Potential Roles of H2A.Z Ubiquitination

Nucleosome is a highly dynamic macromolecular complex that is assembled and remodeled by the replacement of major histone variant H2A.Z, thereby affecting nucleosome structure and stability.Here we established two cell lines stably overexpressing wild-type H2A.Z and mutant H2A.Z with ubiquitination deficiency to explore the potential effects caused by H2A.Z ubiquitination. Intriguingly, H2A.Z ubiquitination was first revealed to enhance the stability of H2A-H2A.Z heterotypic nucleosomes. Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-Seq) further revealed that H2A.Z ubiquitination played a crucial role in the transcriptional regulation. Overall design: (1) we successfully established two cell lines with overexpression of wild-type H2A.Z and mutant H2A.Z with ubiquitination deficiency. (2) The clonogenic ability of established H2A.Z-knockout (H2A.Z-KO) cell line was overexpressed with the H2A.Z (wt) and H2A.Z (mut) vector to evaluate the rescue effects of clonogenic ability. (3) Separation of Mononucleosomes and Identification of H2A-H2A.Z Heterotypic Nucleosomes. (4) H2A.Z Ubiquitination Promotes the Formation and Stability of H2A-H2A.Z Heterotypic Nucleosomes using Co-IP. (5) Characterization of non-UbH2A.Z and UbH2A.Z-interacting Proteins using silver staining and mass spectrometry. (6) Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-Seq) further revealed that H2A.Z ubiquitination played a crucial role in the transcriptional regulation.