During animal embryonic development, gene expression must be tightly regulated in space and time to generate the plethora of cell types that compose animal tissues. Gene regulation occurs largely at the transcriptional level through cis-regulatory elements (CREs), including promoters and enhancers, where transcription factors (TFs) bind and stimulate transcription. Pioneer TFs can bind chromatin in a closed configuration and promote chromatin accessibility through the displacement of nucleosomes. They can alter the expression of many genes and promote differentiation processes, being therefore considered as lineage-determining TFs. How these factors interact among them at developmental branches is key to understand cell decision making.
Regulation of developmental genes by CREs commonly imply physical enhancer-promoter (E-P) interactions that are established by looping mechanisms. In this sense, the genome is organized at the 3D level leading to the formation of topologically associating domains (TADs). These have been proposed to serve as structural scaffolds for regulatory landscapes, within which E-P interactions would operate. However, the link between TADs and gene expression remains controversial and we know very little about the dynamics of TADs, E-P interactions and gene expression during cell fate decisions in differentiation and development.
Figure 1 - a, Genomic tracks showing ChIP-seq of p63, H3K27ac, H3K4me1 and H3K4me3, and ATAC-seq experiments in zebrafish embryos at 80% epiboly and 24 hpf stages. CREs pioneered by p63 are highlighted. b, H3K4me3 HiChIP experiments in wild-type and ctcf mutant embryos at 48 hpf. ChIP-seq of CTCF, RNA-seq and computationally called HiChIP loops are shown below.
My goal is to shed light into the relationship between the 3D structure of the genome and gene regulation during cell fate decisions, as well as the role that lineage-determining TFs play in this connection. Using zebrafish as the main model system, we combine cutting-edge epigenomic, transcriptomic, chromosome conformation capture techniques (ChIP-seq, ATAC-seq, RNA-seq, HiC, HiChIP, UMI-4C) and single-cell technologies with functional studies using CRISPR-Cas systems, to understand these processes in an in vivo context. Using this approach, in recent works we have generated knockout mutants of pioneer TFs and architectural proteins, including the master regulator of epidermal development p63 and the zinc-finger protein CTCF, respectively, to elucidate gene regulation mechanisms and the link between chromatin structure and function.