Abstract
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.
Highlights
The SWI/SNF chromatin remodeling complex in yeast is a large multisubunit complex, required for regulation of mating type switching, sucrose-dependent growth and transcription [1]
When the other prominent domains in INI1 viz Repeat 1 (Rpt1) and Repeat 2 (Rpt2) are considered, it was found that these domains are present in both lower eukaryotes and higher eukaryotes while the proposed coiled coil domain is absent in A. thaliana and Z. mays (Figure 1A)
INI1/hSNF5 is a component of transcription complexes like the chromatin remodeling hSWI/SNF transcription activator complex and the Sin3A-HDAC1 transcription repressor complex
Summary
The SWI/SNF chromatin remodeling complex in yeast is a large multisubunit complex, required for regulation of mating type switching, sucrose-dependent growth and transcription [1]. Deletion or mutation of INI1 can potentially affect other functions of INI1, including its role through the SWI/SNF complex, as mutations throughout the gene have been found in patients with rhabdoid cancers. Earlier studies have shown that INI1 stimulates as well as inhibits IN activity depending on the concentration of IN and the ratio of IN:INI1 [10] These studies demonstrate that INI1 is a multimer and that multimerization of INI1 is essential for its ability to bind IN to form a high MW complex that is required for inhibition of IN activity [10]. INI1 exhibits non-specific DNA binding activity at the minor groove [10], a characteristic of transcription factors that are architectural proteins
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