Characterization of the Stoichiometry of HMGA1/DNA Complexes
Miki Watanabe, Shuisong Ni, Amy L Lindenberger, Junho Cho, Stuart L Tinch, Michael A Kennedy*
Identifiers and Pagination:Year: 2013
First Page: 73
Last Page: 81
Publisher ID: TOBIOCJ-7-73
Article History:Received Date: 5/11/2011
Revision Received Date: 9/12/2011
Acceptance Date: 20/12/2011
Electronic publication date: 4/9/2013
Collection year: 2013
open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
High-mobility group A1 (HMGA1) non-histone chromatin architectural transcription factors regulate gene expression, embryogenesis, cell differentiation, and adaptive immune responses by binding DNA and other transcription factors. HMGA1 has also been shown to be highly over-expressed in many human cancers and is considered to be a valuable cancer biomarker. Elevated HMGA1 expression levels also make cancer cells resistant to chemotherapy. Here, HMGA1/DNA complex formation was investigated using electrophoretic mobility shift assays (EMSA). Collectively, the EMSA results indicated that full length HMGA1 mixed with DNA containing three AT-hook binding sites formed four distinct HMGA1/DNA complexes ranging in stoichiometry from 1:2 to 3:1 in HMGA1:DNA ratio. The data indicated that the distribution of complexes with different HMGA1 to DNA stoichiometries depended on the molar ratio of HMGA1 to DNA in solution, which could have significant biological implications given that HMGA1 is highly over-expressed in human cancer cells. The two naturally occurring isoforms of HMGA1, HMGA1a and HMGA1b, the latter containing an 11 amino acid deletion between the first and second AT-hooks, were observed to have slightly different DNA binding profiles. Finally, HMGA1 binding affinity to DNA was found to be influenced by the DNA A:T segment sequence context, with higher specificity be observed in HMGA1 binding to TnAn segments, which have two local minor groove minima on either side of the TpA step, compared to An:Tn segments, which have a single minor groove minimum at the 3' end of the An run, implying AT-hook binding favors narrow minor groove structure.