Polycistronic Coexpression and Copurification of the Human MLL MWRA Histone Methyltransferase Complex

Open Access
Author:
Santivasi, Wil Leonard
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Song Tan, Thesis Supervisor
  • Chen Pei David Tu, Honors Advisor
  • Wendy Hanna Rose, Faculty Reader
Keywords:
  • epigenetics
  • histone methyltransferase
  • polycistronic coexpression
  • gene regulation
  • MLL
  • MWRA
Abstract:
Eukaryotic organisms have highly-structured and complex gene regulatory pathways. Histone methyltransferases such as the MLL complex play a prominent role in the transcriptional activation of genes via mono-, di-, and tri-methylation of lysine 4 on the N-terminal tail of histone H3. These modifications allow for the regulation of genes downstream of the methylated nucleosome. The MWRA complex is the smallest catalytically active dimethylase complex of the MLL complex, and is composed of MLL1, WDR5, RbBP5, and Ash2L. Previously, the MWRA complex was expressed as discrete subunits and reconstituted in vitro. Using a system developed by Dr. Song Tan, the four subunits of the MWRA subcomplex were successfully subcloned into a polycistronic expression vector and expressed as a complex in E. coli. The complex was copurified using Talon® metal affinity purification and SourceTM S cation-exchange chromatography, yielding milligram quantities. Single-turnover kinetic experiments performed by Dr. Michael Cosgrove and Dr. Anamika Patel at Syracuse University demonstrated that bacterially coexpressed MWRA subcomplex displays the same specificity and activity as MWRA subcomplex that was reconstituted in vitro. The bacterially coexpressed MLL MWRA complex will facilitate biophysical characterizations including crystallization trials of this chromatin complex.