Identification of direct forkhead box O1 targets involved in palmitate-induced apoptosis in clonal insulin-secreting cells using chromatin immunoprecipitation coupled to DNA selection and ligation.
OBJECTIVE
The transcription factor, forkhead box (FOX)O1, is involved in fatty acid-induced apoptosis in pancreatic beta cells, but the precise mechanism is poorly understood. We aimed to identify which direct downstream targets of FOXO1 are involved in palmitate-induced apoptosis in the pancreatic beta cell line MIN6.


METHODS
Chromatin immunoprecipitation (ChIP) coupled to a DNA selection and ligation technique (ChIP-DSL) was used to identify the direct targets of FOXO1. The mRNA level was examined by real-time PCR assay. The ChIP-DSL results were verified using ChIP-PCR and luciferase assay, respectively. The cell apoptosis rate was determined by TUNEL assay and by scoring cells with pycnotic nuclei.


RESULTS
We identified 189 target genes and selected 106 targets for expression analysis in MIN6 cells treated with palmitate. The results showed that six genes were significantly upregulated and four were downregulated. Binding of FOXO1 to the promoters was determined by ChIP-PCR and confirmed by luciferase assay. Among the ten up- and downregulated genes, mRNA expression of A930038C07Rik was significantly decreased and that of Ppa1 was increased in 8-week-old db/db mice. The apoptosis assay showed that overproduction of the protein 'RIKEN cDNA A930038C07' (A930038C07Rik) drastically enhanced palmitate-induced apoptosis, while pyrophosphatase (inorganic) 1 (PPA1) partially protected the cells from apoptosis. Knockdown of PPA1, moreover, significantly increased apoptosis.


CONCLUSIONS
We identified for the first time FOXO1 targets in MIN6 cells treated with palmitate, thus revealing the important roles of A930038C07Rik and PPA1 in palmitate-induced cell apoptosis. These results shed light on the mechanisms of palmitate-induced apoptosis in pancreatic beta cells.
Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, People's Republic of China.
H Y
HY
Lin
Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, People's Republic of China.
Y
Y
Yin
Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, People's Republic of China.
J X
JX
Zhang
Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, People's Republic of China.
H
H
Xuan
Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, People's Republic of China.
Y
Y
Zheng
Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, People's Republic of China.
S S
SS
Zhan
Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, People's Republic of China.
Y X
YX
Zhu
Key Laboratory of Human Functional Genomics of Jiangsu Province, Department of Biochemistry and Molecular Biology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 140 Hanzhong Road, Nanjing, 210029, People's Republic of China. hanxiao@njmu.edu.cn.
X
X
Han
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004247:DNA / Q000235:genetics*; D003924:Diabetes Mellitus, Type 2; D004195:Disease Models, Animal; D015536:Down-Regulation; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors / Q000502:physiology*; D043564:Inorganic Pyrophosphatase / Q000235:genetics / Q000502:physiology; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000473:pathology*; D007340:Insulinoma / Q000473:pathology; D008156:Luciferases; D051379:Mice; D008815:Mice, Inbred Strains; D009414:Nerve Growth Factors / Q000235:genetics / Q000502:physiology; D010168:Palmitates / Q000494:pharmacology*; D010190:Pancreatic Neoplasms / Q000473:pathology; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology; D015854:Up-Regulation
D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; C561304:NDNF protein, mouse; D009414:Nerve Growth Factors; D010168:Palmitates; D014157:Transcription Factors; D004247:DNA; D008156:Luciferases; D043564:Inorganic Pyrophosphatase