Characterization of DNA binding property of the HIV-1 host factor and tumor suppressor protein Integrase Interactor 1 (INI1/hSNF5).

Integrase Interactor 1 (INI1/hSNF5) is a component of the hSWI/SNF chromatin remodeling complex. The INI1 gene is either deleted or mutated in rhabdoid cancers like ATRT (Atypical terratoid and rhabdoid tumor). INI1 is also a host factor for HIV-1 replication. INI1 binds DNA non-specifically. However, the mechanism of DNA binding and its biological role are unknown. From agarose gel retardation assay (AGRA), Ni-NTA pull-down and atomic force microscopy (AFM) studies we show that amino acids 105-183 of INI1 comprise the minimal DNA binding domain (DBD). The INI1 DBD is absent in plants and in yeast SNF5. It is present in Caenorhabditis elegans SNF5, Drosophila melanogaster homologue SNR1 and is a highly conserved domain in vertebrates. The DNA binding property of this domain in SNR1, that is only 58% identical to INI1/hSNF5, is conserved. Analytical ultracentrifugation studies of INI1 DBD and INI1 DBD:DNA complexes at different concentrations show that the DBD exists as a monomer at low protein concentration and two molecules of monomer binds one molecule of DNA. At high protein concentration, it exists as a dimer and binds two DNA molecules. Furthermore, isothermal calorimetry (ITC) experiments demonstrate that the DBD monomer binds DNA with a stoichiometry (N) of ∼0.5 and Kd  = 0.94 µM whereas the DBD dimer binds two DNA molecules sequentially with K'd1 = 222 µM and K'd2 = 1.16 µM. Monomeric DBD binding to DNA is enthalpy driven (ΔH = -29.9 KJ/mole). Dimeric DBD binding to DNA is sequential with the first binding event driven by positive entropy (ΔH'1 = 115.7 KJ/mole, TΔS'1 = 136.8 KJ/mole) and the second binding event driven by negative enthalpy (ΔH'2 = -106.3 KJ/mole, TΔS'2 = -75.7 KJ/mole). Our model for INI1 DBD binding to DNA provides new insights into the mechanism of DNA binding by INI1

Determination of stoichiometries, binding constants and thermodynamic parameters of INI1 DBD-DNA binding from isothermal calorimetry studies.

<p>N = stoichiometry, ± = SE (standard error).</p

Phylogenetic conservation of the region corresponding to amino acids 105-183 of INI1.

<p>(A) Scheme showing presence and absence of different domains in INI1 from yeast to humans. (B) Neighbor-joining tree using MEGA 2.0 software showing conservation of the region corresponding to amino acids 105-183 of INI1. Percentage identity of the region in different species is shown in parenthesis.</p

DNA binding studies of INI1 DBD.

<p>(A) Coomassie blue stained SDS-PAGE (left panel) and Western blot analysis (right panel) of recombinant, purified INI1 DBD using α-His antibodies as probes. (B) Agarose gel retardation assay (AGRA) of INI1 DBD using 100 ng of pET28a as substrate and indicated amount of polypeptide; RC: relaxed circular DNA, CCC: covalently closed circular DNA. (C) Ni-NTA pull-down assay of INI1 DBD:U5 HIV-1 LTR complex (lane a) and of DNA alone (lane b). The precipitated complex was analyzed by western blot using α-His antibodies as probes to detect INI1 DBD and the co-precipitated DNA was analyzed by ethidium bromide (EtBr) staining following 10% urea-PAGE. The loading controls are shown. (D) Atomic force microscopy (AFM) images of pNEB206A DNA alone (left panel) and in complex with INI1 DBD (right panel). Regions of DNA coated with protein (arrows) and free DNA (arrowhead) is shown.</p

Sedimentation equilibrium analysis of INI1 DBD and INI1:DNA complex.

<p>(A) Representative sedimentation equilibrium (SE) profiles of 10 µM (A) and 60 µM (B) of INI1 DBD generated from data collected at 280 nm. Representative sedimentation equilibrium (SE) profiles of a reaction mixture of 10 µM INI1 DBD and 10 µM U5 HIV-1 LTR DNA diluted 1∶7.5 x with buffer (C) and a reaction mixture of 60 µM INI1 DBD and 60 µM U5 HIV-1 LTR DNA diluted 1∶40 x with buffer (D) generated from data collected at 260 nm. Lower panels: Radial distribution of the concentration of INI1 DBD (A and B) and DNA (C and D) both free and in complex with INI1 DBD at sedimentation equilibrium. The solid line represents best fit. Upper panels: Distributions of the residuals around a zero mean.</p

Sequence conservation of the putative DNA binding domain (DBD) of vertebrate INI1/hSNF5, Drosophila SNR1 and <i>C.</i><i>elegans</i> SNF5.

<p>(A) Sequence comparison of the putative DNA binding domain of Drosophila SNR1 with the domains in vertebrates. Conserved residues are shown by (*) and partially conserved residues by (#). The HH and KKR motifs are shown with dash and broken dash, respectively. The conserved cysteine residues are shown by an arrow. The conserved lysine, arginine and histidine residues are shown with an arrowhead. (B) Sequence comparison of the putative DNA binding domain of <i>C. elegans</i> SNF5 with the human domain.</p

Isothermal calorimetry.

<p>ITC of INI1 DBD binding to U5 HIV-1 LTR DNA using 10 µM INI1 DBD (monomer) and 55 µM U5 HIV-1 LTR DNA (A) and 30 µM INI1 DBD (dimer) and 325 µM U5 HIV-1 LTR DNA (B). Top panel: Raw data of heat associated with mixing of DNA and protein. Heat of dilution is shown in the inset (not to scale). Bottom panel: Heat associated with each injection is obtained by integration of the area under the peak as a function of time. Binding curve (fitted) obtained by subtracting heat of dilution from heat associated with mixing of protein and DNA. Data is representative of multiple experiments.</p

Determination of stoichiometries of INI1 DBD binding to U5 HIV-1 LTR DNA from analytical ultracentrifugation studies.

a<p>Calculated from amino acid or nucleotide composition, <i>^b</i>Based upon sedimentation equilibrium data, ± = SD (standard deviation).</p

Model showing different modes of INI1 DNA binding.

<p>The INI1 DBD undergoes concentration dependent multimerization. Two molecules of monomeric DBD binds to one molecule of U5 HIV-1 LTR DNA whereas one molecule of dimeric DBD binds to two molecules of U5 HIV-1 LTR DNA.</p