chromatin-immunoprecipitation-assay-using-micrococcal-nucleases

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Beckman Research Institute of City of Hope

This study demonstrated that ARID5B mRNA is present in mouse cardiomyocyte HL-1 cells, and that ARID5B siRNA constantly knocked down ARID5B gene expression to the 40% level of control. AMPKα2 protein was elevated in such ARID5B knockdown HL-1 cells, and this was accompanied by an increase in the level of phosphorylated AMPKα. Since AMPKα2 mRNA levels did not change in ARID5B knockdown cells, the stability of AMPKα2 protein was investigated using inhibitors for protein synthesis and proteasomal degradation. Treatment of HL-1 cells with either cycloheximide or MG132 caused an appreciable increase in the amount of AMPKα2 protein in ARID5B knockdown cells, which suggests that knockdown of ARID5B mRNA extends the half-life of AMPKα2 protein in HL-1 cells via yet unidentified mechanisms. As for the expected downstream consequences of AMPKα2 activation, we found thus far that glucose uptake, fatty acid uptake, or fatty acid oxidation remained unchanged in HL-1 cells after knockdown of ARID5B. Further studies are required to understand the mechanisms for ARID5B knockdown and resulting AMPKα2 activation, and also to identify which metabolic pathways are affected by AMPKα2 activation in these cells. In summary, this study provided the foundation for an in vitro cell culture system to study possible roles of ARID5B in cardiomyocytes. 

Knockdown of AT-rich interaction domain (ARID) 5B gene expression induced AMPKα2 activation in cardiac myocytes.

The androgen receptor (AR) is one of the most important and dynamically regulated factors in prostate cancer (PCa) progression. Despite the importance of AR expression regulation, the precise mechanisms are not fully understood. ARID5B, an AT-rich interaction domain DNA-binding motif-containing transcription factor, is expressed higher in primary PCa than normal prostate, and correlated with AR expression. We therefore hypothesized that ARID5B could regulate AR expression.

Takahiro Yamakawa

Department of Molecular and Cellular Biology

Beckman Research Institute of City of Hope

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells