Developmental gene regulation depends on the coordinated action of transcription factors, chromatin regulators, and histone modifications that establish and maintain cell-type-specific transcriptional states. In this thesis, I investigate how activating and repressive chromatin factors shape lineage specification in Drosophila melanogaster embryogenesis, with a particular focus on the histone acetyltransferase CREB-binding protein (CBP) and Polycomb-mediated repression. Using genomic, genetic, and single-cell approaches, this work addresses how chromatin states are established during zygotic genome activation (ZGA), how CBP controls distinct steps of transcription, and how active and repressive histone modifications together define developmental trajectories.

The first paper examines the regulation of dorsoventral patterning genes. We demonstrated that RNA polymerase II is recruited to their promoters independently of whether the gene is expressed. Chromatin profiling revealed that H3K27ac closely correlates with gene and enhancer activity, while CBP occupancy at promoters remains invariant. These results identify promoter-proximal pausing as a central regulatory step in early developmental patterning and suggest that inactive CBP can remain associated with silent but poised promoters.

The second paper of the thesis dissects the catalytic and non-catalytic functions of CBP during ZGA. By combining catalytic inactivation and targeted protein degradation, we show that CBP has separable roles in transcriptional elongation and initiation. Its catalytic activity is required for pause release, whereas its non-catalytic activity supports pre-initiation complex stability and RNA polymerase II recruitment. CBP is dispensable for chromatin opening itself and therefore acts downstream of pioneer factors such as Zelda. 

The third paper addresses how CBP activity is regulated across the genome. We show that CBP activity enhances its own recruitment and affects transcription factor binding at regions pioneered by the transcription factor Zelda. Catalytic functions of CBP are shown to be crucial for proper expression of early patterning genes in the Drosophila embryo. Inactive CBP persists at a subset of paused promoters primed by the pioneer factor GAF, and may contribute to Polycomb-associated repression. Together, these results establish CBP as a transcription factor-dependent regulator whose catalytic and non-catalytic functions differently contribute to early development.

The final part of the thesis extends this analysis to later embryogenesis using single-cell nanoCUT&Tag to co-profile H3K27ac and H3K27me3 in individual nuclei. We suggest a new way for epigenetic potential visualization, and found genes where Polycomb repression and gene activity co-occur in one cell lineage. The data distinguishes between two repressive states: active Polycomb-mediated repression and passive chromatin inaccessibility. H3K27me3 associates with genes exposed to activation cues rather than with all silent developmental genes. We further corroborate this conclusion by showing preferential upregulation of lineage-matching genes when H3K27me3 was partially depleted in a mesoderm lineage. 

Together, the studies in this thesis show that developmental epigenetic regulation occurs by the controlled and context-dependent action of co-activators, co-repressors, and pioneer factors that together ensure lineage commitment and maintain robust boundaries between cell identities.