Aberrant active cis-regulatory elements associated with downregulation of RET finger protein overcome chemoresistance in glioblastoma

The aim of this study is to investigate whether RET finger protein (RFP)-knockdown (KD) causes chemosensitivity in temozolomide-resistant gliomas and whether the disruption of the RFP-HDAC1 complex can disorder active cis-regulatory elements controlled by H3K27ac. [ChIP-seq] U87-MGMT and T98 cells plated in a 100mm-dish and transfected with siRFP or scramble siRNA (siCont) were cross-linked in 1% paraformaldehyde for 8 min, collected with a cell scraper, and washed three times with PBS. Chromatin was sheared with a Covaris sonicator (adaptive focused acoustics; AFA technology, Melaka, Malaysia). H3K27ac antibody (Active Motif, Tokyo, Japan), rabbit polyclonal anti-RFP antibody and anti-HDAC1 antibody (Cell Signaling TECHNOLOGY, MA, USA) were used for immunoprecipitation of sheared chromatin. Protein G dynabeads (Life Technologies, Carlsbad, CA, USA) were used to pull down the chromatin complexes. Complexes were digested with proteinase K, reverse cross-linked, and purified using Agencourt AMPure XP beads (BECKMAN COULTER, Brea, CA, USA). Fragmented ChIP DNA (1ng to 5 ng) was used to prepare DNA libraries for paired-end sequencing on the Miseq or Hiseq2000 (Illumina, San Diego, CA, USA) system, using the NEBNext Ultra II DNA library Prep kit for Illumina (New England Biolabs, Ipswich, MA, USA). [RNA-seq] TRIzol Reagent (Life Technologies) was used to extract RNA from U87-MGMT and T98 cells transfected with siRFP or siCont from a 24-well plate, in triplicate. The quality and quantity of RNA were evaluated using NanoDrop 1000 spectrophotometer and Bioanalyzer 2100 (Agilent Technologies, CA, USA). In this study, all the RNA quality (RIN) scores were higher than 9.7. cDNA libraries for paired-end sequencing on the Miseq or Hiseq2000 (Illumina) system were prepared using the NEBNext Ultra RNA library Prep Kit for Illumina (New England Biolabs), with the NEBNext Poly(A) mRNA Magnetic Isolation Module.