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. 1998 Apr 14;95(8):4152-7.
doi: 10.1073/pnas.95.8.4152.

The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex

O Rozenblatt-Rosen  1 , T Rozovskaia, D Burakov, Y Sedkov, S Tillib, J Blechman, T Nakamura, C M Croce, A Mazo, E Canaani
Affiliations

The C-terminal SET domains of ALL-1 and TRITHORAX interact with the INI1 and SNR1 proteins, components of the SWI/SNF complex

O Rozenblatt-Rosen et al. Proc Natl Acad Sci U S A. .

Abstract

The ALL-1 gene was discovered by virtue of its involvement in human acute leukemia. Its Drosophila homolog trithorax (trx) is a member of the trx-Polycomb gene family, which maintains correct spatial expression of the Antennapedia and bithorax complexes during embryogenesis. The C-terminal SET domain of ALL-1 and TRITHORAX (TRX) is a 150-aa motif, highly conserved during evolution. We performed yeast two hybrid screening of Drosophila cDNA library and detected interaction between a TRX polypeptide spanning SET and the SNR1 protein. SNR1 is a product of snr1, which is classified as a trx group gene. We found parallel interaction in yeast between the SET domain of ALL-1 and the human homolog of SNR1, INI1 (hSNF5). These results were confirmed by in vitro binding studies and by demonstrating coimmunoprecipitation of the proteins from cultured cells and/or transgenic flies. Epitope-tagged SNR1 was detected at discrete sites on larval salivary gland polytene chromosomes, and these sites colocalized with around one-half of TRX binding sites. Because SNR1 and INI1 are constituents of the SWI/SNF complex, which acts to remodel chromatin and consequently to activate transcription, the interactions we observed suggest a mechanism by which the SWI/SNF complex is recruited to ALL-1/trx targets through physical interactions between the C-terminal domains of ALL-1 and TRX and INI1/SNR1.

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Figures

Figure 1
Figure 1
Deletion mapping of interacting fragments. Assays of β-galactosidase were made in SFY526 yeast cells. Cotransforming fragments in A, B, C, and D were SNR1 (amino acid residues 1–370), INI1 (amino acid residues 1–385), TRX (amino acid residues 3540–3759), and ALL-1 (amino acid residues 3745–3969), respectively. ++, strong interaction; –/+, very weak interaction.
Figure 2
Figure 2
In vitro (A) and in vivo (B and C) binding of TRX/ALL-1 polypeptides to SNR1/INI1. (A) TRX and ALL-1 segments spanned amino acid residues 3375–3759 and 3745–3969, respectively. Full length SNR1 and INI1 were synthesized and radiolabeled in a coupled transcription–translation system. Amount in input is 12% of the amount applied to beads. (B and C) Coimmunoprecipitation from COS cells of overproduced SNR together with TRX fragments and overproduced INI1 together with ALL-1 fragment, respectively. Equivalent amounts of extracts (including those analyzed in lanes 1–4 were immunoprecipitated, and proteins in the pellets were detected with anti-T7 or anti-HA mal.
Figure 3
Figure 3
Endogenous ALL-1 is coimmunoprecipitated from K562 cells together with overproduced INI1 (A). The product of the snr1 transgene is coimmunoprecipitated from fly extracts with endogenous TRX (B, Left) and such SNR1 coimmunoprecipitation is TRX-dependent (B, Right). (A) T7-tagged INI1 was transiently expressed in human K562 cells (K) or in simian COS cells (C). Equivalent amounts of protein were subjected to IP. Input represents an aliquot of the nuclear extract analyzed directly by Western blotting (corresponds to 16% of the amount used for immunoprecipitation). (B, Left) Synthesis of HA-tagged SNR1 was induced by heat shock, and fly extracts were incubated with either anti-HA mAb, or with anti-TRX Ab and the corresponding preimmune sera (PRE), or with Ab against the E(Z) or PC proteins. Following precipitation, SNR1 was monitored by immunoblotting with anti-HA mAb. (B, Right) HA-SNR1 was expressed transiently in COS cells and subjected to immunoprecipitation and detection with the same Ab as in B, Left.
Figure 4
Figure 4
Endogenous TRX and epitope-tagged SNR1 proteins colocalize on salivary gland polytene chromosomes of third instar larvae. Merging of green and red signals representing SNR1 and TRX respectively, identify the sites (yellow bands at the bottom) where the two proteins colocalize. Arrows indicate colocalization of both proteins at 98D1, the site of the endogenous forkhead gene.
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