PPM: Functional asymmetries of trans-histone and histone-DNA crosstalk in development

Understanding how cells control gene expression is a central challenge in biology. Genes are packaged in chromatin and their expression is controlled by chemical modifications on histone proteins and DNA in nucleosomes, the fundamental building blocks of chromatin. This project examines how complex combinations of histone or DNA modifications that occur asymmetrically within individual nucleosomes work together to determine gene expression during cell type specification. The molecular rules for how these patterns are established and interpreted remain poorly understood because of longstanding technical barriers. This project will use new experimental tools that allow precise control of the geometry of histone and DNA modifications in individual nucleosomes, and thereby enable fundamental research into how different combinations of these modifications elicit distinct outcomes. The findings will advance understanding of epigenetic regulation, developmental biology, and genome organization that constitute foundational knowledge to advance biotechnology and biomedicine. Broader impacts include interdisciplinary training for students and postdoctoral researchers in structural biology and epigenetics, outreach activities for high school biotechnology education programs, and public engagement efforts aimed at increasing understanding of gene regulation. Proper cellular differentiation depends on precisely patterned histone and DNA modifications that regulate developmental gene expression. Mounting evidence indicates that these modifications are often deposited asymmetrically within single nucleosomes, with distinct marks positioned on opposite histone tails. This project will define how these asymmetries are generated and interpreted through trans-histone and histone-DNA crosstalk. The investigators will build on their technological breakthroughs to assemble fully asymmetric nucleosomes with precisely defined histone and DNA geometries. These asymmetric nucleosomes will be used to (i) establish the geometric rules by which H2BK120-Ub, H2BK34-Ub, and H4K16ac direct Dot1L methyltransferase to methylate H3K79 on one side of the nucleosome or the other, (ii) determine how H2BK120-Ub-dependent conformational switching biases MLL2 methyltransferase toward a specific H3 tail, and (iii) define whether H3K4 and H3K36 methylation direct DNMT3A2/3B3 methyltransferase to methylate DNA in a directional manner. In addition, the project will determine structures of the active complex formed between the nucleosome and DNMT3A2/3B3. This work integrates asymmetric chromatin engineering biochemistry and cryo-electron microscopy to determine how modification asymmetries within nucleosomes modulate histone and DNA crosstalk interactions. By revealing how the spatial organization of chromatin modifications encodes regulatory information, this research will establish fundamental principles linking the geometry of epigenetic modifications to gene regulation. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria. NSF Award ID: 2603280 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Evan Worden | Institution: Van Andel Research Institute, Grand Rapids, MI | Award Amount: $1,500,000 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2603280 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2603280.html