widespread rather than restricted to early developmental genes, bivalency does not resolve with differentiation, but increases instead, and is neither sensitive nor specific for identifying poised developmental genes or gene expression status more broadly. Our findings caution against imbuing bivalent domains as meaningful markers of developmentally poised genes.
Bio
Alex Ruthenburg is an Associate Professor in the Departments of Molecular Genetics and Cell Biology as well as Biochemistry and Molecular Biology at the University of Chicago. He received a B.A. in Chemistry at Carleton College, then performed doctoral research in Chemical Biology at Harvard University under the tutelage of Dr. Gregory Verdine, and received post-doctoral training in chromatin biochemistry the lab of Dr. C. David Allis at the Rockefeller University. His research program spans a host of traditional disciplines (discovery biochemistry, chemical biology, biophysics, technology development, quantitative genomics and cell biology) with the goal of developing fundamental mechanistic understanding of epigenetic information systems through three main avenues. 1.) Accurate measurement of the individual chromatin marks, patterns and structures is a crucial prerequisite to deciphering how epigenetic systems work. To address this challenge, they developed internally calibrated chromatin immunoprecipitation (ICeChIP), which uses barcoded semisynthetic nucleosomal calibrants to enable absolute quantification of histone marks and variants and access finer scale chromatin features in cells, revealing many new insights and overturning existing dogma. 2.) A second major effort in the lab is the biochemical discovery of new epigenetic pathways that regulate gene expression, centered on orphaned histone marks and DNA-modifications that have resisted elucidation. 3.) His lab has discovered a new class of noncoding RNA molecules, which display potent enhancer activity and are defined by tight attachment at their site of transcription.