Transcriptional Regulation by ATOH1 and its Target SPDEF in the Intestine.
OBJECTIVE
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.
Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, Texas.
Yuan-Hung
YH
Lo
Division of Pediatric Urology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
Eunah
E
Chung
Jan and Dan Duncan Neurological Research Institute, Houston, Texas.
Zhaohui
Z
Li
Jan and Dan Duncan Neurological Research Institute, Houston, Texas.
Ying-Wooi
YW
Wan
Department of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
Maxime M
MM
Mahe
Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, Texas.
Min-Shan
MS
Chen
Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
Taeko K
TK
Noah
Graduate Program in Molecular Developmental Biology, University of Cincinnati, Cincinnati, Cincinnati, Ohio.
Kristin N
KN
Bell
Jan and Dan Duncan Neurological Research Institute, Houston, Texas.
Hari Krishna
HK
Yalamanchili
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas.
Tiemo J
TJ
Klisch
Department of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
Zhandong
Z
Liu
Division of Pediatric Urology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio; Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.
Joo-Seop
JS
Park
Integrative Molecular and Biomedical Sciences Graduate Program, Baylor College of Medicine, Houston, Texas; Division of Medicine, Section of Gastroenterology and Hepatology, Baylor College of Medicine, Houston, Texas.
Noah F
NF
Shroyer
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