Deciphering the Molecular Mechanisms of H2BE76K Mutation in Cancer
DescriptionCancer arises when cells begin to grow and divide in an uncontrolled manner. Recent genomics studies have shown that somatic copy number alterations and DNA mutations in oncogenes and tumor suppressor genes are key drivers of tumorigenesis. Interestingly, a significant number of mutations in genes that encode chromatin modifiers and epigenetic factors have also been identified in many types of carcinomas, highlighting the crucial role of epigenetics in cancer development.We have previously elucidated the role of a driver mutation; histone H3 K27-to-M missense mutation in pediatric brain cancer. The H3K27M mutant histone exhibits a dominant negative effect which inhibits the di- and tri-methylation of H3 at lysine 27, leading to dramatic changes in gene expression including the epigenetic silencing of tumor suppressorp16INK4a. To explore whether additional histone mutations might play a role in other diseases, we analyzed the “The Cancer Genome Atlas” (TCGA) and “cBioPortal” databases and have identified a histone H2B E76-to-K missense mutation in some cancers including breast and lung carcinomas. Glutamic acid 76 of H2B (H2BE76) is evolutionarily conserved from yeast to human, and structural analysis of human nucleosome revealed that the H2B E76 residue is required for its direct inter-histone interactions with histone H4. Using a transient transfection approach in 293T our co-immunoprecipitation assay confirmed that the H2BE76K mutation weakens the interaction between H2B with histone H4. Intriguingly, we also showed that this mutation dramatically increased the H2B binding with histone chaperones NAP1L1/2 but reduced the interaction with SPT16. To express the mutant histone at physiological level and mimic the patient situations, we used CRISPRCas9 approach and knocked in the H2BE76K mutation in breast cancer cell lines MDA-MB-231 and MCF7. Our cell fractionation assay and mononucleosome IP revealed that the E76K mutation alters the nuclear import and deposition of H2B to the chromatin. In addition, CRISPR knockin lines expressing the H2BE76K have increased colony formation and cell migration abilities compared to isogenic wildtype CRISPR controls. More importantly, our RNA-seq data uncovered 300 differentially expressed genes in the H2BE76K mutant cells. Gene ontology analysis demonstrated that many of these genes are implicated in cancer pathways, further supporting the idea that the H2BE76K mutation might play a crucial role in cancer. Interestingly, we found that cells expressing the H2BE76K mutant histones are sensitive to DNA damage agents and inhibitors against DNA replication in our yeast model. This data revealed that in addition to altering the expression of genes in cancer pathways, the H2BE76K mutation might also lead to genomic instability and to possibly further drive cancer progression.We hypothesize that H2BE76K is one of the driver mutations of cancer by altering the biological activities on the chromatin: DNA replication, transcription and DNA damage repair. In this proposal we aim to define how this mutation affects chromatin functions and decipher the mechanisms of H2BE76K mutation in cancer. Our objectives are to 1) Elucidate the effect of H2BE76K on H2B nucleosome position and transcription, 2) Investigate the effect of H2BE76K in DNA damage repair and DNA replication 3) Decipher the genes and pathways that are responsible for the oncogenic properties in H2BE76K expressing cells.By completing this project we will uncover the clinical relevance and molecular mechanisms of H2BE76K mutation in cancer as well as making a significant breakthrough in the field by linking genetic mutation and epigenetic gene regulation in cancer development. Knowledge gained from this work will be translated to the development of new strategies in treating patients with this missense mutation.
|Effective start/end date||1/09/19 → …|