Role of BEND3 in transcription repression
We have uncovered that BEND3 is a heterochromatin-associated protein that functions as a transcription repressor. Expression of BEND3 correlates with the state of pluripotency, with a significant decline in BEND3 levels in differentiated cells. Cells lacking BEND3 show defects in cell cycle progression with failure to enter S-phase. BEND3 affects the transcription of cellular genes that modulate chromatin organization and cellular differentiation and that downregulation of BEND3 is critical for cellular differentiation. Overall design: Chromatin immunoprecipitation was performed using chromatin isolated from NTera2 cell line as explained in Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):8338-43. doi: 10.1073/pnas.1424705112. Anti-BEND3 (S3242-2) and rabbit IgG antibodies were used to pull the chromatin. The ChIPseq libraries were prepared with the Hyper Library Construction Kit from Kapa Biosystems with two modifications: adaptors were diluted 1:20 and adaptored DNA were amplified for 10 cycles. The libraries were quantitated by PCR and sequenced on one lane for 101 cycles from one end of the fragments on a HiSeq 4000 using a HiSeq 4000 sequencing kit version 1. Fasta files were generated and demultiplexed with the bcl2fast v2.17.1.14 Conversion Software (Illumina). Adaptors have been trimmed from the 3'-end of the reads. Construction of ChIP-Seq libraries and sequencing on the HiSeq 4000 was carried out at the Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign (UIUC).