Bo Hyun "Cindy" Kim

Topic

Characterization of ReNCell for studying chromatin associated proteins MeCP2 and histone H1

Department of Biochemistry and Microbiology

Date & location

• Wednesday, July 27, 2022
• 10:00 A.M.
• Virtual Defence

Reviewers

Supervisory Committee

• Dr. Juan Ausio, Department of Biochemistry and Microbiology, University of Victoria (Supervisor)
• Dr. Chris Nelson, Department of Biochemistry and Microbiology, UVic (Member)
• Dr. Kerry Delaney, Department of Biology, UVic (Outside Member)

External Examiner

• Dr. Raad Nashmi, Department of Biology, UVic

Chair of Oral Examination

• Dr. Yu-Ting Chen, Department of Mathematics and Statistics, UVic

Abstract

Methyl-CpG binding protein 2 (MeCP2) and histone H1 are important chromatin associated proteins. Both exhibit their own extent of complexity as MeCP2 is an intrinsically disordered protein (IDP) that interacts with many different partners involved in several cellular processes and histone H1 consists of 11 different subtypes each of them associated with different PTMs. An interesting avenue of studying these proteins is in the neuron where MeCP2 is very abundant and histone H1 level is half that observed in other somatic tissues. Several reports in the past have proposed that this lower level of histone H1 is due to the abundance of MeCP2 which displaces histone H1. However, this notion has been debated and there is no clear consensus on this. In an attempt to study this controversy, a cell model system ReNCell WT and MeCP2-KO was used that can be induced to neuronal differentiation. The protein levels, transcript levels and localization of histone H1 subtypes in these cells were analyzed using HPLC, RT-qPCR and immunofluorescence, respectively. The results show that ReNCell WT and MeCP2-KO do not exhibit significant differences in their relative amount of histone H1 protein and transcript level neither at the proliferative nor at the later differentiated stages. However, HPLC analyses show that the histone H1 subtypes of these two cell types exhibit significant elution differences probably resulting from differences in their PTM content. Immunofluorescence analyses show that ReNCell WT differentiation proceeds normally while the lack of MeCP2 hinders differentiation. Interestingly, there is a significant difference in the nuclear area of these two cells at 8 DIV. This study provides important preliminary data for future research in MeCP2 and histone H1 using this cell model system and show that MeCP2 may have a bearing on histone H1 PTMs.