Cis acting variation is common, can propagates across multiple regulatory layers, but is often buffered in developmental programs

Precise patterns of gene expression are driven by interactions between transcription factors, regulatory DNA sequence, and chromatin. How DNA mutations affecting any one of these regulatory ‘layers’ is buffered or propagated to gene expression remains unclear. To address this, we quantified allele-specific changes in chromatin accessibility, histone modifications, and gene expression in F1 embryos generated from eight Drosophila crosses, at three embryonic stages, yielding a comprehensive dataset of 240 samples spanning multiple regulatory layers. Genetic variation in cis -regulatory elements is common, highly heritable, and surprisingly consistent in its effects across embryonic stages. Much of this variation does not propagate to gene expression. When it does, it acts through H3K4me3 or alternatively through chromatin accessibility and H3K27ac. The magnitude and evolutionary impact of mutations is influenced by a genes’ regulatory complexity ( i . e . enhancer number), with transcription factors being most robust to cis -acting, and most influenced by trans -acting, variation. Overall, the impact of genetic variation on regulatory phenotypes appears context-dependent even within the constraints of embryogenesis.