Transcription elongation factor Brd4-P-TEFb accelerates intestinal differentiation-associated SLC2A5 gene expression

Yuko Inamochi,Anup Dey,Akira Nishiyama,Takeo Kubota,Keiko Ozato,Toshinao Goda,Kazuki Mochizuki

doi:10.1016/j.bbrep.2016.05.016

Abstract

BackgroundExpression of the fructose transporter gene SLC2A5 and histone acetylation in the transcribed region are induced by differentiation associated-signals such as glucocorticoids and p44/42 mitogen-activated protein kinase (MAPK) inhibition in small intestinal Caco-2 cells. MethodsWe co-treated with glucocorticoid receptor agonist dexamethasone (Dex) and p44/42 MAPK inhibitor PD98059 (PD) in Caco-2 cells with or without Brd4 small hairpin (sh) RNA expression vector, and the cells were analyzed by qRT-PCR and chromatin immunoprecipitation assays. The small intestine of wild-type mice and Brd4+/− mice during weaning period were analyzed by qRT-PCR. ResultsCo-treatment with Dex and PD increased binding of the bromodomain-containing protein-4 (Brd4)–positive transcriptional elongation factor-b (P-TEFb)–RNA polymerase II complex to acetylated histones in the transcribed region of SLC2A5. Brd4-protein depletion by shRNA revealed that the association of these proteins on the transcribed region of SLC2A5 promoted gene expression in a Brd4-dependent manner. Expression of small-intestine Slc2a5, but not another intestinal gene sucrase-isomaltase, during weaning period, was significantly lower in Brd4+/− mice compared with wild-type mice. ConclusionsBrd4-P-TEFb plays a crucial role in differentiation-associated transcription of SLC2A5 gene in intestinal Caco-2 cells and in the small intestine of mice during weaning period. General significanceHistone acetylation and the transcription elongation factor Brd4 are important for SLC2A5 expression in the small intestine.

Highlights

Stem cells in the crypt of the small intestine rapidly differentiates to absorptive cells in the villus, and start to express many genes related digestion and absorption of nutrients
After 2 days, cells had reached approximately 70% confluence and normal Caco-2 and control/bromodomain-containing protein-4 (Brd4)-shRNA-expressing cells were continuously cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FCS) stripped of glucocorticoids by treatment with charcoal/dextran, with 1 mM Dex and 50 mM PD (p44/42 mitogen-activated protein kinase (MAPK) kinase inhibitor), or vehicle (DMSO) alone for 48 h
We demonstrated that co-treatment with Dex and PD induced SLC2A5 expression as well as acetylation of histones around the SLC2A5 gene in Caco-2 cells

Summary

Introduction

Stem cells in the crypt of the small intestine rapidly differentiates to absorptive cells in the villus, and start to express many genes related digestion and absorption of nutrients. Inhibition of p44/42 MAPK was recently shown to enhance glucocorticoid-mediated SLC2A5 expression in human intestinal Caco-2 cells [6]. Expression of the fructose transporter gene SLC2A5 and histone acetylation in the transcribed region are induced by differentiation associated-signals such as glucocorticoids and p44/42 mitogen-activated protein kinase (MAPK) inhibition in small intestinal Caco-2 cells. Methods: We co-treated with glucocorticoid receptor agonist dexamethasone (Dex) and p44/42 MAPK inhibitor PD98059 (PD) in Caco-2 cells with or without Brd small hairpin (sh) RNA expression vector, and the cells were analyzed by qRT-PCR and chromatin immunoprecipitation assays. Expression of small-intestine Slc2a5, but not another intestinal gene sucrase-isomaltase, during weaning period, was significantly lower in Brd þ /À mice compared with wild-type mice. Conclusions: Brd4-P-TEFb plays a crucial role in differentiation-associated transcription of SLC2A5 gene in intestinal Caco-2 cells and in the small intestine of mice during weaning period. General significance: Histone acetylation and the transcription elongation factor Brd are important for SLC2A5 expression in the small intestine

Methods

Results

Conclusion