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. 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).