Transcriptional Regulation by ATOH1 and its Target SPDEF in the Intestine

Abstract

Background & AimsThe 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.MethodsWe 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.ResultsBy 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.ConclusionsThis 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.

Drawing Details into the Lineage Tree

“…Although the genetic functions of these transcription factors are well documented, their regulatory mechanisms are less clear. In this issue, Lo et al 1 leverage a series of mouse genetic tools and cell isolation and sorting techniques to explore the epigenomic regulatory underpinnings of how Atoh1 and Spdef mediate secretory cell fate.…”

“…To look under the hood at how ATOH1 mediates lineage fate, Lo et al 1 took advantage of a green fluorescent protein-tagged Atoh1 locus, which provides both an epitope tag for chromatin immunoprecipitation (ChIP) and a fluorescent marker for cell isolation. The model provides a robust signal in goblet and Paneth cells, and a detectable signal in enteroendocrine cells.…”

“…They also use Atoh1 flox/flox mice and the Cre ERT2 driven by the villin promoter to conditionally inactivate Atoh1 in intestinal epithelia. These genetic tools allowed Lo et al 1 to compare the transcriptional profiles of whole crypts, GFP-expressing ATOH1-expressing secretory cells isolated from the crypts, or whole crypts that received conditional inactivation of ATOH1. Subsequent analysis confirmed the expected depletion of secretory lineage genes in ATOH1-deficient cells, and the enrichment of secretory genes in GFP + cells.…”

“…Subsequent analysis confirmed the expected depletion of secretory lineage genes in ATOH1-deficient cells, and the enrichment of secretory genes in GFP + cells. More interestingly, Lo et al 1 took the next step to identify which of these regulated transcripts had loci bound by ATOH1–GFP using ChIP-sequencing in the ileum and colon. ATOH1 genomic binding events showed strong concordance in the ileum and colon, were enriched at transcriptional start sites, and at regions showing the active chromatin mark H3K27ac.…”

“…It also will help refine sets of genes specific to secretory lineages. In addition, using an impressive knockout-plus-overexpression model, Lo et al 1 began to describe the co-regulatory relationship between ATOH1 and its downstream partner SPDEF. They observed that SPDEF overexpression can cooperate with ATOH1 to amplify its target gene expression levels, but cannot stimulate transcription of these co-targets in the absence of ATOH1.…”