Abstract 3596: KLLN protects genomic stability by maintaining H3K9 trimethylation (H3K9me3) at the pericentric heterochromatin

Abstract

Abstract Dysregulation in the maintenance of chromatin organization resulting in genomic instability is a major driving force for inappropriate development and carcinogenesis. Tumor suppressor genes play a critical role in this regulation through the maintenance of epigenetic marks. Our study focuses on the role of KLLN in the maintenance of pericentric H3K9 trimethylation (H3K9me3) and genomic stability. Germline hypermethylation of KLLN resulting in decreased KLLN expression has been linked to Cowden cancer-predisposition syndrome (CS) in PTEN mutation negative patients. KLLN is a tumor suppressor gene necessary for p53-mediated apoptosis. The protein mediates S-phase arrest and is known to have DNA binding ability. Here, we first used chromatin immunoprecipitation-based sequencing (ChIP-seq) to investigate regions of KLLN binding on the genome and compared it to regions of H3K9me3 enrichment. We used H3K9 specific histone methyltransferase (HMT) activity assay and immunoblotting to measure levels of H3K9me3. Immunostaining was used to study the localization of KLLN, and micronuclei frequency and aberrations in chromosome number was used to assess chromosomal instability. Analysis of ChIP-seq shows enrichment of KLLN binding in regions of H3K9me3. Overexpression of KLLN using plasmid-based transfection correlates with increased H3K9 methyltransferase activity and increased global H3K9me3, while loss of KLLN expression through siRNA-mediated knockdown had an opposite effect. We also established that KLLN localizes to pericentric regions, and loss of KLLN results in dysregulation of pericentric heterochromatin, with consequent chromosomal instability manifested by increased micronuclei frequency and numerical chromosomal aberrations. KLLN regulation of H3K9me3 could be correlated with its interaction with deleted in breast cancer (DBC1). DBC1 is a known inhibitor of SUV39H1, a H3K9 specific histone methyltransferase responsible for maintenance of pericentric heterochromatin. We hypothesize that KLLN sequesters DBC1 through their interaction, thereby abrogating DBC1 inhibition of SUV39H1, resulting in maintenance of pericentric heterochromatin and genomic stability. Our study therefore suggests a critical role for KLLN in the deterrence of tumor initiation and progression. SIGNIFICANCE: Our study demonstrates a clear role for KLLN in maintenance of chromatin organization thereby maintaining genomic stability, which begins to uncover the basis of KLLN as a tumor suppressor and as a susceptibility gene for inherited cancers. Citation Format: Madhav Sankunny, Emily Nizialek, Farshad Niazi, Charis Eng. KLLN protects genomic stability by maintaining H3K9 trimethylation (H3K9me3) at the pericentric heterochromatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3596.