Nascent CUT&Tag captures transcription factor binding after chromatin duplication

DNA replication strips off all chromatin proteins, which must be reassembled behind the replication fork. To track chromatin reassembly on newly synthesized DNA, we developed Nascent CUT&Tag, a chromatin profiling method that uses antibody-targeted in situ tagmentation to directly measure transcription factor binding on nascent chromatin. Using Nascent CUT&Tag, we tracked the recovery of GAGA factor (GAF) in Drosophila Kc cells. We find that GAF is displaced from chromatin during DNA replication and shows a broad spectrum of recovery times, ranging from minutes to hours. Early recovering peaks are characterized by shorter GAF motifs and are associated with functions related to cell cycle progression. Conversely, late recovering peaks are characterized by longer, degenerate GAF motifs and are associated with developmental functions. We also show that GAF recovery on newly synthesized DNA requires chromatin remodeling by Brahma Associated Factor (BAF), implying that nucleosome eviction is critical to fully reestablish GAF binding.