Cyril S. Anyetei-Anum1, Mary P. Leatham-Jensen2, Geoffrey C. Fox1, B. Rutledge Smith3, Venkat R. Chirasani4, Krzysztof Krajewski4, Brian D. Strahl1,4,5, Jill M. Dowen1,2,3,4,5, A. Gregory Matera1,2,3,5,6, Robert J. Duronio1,2,3,5,6 and Daniel J. McKay1,2,3,6 1Curriculum in Genetics and Molecular Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; 2Integrative Program for Biological and Genome Sciences, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; 3Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; 4Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; 5Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; 6Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA Corresponding author: dmckay1email.unc.edu Abstract

Tight control over cell identity gene expression is necessary for proper adult form and function. The opposing activities of Polycomb and trithorax complexes determine the on/off state of cell identity genes such as the Hox factors. Polycomb group complexes repress target genes, whereas trithorax group complexes are required for their expression. Although trithorax and its orthologs function as methyltransferases specific to histone H3 lysine 4 (H3K4), there is no direct evidence that H3K4 regulates Polycomb group target genes in vivo. Using histone gene replacement in Drosophila, we provide evidence of two key roles for replication-dependent histone H3.2K4 in Polycomb target gene control. First, we found that H3.2K4 mutants mimic H3.2K4me3 in antagonizing methyltransferase activity of the PRC2 Polycomb group complex. Second, we found that H3.2K4 is also required for proper activation of Polycomb targets. We conclude that H3.2K4 directly regulates Polycomb target gene expression.

Received August 3, 2024. Accepted November 5, 2024.