The homeobox transcription factor Hlxb9 controls pancreatic β-cell differentiation during the early and late stages of endocrine pancreas development. Recently, increased expression of Hlxb9 was observed in β-cell tumors associated with multiple endocrine neoplasia type 1 (MEN1), a tumor syndrome caused by germline mutation in the MEN1 tumor suppressor gene encoding menin. In MIN6 cells, a mouse pancreatic β-cell line, overexpression of Hlxb9 has been shown to cause apoptosis in a menin-dependent manner. Although Hlxb9 is known to act as a transcriptional repressor, its DNA-binding site at its target genes in pancreatic β-cells has not been elucidated. To identify targets of Hlxb9 and genomic binding sites in β-cells (MIN6 cells) we used chromatin immunoprecipitation and deep sequencing (ChIP-seq) followed by bioinformatics analysis of the ChIP-seq data. The Hlxb9 specific ChIP-seq libraries and corresponding input controls were sequenced on the Illumina Hi Seq 2000. The ChIP-seq reads were subjected to peak calling tools, and upon stringent filtering 2569 peaks were obtained. After assigning chromosomal positions to the peaks, 24 statistically significant (FDR-P<0.05) targets were considered as potential Hlxb9-binding regions for further analysis. The ChIP-seq targets were validated by qPCR. We identified a potential Hlxb9 consensus-binding motif ‘NCNNNNGCTCCTGN’ de novo using the Genomatix Genome Analyzer suite that appeared to be common in most of the targets (Re-value: 1.88). Further analysis revealed that this motif was similar to binding sequences of the following proteins: Tcfe2a (involved in brain development), Ascl2 (involved in nervous system), REI1 (involved in mitotic signal network), ZFp691 (zinc finger protein), myf (involved in mitosis), and Bcl6b (involved in apoptosis). Gene Ontology analysis of the identified targets showed that 18.3% were associated with the Wnt signaling pathway, 27.3% with cell proliferation, 54.5% were involved in cellular response to stimulus, 13.6% in MAPK cascade and 31.8% in negative regulation of biological processes. Our genome-wide analysis of Hlxb9 target genes demonstrates the presence of a potential Hlxb9-binding site in target genes associated with important processes relevant for cellular differentiation and neoplasia. Further studies investigating these targets for menin-dependent and menin-independent actions of Hlxb9 will provide insights into the role of menin-Hlxb9 interaction in normal physiology and in the development of pancreatic β-cell tumors.
Citation Format: Shruti S. Desai, Sita D. Modali, Weiping Chen, Vaishali I. Parekh, Sunita K. Agarwal. ChIP-seq based identification of genes regulated by a pancreatic β-cell differentiation factor, Hlxb9, that is dysregulated in β-cell tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-118. doi:10.1158/1538-7445.AM2013-LB-118