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CHD1 Is Concentrated in Interbands and Puffed Regions of Drosophila Polytene Chromosomes [Chromatin/Transcription/Chromo Domain/Atpase (Helicase)] DAVID G

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CHD1 Is Concentrated in Interbands and Puffed Regions of Drosophila Polytene Chromosomes [Chromatin/Transcription/Chromo Domain/Atpase (Helicase)] DAVID G Proc. Natl. Acad. Sci. USA Vol. 93, pp. 7137-7142, July 1996 Genetics CHD1 is concentrated in interbands and puffed regions of Drosophila polytene chromosomes [chromatin/transcription/chromo domain/ATPase (helicase)] DAVID G. STOKES, KENNETH D. TARTOF, AND ROBERT P. PERRY* Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111 Contributed by Robert P. Perry, March 11, 1996 ABSTRACT Previously, we reported on the discovery and homeotic genes in Drosophila, presumably by participating in characterization of a mammalian chromatin-associated pro- locus-specific chromatin condensation or gene sequestration (5). tein, CHD1 (chromo-ATPase/helicase-DNA-binding do- Another domain in CHD1 is a '500-aa ATPase/helicase main), with features that led us to suspect that it might have (H) domain, which is present in a huge superfamily of proteins an important role in the modification of chromatin structure. with a broad range of biochemical functions including tran- We now report on the characterization of the Drosophila scriptional control, DNA recombination/repair, translation, melanogaster CHD1 homologue (dCHD1) and its localization and RNA processing. The CHD1 H domain is most closely on polytene chromosomes. A set of overlapping cDNAs en- related to the SNF2 family of proteins. Genetic and biochem- codes an 1883-aa open reading frame that is 50% identical and ical studies in yeast, Drosophila, and mammalian cells have 68% similar to the mouse CHD1 sequence, including conser- implicated the SNF2 family of proteins as pleiotropic activators vation of the three signature domains for which the protein of transcription, presumably via their effect on chromatin was named. When the chromo and ATPase/helicase domain structure (reviewed in refs. 6 and 7). These proteins are found sequences in various CHD1 homologues were compared with in large multiprotein complexes with a combined molecular the corresponding sequences in other proteins, certain dis- mass of >106 Da (8, 9). Recent evidence has indicated that tinctive features of the CHD1 chromo and ATPase/helicase these multiprotein complexes can elicit an ATP-dependent domains were revealed. The dCHD1 gene was mapped to alteration in the nucleosomal organization of certain promot- position 23C-24A on chromosome 2L. Western blot analyses ers, thereby facilitating the entry of both gene-specific and with antibodies raised against a dCHD1 fusion protein spe- general transcription factors (10-12). cifically recognized a '210-kDa protein in nuclear extracts The third signature of CHD1 is its DNA-binding (D) from Drosophila embryos and cultured cells. Most interest- domain, which has been localized to a 229-aa segment on the ingly, these antibodies revealed that dCHD1 localizes to sites C terminal side of the H domain in mouse CHD1 (mCHD1; of extended chromatin (interbands) and regions associated ref. 13). We have shown that CHD1 preferentially binds via with high transcriptional activity (puffs) on polytene chro- minor groove interactions to DNA that contains (A+T)-rich mosomes from salivary glands of third instar larvae. These tracts, including those in a matrix attachment region. The D observations strongly support the idea that CHD1 functions domain contains sequence motifs known to be essential for the to alter chromatin structure in a way that facilitates gene (A+T)-minor groove binding of other chromatin-associated expression. proteins, such as histone Hi, HMGI/Y, Dl, and datin (14, 15). Given the established or inferred functions of proteins that It has become increasingly apparent that eukaryotic cells have share one or another of the CHD1 signatures, it seems evolved elaborate systems to enable gene expression to occur reasonable to assume that CHD1 plays an equally important in the context of chromatin. Alteration of chromatin structure role in the organization of chromatin structure and the regu- in the form of compaction or extension is associated with many lation of gene activity. To study the function of CHD1 in a inducible genes and with the heritable expression patterns of genetically pliable organism, we have isolated cDNA clones genes involved in development. Proteins that implement these that encode the Drosophila melanogaster CHD1 homologue changes in chromatin structure and their mechanisms of action (dCHD1). When antibodies raised against a portion of dCHD1 are beginning to be elucidated. However, there is still much to were used to investigate the distribution of the protein on be learned about the process of chromatin remodeling and the polytene chromosomes, we observed that CHD1 is concen- interactions of chromatin constituents with other components trated in transcriptionally active regions of the genome. of the gene expression machinery. Our interest in this problem arose when we discovered a MATERIALS AND METHODS novel mouse protein, called CHD1 (chromo-ATPase/helicase- cDNA Cloning and Sequencing. A set of five overlapping DNA-binding protein 1), which contains three identifiable a D. mela- to determinants cDNA clones encoding dCHD1 was isolated from domains that are present in proteins known be nogaster 3- to 12-hr embryonic library obtained from T. of chromatin structure (1). One of these domains, the 52-aa Kornberg (University of California, San Francisco) and from chromo (C) domain, is present in proteins that have been 4- to 8-hr and 12- to 24-hr embryonic libraries constructed by implicated in the process of chromatin compaction and the Brown and Kafatos (16). The library screens were initiated repression of gene expression (reviewed in refs. 2 and 3). with a fortuitously cloned 494-bp cDNA fragment (generously Among these proteins are HP1, a 25-kDa protein that is mainly provided by Jonathan M. Rothberg, Yale University), which associated with pericentric/constitutive heterochromatin in has strong sequence similarity to a region of the domain of Drosophila (2) and mammalian cells (4), and Polycomb, a 44-kDa protein, which has been implicated in the repression of Abbreviations: C, chromo; H, ATPase/helicase; D, 5NA-binding; dCHD1, Drosophila CHD1; mCHD1, mouse CHD1. The publication costs of this article were defrayed in part by page charge Data deposition: The sequence reported in this paper has been payment. This article must therefore be hereby marked "advertisement" in deposited in the GenBank data base (accession no. L7790,). accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 7137 Downloaded by guest on September 26, 2021 7138 Genetics: Stokes et al. Proc. Natl. Acad. Sci. USA 93 (1996) mCHD1, and completed with probes derived from subse- 1 MSQALNESAN SIGSDEQDDT REEANGTDHS GSGSGSGSSG SDSDSDSSSG quently isolated clones. In total, the clones span a region of 51 NSSDGRSSPE PEDKSLSVAG FPPTAAAAQA DSKTNGFTDD QEDSSSDGSS 6242 bp. All of the clones were sequenced on both strands by 1)1 GSDSDSDAEG PSDQRNQSIN NANTSSSLPK PEQNEEEDNE TEAGQQQPAS a combination of manual and automated protocols [Sequenase 151 DASADESSDS SANVSPTSSS SSSEEEEEDY RPKRTRQARK PPTAAEKSKK 7-deaza-GTP kit (United States Biochemical); and ABI Prism 201 APAPKNKKKT WDSDESDESE DSDDEVSTAQ KRKPAATTSR SKLAQQQQRR 377 DNA sequencer (Perkin-Elmer)]. Sequence analysis was 251 RVKPFSSEDS DDDDASKRCA TRRKGAAVSY KEASEDEATD SEDLLEFEYD performed with the Applied Biosystems 373A DNA Sequencer 301 ESQAATTAAT AEEEEKCETI ERILAQRAGK RGCTGNQTTI|YAIEENGFDP c Analysis Program and the Wisconsin Sequence Analysis Pack- 351 HAGFDEKQTP DAETEAQFLI KWKGWSYIHN TWES1ATLRD MIKGMKKLD age (Genetics Computer Group, Madison, WI). 401 NFIKKEKEQA YWRRYAGPED IDYFECQLEL QHELLKSYNN VDRIIAKGSK Fusion Protein Production and Antibody Purification. Rab- 451 PDDGTEEYLC KWQSLPYAES TWEDAALVLR KWQRCAEQFN DRESSKCTPS bit polyclonal antibodies were raised against a glutathione 501 RHCRVIKYRP KFSRIKNQPE FLSSpLTLRe YQMDGLNWLL HSWCKENSVI Ia S-transferase-ClaI-EcoRI fragment fusion protein (GST-CE) 551 LADEMGLGKT FKIHHLYGPF LCWPLSTMT amino acid residues 1419-1644 of dCHD1. The IQTICFLYSL AWQREFDLWA containing 601 PDMNVVTYLG DIKSRELIQQ YEWQFESSKR LKFNCILTTY EIVLKDKQFL fusion was as described and II III protein produced previously (13) 651 GTLQWAALLV DEAHRLKNDD SLLYKSLKEF DTNHRLLITG TPLQNSLKEL purified by SDS/PAGE. Affinity-purified antibodies were IV H isolated from the polyclonal antisera as described (13) with a 701 WALLHFIMPD KFDTWENFEV QHGNAEDKGH TRLHQQLEPY ILRRVKKDVE column containing GST-CE fusion protein. GST-CE fusion 751 KSLPAKVEQI LRVEMTSLQK QYYKWILTKN FDALRKGKRG STSTFLNIVI protein (20 mg), purified by standard procedures (17) and 801 ELKKCCNHAA LIRPSEFELM GLQQDEALQT LLKGSGKLVL LDKLLCRLKE 851 TGHRVLIFSQ MVRMLDVLAD YLQKRHFPFQ RLDGSIKGEM RRQALDHFNA according to manufacturer's specifications (Pharmacia), was V VI combined with CnBr-activated Sepharose-4B (Pharmacia) to 901 EGSQDFCFLL STRAGGLGIN LATADTVIIF DSDWNPQNDL QAQARAHRIG make the antigen column. 951 QKNQVNIYRL VTARSVEEQI VERAKQKMVL DHLVI+MDT TGRTVLDKSG Extracts, Immunoblotting, and Immunolocalization of Pro- 1001 NGHSSNSNPF NKDDLSAILK FGAEELFKDE QEHDDDLVCD IDEILRRAET teins on Polytene Chromosomes. Nuclei were isolated from 1051 RNEDPEMPAD DLLSAFKVAS IAAFEEEPSD SVSKQDQNAA GEEDDSKDWD Oregon-R 0- to 24-hr Drosophila embryos obtained from 1101 DIIPEGFRKA IDDQERAKEM EDLYLPPRRK TAANQNEGKR GAGKGGKGKQ overnight egg lays by the method of Franke et al. (18) or the 11511.1...1. uADDSuGDLi---JlYELGSDGSGD- ---- I.PERKRt...........----UF-_ PITMKEKITGFT DAELRRFIRSD-E-RR ....I...........- method of Kamakaka and The nuclei were 1201 YKKFPAPLHR
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