Background & aims: The transcription factor atonal homolog 1 (ATOH1) controls the fate of intestinal progenitors downstream of the Notch signaling pathway. Intestinal progenitors that escape Notch activation express high levels of ATOH1 and commit to a secretory lineage fate, implicating ATOH1 as a gatekeeper for differentiation of intestinal epithelial cells. Although some transcription factors downstream of ATOH1, such as SPDEF, have been identified to specify differentiation and maturation of specific cell types, the bona fide transcriptional targets of ATOH1 still largely are unknown. Here, we aimed to identify ATOH1 targets and to identify transcription factors that are likely to co-regulate gene expression with ATOH1.
Methods: We used a combination of chromatin immunoprecipitation and messenger RNA-based high-throughput sequencing (ChIP-seq and RNA-seq), together with cell sorting and transgenic mice, to identify direct targets of ATOH1, and establish the epistatic relationship between ATOH1 and SPDEF.
Results: By using unbiased genome-wide approaches, we identified more than 700 genes as ATOH1 transcriptional targets in adult small intestine and colon. Ontology analysis indicated that ATOH1 directly regulates genes involved in specification and function of secretory cells. De novo motif analysis of ATOH1 targets identified SPDEF as a putative transcriptional co-regulator of ATOH1. Functional epistasis experiments in transgenic mice show that SPDEF amplifies ATOH1-dependent transcription but cannot independently initiate transcription of ATOH1 target genes.
Conclusions: This study unveils the direct targets of ATOH1 in the adult intestines and illuminates the transcriptional events that initiate the specification and function of intestinal secretory lineages.
Keywords: ATOH1; ATOH1, atonal homolog 1; Atoh1Flag; Atoh1GFP; CRC, colorectal cancer; ChIP, chromatin immunoprecipitation; ChIP-seq, chromatin immunoprecipitation sequencing; DBZ, dibenzazepine; FACS, fluorescence-activated cell sorting; FDR, false-discovery rate; GFP, green fluorescent protein; GO, gene ontology; Gfi1, growth factor independent 1; ISC, intestinal stem cell; Intestinal Epithelium; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; QES, Q-enrichment-score; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; SPDEF; Spdef, SAM pointed domain containing ETS transcription factor; TRE-Spdef; TSS, transcription start site; Transcription; Villin-creER; mRNA, messenger RNA.