Biochimica et Biophysica Acta 1591 (2002) 63–68 www.bba-direct.com MacroH2A1.2 binds the nuclear protein Spop Ichiro Takahashi *, Yosuke Kameoka, Katsuyuki Hashimoto Division of Genetic Resources, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan Received 27 September 2001; received in revised form 18 April 2002; accepted 27 May 2002 Abstract X-chromosome inactivation is a phenomenon by which one of the two X chromosomes in somatic cells of female mammals is inactivated for life. The inactivated X chromosomes are covered with Xist (X-inactive specific transcript) RNA, and also enriched with the histone H2A variant, macroH2A1.2.The N-terminal one-third of macroH2A1.2 is homologous to core histone H2A, but the function of the C-terminal two-thirds, which contains a basic, putative leucine zipper domain, remains unknown.In this study, we tried analyzing protein–protein interaction with a yeast two-hybrid system to interact with the nonhistone region of mouse macroH2A1.2. The results showed that macroH2A1.2 interacts with mouse nuclear speckled type protein Spop. The Spop protein has a unique composition: an N-terminal MATH, and a C-terminal BTB/POZ domain. Further binding domain mapping in a glutathione-S-transferase (GST) pull-down experiment revealed that macroH2A1.2 binds the MATH domain of Spop, which in turn binds to the putative leucine zipper domain of macroH2A1.2. D 2002 Elsevier Science B.V. All rights reserved. Keywords: MacroH2A1.2; Spop 1. Introduction reported to be physically associated with Xist RNA, no specific role of macroH2A1.2 in X inactivation has yet Macrohistone H2A (macroH2A) is a histone H2A var- been identified. iant. The N-terminal one third of macroH2A is homologous X-chromosome inactivation occurs in three steps: ini- to histone H2A, but the remaining two-thirds is a novel tiation, spreading, and maintenance [5], and a noncoding polypeptide with unknown function [1,2]. large RNA, Xist, is involved in each step [5]. The histone- The recently demonstrated subtype of macroH2A1, H2A homologous region of macroH2A plays a crucial role macroH2A1.2 localizes on the inactive X chromosomes in the association between macroH2A molecules and inac- of female mammals [3]. The specific association between tive X chromosomes according to the results of chromatin macroH2A1.2 histone and the inactive X chromosome immunoprecipitation using anti-macroH2A antibody fol- suggests that macroH2A may function in the inactivation lowed by reverse transcription-coupled polymerase chain process. The association between macroH2A1.2 and the reaction (RT-PCR) with an extract of female cells [6].A inactive X chromosome depends on the continued presence recent study has shown that the association between mac- of the X-inactive specific transcript (Xist), the large roH2A and the inactive X, which seems to depend on the untranslated RNA that associated in cis along the length histone H2A domain of the macroH2A molecule rather of the inactive X [4]. Although macroH2A1.2 has been than the nonhistone domain, may reflect the higher density of several histones on the inactive X [7]. Study of its Abbreviations: MacroH2A, macrohistone H2A; SPOP (Spop), nuclear genomic structure has revealed that the gene at the mac- speckled type protein; MATH, meprin and TRAF homology domain; POZ, roH2A locus contains 10 exons [8]. The macroH2A locus poxvirus and zinc finger domain; SDS, sodium dodecyl sulfate; PAGE, produces two protein isoforms, 1.1 and 1.2, by alternative polyacrylamide gel electrophoresis; RT-PCR, reverse transcription-coupled splicing. Exon 6 has been found to be a macroH2A1.2- polymerase chain reaction; GST, glutathione-S-transferase; EGFP, enhanced specific exon, while exon 7 is macroH2A1.1-specific. green fluorescent protein * Corresponding author. Tel.: +81-3-5285-1111x2121; MacroH2A1.2 is preferentially associated with the inactive fax: +81-3-5285-1181. X chromosome since exon 6 contains a putative leucine E-mail address: [email protected] (I. Takahashi). zipper motif [8]. 0167-4889/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S0167-4889(02)00249-5 64 I. Takahashi et al. / Biochimica et Biophysica Acta 1591 (2002) 63–68 In order to understand the function of macroH2A1.2 on NP40, a proteinase inhibitor cocktail (Roche). After 1-h inactive X chromosomes, we used a yeast two-hybrid screen incubation on ice, the beads were washed four times with to search for a protein module(s) that is(are) associated with GST buffer. The bound proteins were eluted with SDS the nonhistone H2A domain of macroH2A1.2. sample buffer and analyzed on SDS-PAGE followed by autoradiography [10]. 2. Methods 2.4. Immunofluorescence analysis 2.1. Plasmid C127 cells were plated onto coverslips and transfected with pEGFP-Spop using the lipofectamine PLUS regent We amplified either full-length cDNA sequences or (Gibco BRL), according to the manufacturer’s instructions. selected regions of their sequences by polymerase chain After 24 h, the cells were fixed with a paraformaldehyde reaction (PCR) using restriction enzyme-site-tagged primers solution (4% paraformaldehyde in PBS) for 15 min fol- and AmpliTaq DNA polymerase (PE Applied Biosystems). lowed by a 5-min incubation in 0.2% Triton X100/PBS. The The PCR products were digested with appropriate cells were subsequently blocked with 5% skim milk (Difco, enzymes and usually cloned into a pcDNA vector (Invitro- MI) in 2 Â SSC for 1 h. After blocking, anti-macroH2A1.2 gen) and sequenced for PCR errors (ABI Sequencer). rabbit antibody (Upstate Biotech, NY) was added to the We cloned various domains of mouse Spop or mouse cells and incubated at room temperature for 3 h. After macroH2A1.2 into the pGEX vector (Amersham Pharmacia washing, the coverslips were incubated with Texas red Biotech) by PCR using appropriate sets of primers, and also conjugated anti-rabbit goat antibody (Molecular Probes) cloned mistake-free products in-frame into the appropriate for 1 h at room temperature and finally mounted in Vecta- vector(s). shield anti-fade (Vector Laboratories, CA) containing DAPI. 2.2. Two-hybrid screening 2.5. Confocal microscopy Two-hybrid screening in yeast was performed with the Samples were analyzed with the laser confocal scanning system of Gyunis et al. [9]. Briefly, the yeast strain used was microscope IX81 (Olympus). UV–Ar (351 nm), Ar (488 EGY48/LacZ, which carries two reporters whose expression nm), and HeNe (543 nm) lasers were used to excite DAPI, is regulated by LexA-responsive promoters: a chromoso- enhanced green fluorescent protein (EGFP), Texas red mally integrated LEU2 reporter gene (LexA:LEU2) and the fluorescence, respectively. The lens was an Olympus 2ALacZ reporter plasmid pSH18-34. pEG202-macroH2A Apo40XWLSM/UV objective. (aa110–372) was used as bait in EGY48/LacZ to screen the mouse embryo Day 19 pJG4-5 prey library (OriGene 2.6. EGFP-Spop MD). A total of 2 Â 106 yeast cotransformants were selected for galactose-induced reporter-dependent leucine prototro- The full coding sequence from Spop was PCR-amplified phy. Prey plasmids were rescued from positive clones and with primers incorporating HindIII and SalI restriction retransformed into EGY48/LacZ strains expressing enzyme recognition sites and subcloned into pEGFP-C1 pEG202-macroH2A. Cotransformants were reselected for (Clontech) vector. reporter-dependent leucine prototrophy and h-galactosidase production, and positive clones were sequenced. 3. Results 2.3. Glutathione-S-transferase (GST) pull-downs of in vitro translated proteins 3.1. Isolation of mouse macroH2A1.2 cDNA GST and GST fusion proteins were expressed in E. coli The mouse macroH2A-specific nonH2A region was DH5a by using the pGEX (Amersham Pharmacia) vector amplified by PCR using rat macroH2A-specific primers: system. Bacteria were induced with 0.2 mM IPTG for 4 h at forward, 5VCCA AAA AGG CCA AGT CTC CAT 3V, and 37 jC, and recombinant proteins were purified with gluta- reverse, 5VACA TGT GCT GGC TCT AGG GAA 3V, and thione-Sepharose beads and analyzed on sodium dodecyl random primed cDNA from mouse MC12 cell as the sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to template. PCR products from 30 cycles of amplification at normalize protein amounts. Equivalent amounts of GST 95 jC 1 min and 58 jC 5 min were cloned into pGEM-T fusion proteins were incubated with [35S]methionine- Easy Vector (Promega) to generate a mouse macroH2A1 labeled mouse macroH2A1.2 or Spop proteins, produced clone referred to as pGEM-Macro. by a T7-TNT-coupled transcription/translation system Since the pGEM-Macro had a 6-bp deletion compared (Promega, WI) in 200 Al GST buffer(20 mM Tris–HCl with the rat macroH2A sequence because of a PCR error, we pH 8, 100 mM NaCl, 1 mM EDTA) added with 0.1% tried to isolate a complete mouse macroH2A cDNA clone I. Takahashi et al. / Biochimica et Biophysica Acta 1591 (2002) 63–68 65 from a mouse brain cDNA library (Stratagene, CA) with the 0.7 kb-insert of pGEM-Macro as a probe. The clones picked were identified by their insert-sequences, and one of the clones obtained, Ma-8, was shown to have the entire sequence of mouse macroH2A1.2. 3.2. Isolation of macroH2A1.2 interacting proteins The macrohistone H2A1.2 has been reported to concen- trate on the inactive X chromosomes of female mammals. To better understand the role of macroH2A1.2, we used a yeast two-hybrid approach to identify interacting proteins. Fusion between the LexA DNA binding protein and amino Fig. 2. MacroH2A1.2 interacts with the nuclear protein Spop in vitro. The Spop consisting of an amino-terminal MATH (GST–MATH) domain or the acids 110–372 of the macroH2A protein (Fig. 1A) was used carboxyl terminal BTB/POZ (GST–POZ) domain fused with GST was as bait to screen cDNA from mouse embryo Day 19 DNA used in GST pull-down experiments with [35S]-labeled in vitro translated inserted into an expression cassette consisting of the SV40 non-H2A region of macroH2A1.2.