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. 2013 Sep 4:7:73-81.
doi: 10.2174/1874091X01307010073. eCollection 2013.

Characterization of the Stoichiometry of HMGA1/DNA Complexes

Miki Watanabe  1 , Shuisong Ni, Amy L Lindenberger, Junho Cho, Stuart L Tinch, Michael A Kennedy
Affiliations

Characterization of the Stoichiometry of HMGA1/DNA Complexes

Miki Watanabe et al. Open Biochem J. .

Abstract

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.

Keywords: AT-hook; DNA.; EMSA; HMGA1; architectural transcription factor; enhanceosome.

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Figures

Fig. (1)
Fig. (1)
Schematic representation of the sequences of HMGA1 isoforms and deletion mutants used in this study.
Fig. (2)
Fig. (2)
Some potential HMGA1/DNA complexes that might form between full length HMGA1 containing three AT-hooks and DNA containing three AT-hook binding sites: (a) HMGA1:DNA (2:1), (b) HMGA1:DNA (3:1), (c) HMGA1:DNA (1:1), (d) HMGA1:DNA (1:2), (e) HMGA1:DNA (1:3).
Fig. (3)
Fig. (3)
EMSA of ΔN20C17-HMGA1a DNA binding activity. (A, B) EMSA of ΔN20C17-HMGA1a with A19-DNA, (C, D) A5T5-DNA, (E, F) with A3T3-DNA, (G, H) A9T9-DNA and T9A9-DNA, (I, J) A18-DNA and A5-DNA. All gels were stained either with ethidium bromide (left) or Coomassie blue (right). The molar ratio of ΔN20C17-HMGA1a to DNA in each lane is indicated below the gel on the left.
Fig. (4)
Fig. (4)
EMSA of ΔN20C17-HMGA1b with A19-DNA (A, B), and A5T5-DNA (C). Amino acid sequence of HMGA1a and HMGA1b deletion mutants (D). The AT-hook sequences are underlined and labeled. In ΔN20C17-HMGA1a, the insert sequence is indicated, and in ΔN20C17-HMGA1b, the deletion of the insertion region (boxed) results in a lysine residue (arrow) being to close to first AT-hook (underlined). The molar ratio of ΔN20C17-HMGA1b to DNA in each lane is indicated below the gel on the left.
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