Copyright � 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.107.079038

Note

Investigations of CHD1 Function in Transcription and Development ofDrosophila melanogaster

Ivy E. McDaniel, Jennifer M. Lee, Matthew S. Berger, Cori K. Hanagami andJennifer A. Armstrong1

Joint Science Department, W. M. Keck Science Center, Scripps, Claremont McKenna, and Pitzer Colleges, Claremont, California 91711

Manuscript received July 19, 2007Accepted for publication November 19, 2007

ABSTRACT

In this report we describe chd1 mutant alleles and show that the CHD1 chromatin-remodeling factor isimportant for wing development and fertility. While CHD1 colocalizes with elongating RNA polymerase II(Pol II) on polytene chromosomes, elongating Pol II can persist on chromatin in the absence of CHD1.These results clarify the roles of chromatin remodelers in transcription and provide novel insights intoCHD1 function.

IN eukaryotic cells, RNA polymerase II (Pol II) encoun-ters nucleosomes at each step in transcription. To

deal with these obstacles, Pol II requires the action of aplethora of proteins including chromatin-remodelingfactors, which function as DNA-translocating machinesthat draw waves of DNA around the histone octamer toslide, perturb, or disassemble the nucleosomes (Cairns

2005; Smith and Peterson 2005; Saha et al. 2006).While a number of chromatin-remodeling factors havebeen identified, the relative roles of these proteins in PolII transcription are unclear.

The stages of transcription are largely defined by thephosphorylation status of the C-terminal domain(CTD) of the largest subunit of Pol II. During initiation,the CTD is unphosphorylated (Pol IIa); as Pol II clearsthe promoter, it is phosphorylated at serine 5 within theheptad repeats of the CTD; and later in elongation, PolII is phosphorylated on serine 2 (Pol IIoser2) (Phatnani

and Greenleaf 2006). Recent studies utilizing tran-scriptionally active polytene chromosomes derived fromDrosophila melanogaster larval salivary glands suggest thatthree distinct chromatin-remodeling factors may berequired for a successful round of transcription. PolIIa levels are reduced on polytene chromosomes de-rived from larvae expressing a dominant-negative alleleof brahma (brm), suggesting that the SWI2-like BRMchromatin remodeler is critical for transcription initia-tion (Armstrong et al. 2002). Polytene chromosomes

derived from larvae lacking the Kismet (KIS) chroma-tin-remodeling protein retain Pol IIoser5, but lose PolIIoser2, suggesting that KIS is required for an early step inthe transition to transcription elongation (Srinivasan

et al. 2005). The CHD1 (chromodomain, helicase, DNA-binding protein 1) chromatin-remodeling protein local-izes to active genes of polytenes in a pattern nearlyidentical to that of Pol IIoser2, suggesting a role for CHD1in facilitating elongation (Stokes et al. 1996; Srinivasan

et al. 2005).Taken together, these results lead to a model in which

three Drosophila chromatin-remodeling factors functionsequentially to allow transcription: (1) BRM is requiredfor initiation, (2) KIS is required for the transition frompromoter clearance to the elongation phase, and (3)CHD1 is required for continued elongation by Pol II. Aplace for CHD1 in elongation is also supported by datain yeast. Saccharomyces cerevisiae CHD1 is localized to atranscriptionally active gene and physically interactswith transcription elongation factors (Simic et al. 2003).To determine whether Drosophila CHD1 is required forelongation, we generated loss-of-function alleles of chd1and examined the consequences of the loss of CHD1 onglobal chromosome structure and transcription.

chd1 is not an essential gene: To investigate the func-tion of CHD1, we generated two deletion alleles (chd14

and chd15) by imprecise excision of an EP elementinserted into position -2 of the chd1 promoter (GenExelstock G2213) (Figure 1A). We observed no differencesin the behavior of the two alleles. As described below,chd14 and chd15 homozygous and chd14/chd15 hetero-allelic individuals display phenotypes that are less se-

1Corresponding author: Joint Science Department, W. M. Keck ScienceCenter, 925 N. Mills Ave., Scripps, Claremont McKenna, and PitzerColleges, Claremont, CA 91711. E-mail: jarmstrong@jsd.claremont.edu

Genetics 178: 583–587 (January 2008)

vere than those seen in hemizygous mutants ½usingDf(2L)Exel7014�. These genetic data would suggest thatchd14 and chd15 are hypomorphic alleles. We investi-gated the possibility that the two chd1 alleles couldgenerate proteins with N-terminal truncations. Giventhe location of the earliest in-frame start codon, wepredict that both chd14 and chd15 alleles would gener-ate a 166-kDa protein. However, Western blot analysisof heterozygous embryo extracts failed to detect anN-terminal truncated protein (Figure 1B). While it isformally possible that chd14 and chd15 express an un-stable protein that is not detectable by Western blot,we propose that our alleles are protein nulls and con-clude that chd1 is not an essential gene. chd14 and chd15

homozygous, heteroallelic, and hemizygous mutant in-dividuals are viable, although they display a 1- to 2-daydevelopmental delay, and some marker combinationsreduce viability of chd1 mutants. Given the genetic datadescribed above, we propose that 1 of the other 18 genesuncovered by Df(2L)Exel7014 may dominantly enhancechd1 mutant phenotypes. For example, okra (the RAD54homolog, a SNF2-like helicase) is located 20 kb awayfrom chd1. okra mutant phenotypes include femalesterility (Kooistra et al. 1997), one of the chd1 pheno-types that may be dominantly enhanced by the deficiency.

chd1 mutants reveal unexpected defects in fertility andwing development: We examined the distribution of chd1

mRNA by in situ hybridization and found that chd1 isbroadly expressed throughout embryogenesis and inimaginal discs (data not shown). This broad expressionpattern is similar to that of brm and kis (Elfring et al. 1998;Daubresse et al. 1999) and suggests that, like BRM andKIS,CHD1couldfunctionglobally toregulate transcription.

While chd1 is broadly expressed, we observed specificphenotypes in mutant animals, suggesting that CHD1may function as a tissue-specific chromatin-remodelingfactor. Wing margins in chd14 and chd15 homozygous,heteroallelic, and hemizygous mutant individuals dis-played notching (Figure 2B), with 3.8–36% of heteroallelicindividuals and 75–94% of hemizygous individualsshowing notched wing margins (Table 1). Several con-trol individuals are presented in Table 1 to ensure thatthe cut-in wing margins were not a consequence of thechromosomal markers (which are a result of meioticmapping of the chd1 alleles), although the markers mayaffect how often the phenotype is seen (Table 1). Thevariability of the wing-notching phenotype was notcorrelated with developmental delay or viability. Indi-viduals homozygous for the precise excision did notshow cut-in wing margins, indicating that the phenotypeis due to lack of CHD1. This specific wing phenotype isnot observed in animals lacking BRM or KIS and sug-gests that genes critical for wing-margin formation areespecially sensitive to loss of CHD1.

Figure 1.—The generation of two chd1alleles. (A) The chd14 allele carries a dele-tion from �1 to 11994 with the additionalsequence CATGATGAAATAACATATAGTTAGATATGAAATAA. The chd15 allele car-ries a deletion from �1 to 11871 withthe additional sequence CATGATGAAATAACATCATCATAACATGAAATAAC. Muchof the additional sequence in both alleles isderived from the P element. (B) Westernblot analysis of embryo extracts derivedfrom Oregon-R (WT), flies in which theP element was precisely excised (precise),and chd14/CyO and chd15/CyO heterozy-gotes. Full-length CHD1 is observed ineach lane, and a truncated protein (pre-dicted to be 166 kDa) is not observed inthe mutant heterozygous embryo extracts.The CHD1 rabbit polyclonal antibody wasraised and affinity purified against CHD1amino acids 1706–1721 (CRLNMDRHED

RKKHHRG) (Covance). This peptide antibody recognizes CHD1 on polytenes in a pattern identical to that observed with theantibody raised by Robert Perry (Stokes et al. 1996) (data not shown).

Figure 2.—chd1 mutant individuals displaywing defects that include notched wing margins.In contrast to a wild-type Oregon-R wing (A),wings from chd14 b pr c px sp/chd5 b individuals shownotched wing margins (B). Wings from chd1 mu-tant animals are�80% the size of wild-type wings,consistent with their overall smaller size.

584 I. E. McDaniel et al.

chd14 and chd15 homozygous, heteroallelic, and hemi-zygous males are sterile; CHD1 is therefore required formale fertility (Table 2). chd1 mutant males displayednormal mating behaviors, and there were no obviousdefects in the general morphology of testes of chd14/chd15 mutant males (data not shown). While our mutantmales produced zero progeny, control males producedan average of 101 progeny per single male under thesame conditions. CHD1 is also important for femalefertility as chd1 mutant females produced few offspring(Table 2) (by comparison, a single wild-type femaleproduced 108 progeny under the same conditions). Wepropose that the majority of the fertilized eggs cannotdevelop due to an inability to repackage the spermpronuclear DNA into H3.3-containing chromatin (Konev

et al. 2007). Examination of egg chambers from hemi-zygous mutant females ½chd14/Df(2L)Exel7014� revealsthat, while eggs are occasionally formed, oogenesis oftenfails at stage 8, the start of yolk production (Figure 3B). An8.5-kb genomic chd1 transgene (�456 to 1 8019 relativeto the chd1 start site) fully rescued all mutant phenotypes:male sterility, reduced female fertility, and notching ofwing margins.

CHD1 and transcriptional elongation: CHD1 proteinpersisted into early third instar larval stages in chd1mutant larvae (likely a consequence of maternal per-durance). However, in contrast to CHD1 levels in

control chromosomes (Figure 4A), CHD1 levels weregreatly reduced on chromosomes derived from chd1mutant individuals in mid-to-late third instar larvaldevelopment (Figure 4B), providing us with an excel-lent system in which to dissect the role of CHD1 onchromosomes. Unlike the chromatin-remodeling factorISWI (Deuring et al. 2000), CHD1 is not essential forglobal chromosome structure (Figure 4D).

Given the observation that chromosomes lacking KISare not bound by CHD1 (Srinivasan et al. 2005), weproposed that CHD1 is functionally downstream ofKismet. Alternatively, Kismet and CHD1 could be mu-tually dependent upon each other for chromosomebinding. To distinguish between these two possibilities,we asked whether KIS was found on chromosomeslacking CHD1. KIS was present at wild-type levels inchd1 mutant individuals (Figure 5), indicating that,while CHD1 localization depends upon KIS, KIS local-ization is not dependent upon CHD1.

To test our hypothesis that CHD1 is required for thecontinued elongation of Pol II, we examined the levelsof Pol IIoser2 on chromosomes derived from mutant chd1individuals. We observed chromosomes that lack CHD1protein that still possessed normal levels of Pol IIoser2

TABLE 1

Wing defects in chd1 mutant flies

Genotype

Totalno. offlies

% of fliesdisplaying cut-inwing marginsa

chd14 b pr c px sp/chd15 b 285 3.8chd14 b pr c px sp/chd15 b c sp 115 36chd14 b pr c px sp/b 269 0chd14 b pr c px sp/al b c sp 294 0b pr c px sp/chd15 b c sp 280 0chd14 b pr c px sp/Df(2L)Exel7014 159 78chd15 b/Df(2L)Exel7014 48 94In(2LR) Gla, Bc/Df(2L)Exel7014 209 0

a At least one wing showed a cut-in wing margin.

TABLE 2

chd1 mutant males and females have reduced fertility

Genotype Gender

No. of chd1mutantparents

No. ofadult

offspring

chd14 b pr c px sp/chd15 b Male 120 0chd14 b pr c px sp/chd15 b Female 69 39chd14 b pr c px

sp/Df(2L)Exel7014Female 47 2

Flies were crossed to Oregon-R flies of the opposite gender(males or female virgins) as appropriate.

Figure 3.—chd1 mutant individuals display defects in oo-genesis. Ovarioles derived from wild-type (A) and chd14/Df(2L)Exel7014 hemizygous mutant females (B) were stainedwith DAPI and prepared as described (Verheyen and Cooley

1994). While eggs are occasionally produced, oogenesis ofhemizygous mutant females often fails at stage 8.

Note 585

(Figure 4F). An antibody recognizing all the forms ofPol II (4H8, Abcam) similarly showed normal levels anddistribution of total Pol II on chd1 mutant chromosomes(data not shown). Given that we observed chromosomeslacking observable CHD1 protein that retain elongatingPol II, we conclude that while CHD1 colocalizes withelongating Pol II, it is not absolutely required for theassociation of Pol II with chromatin. We note that lowerlevels of CHD1 protein on polytenes from chd1 mutantlarvae can correlate with reduced levels of Pol IIoser2

(data not shown). Reduced levels of transcription maybe a secondary affect of healthy larvae; alternatively, loss

of CHD1 may affect subsequent rounds of transcription,leading to a reduction of Pol IIoser2 levels over time.

In conclusion, we have generated two chd1 null allelesthat have revealed roles for CHD1 in male fertility,oogenesis, and wing development. While it is formallypossible that transcription is affected in a subtle way, ourexperiments allow us to conclude that CHD1 is notabsolutely required for the association of elongating PolII on chromosomes. However, since Pol IIoser2 levels canoccasionally be reduced on chromosomes derived fromchd1 mutant larvae, CHD1 activity may indirectly impacttranscriptional elongation. Yeast Chd1 is implicated in

Figure 4.—CHD1 binding is not required forthe structure of interphase chromosomes or forelongation by Pol II (IIoser2). Polytene chromo-somes were derived from control individuals thatunderwent a precise excision of the P element (A,C, and E) or from homozygous chd14 b pr c px spindividuals (B, D, and F). Chromosomes were im-munostained with CHD1 (A and B) or Pol IIoser2

(E and F) or were stained with DAPI (C and D).Despite lack of the CHD1 protein on chromo-somes derived from chd1 mutant individuals(B), the chromosomes display an overall normalmorphology (D) and contain normal levels ofelongating Pol II (F). Control and mutant larvaewere immunostained and imaged at the sametime under identical conditions. Immunostainswere carried out as described (Armstrong

et al. 2002; Corona et al. 2004) using an antibodydirected against the C-terminal region of CHD1(Stokes et al. 1996) and the commercially avail-able H5 antibody (Covance). Secondary antibod-ies (Jackson ImmunoResearch Laboratories)were tested with individual primary antibody toensure specificity. Images were obtained on aZeiss Axioskop 2 plus microscope with an Axio-plan HRm camera and Axiovision 4 software.Control and mutant chromosomes were photo-graphed using identical exposure times, and im-ages were processed identically in Photoshop 7.0.

Figure 5.—CHD1 is not required for KIS bind-ing. Polytene chromosomes derived from controlindividuals that underwent a precise excision ofthe P element (A) or homozygous chd14 b pr cpx sp individuals (B) were immunostained forKIS. DAPI stains of the same chromosomes areshown in insets. Immunostains were carried outas described (Armstrong et al. 2002; Corona

et al. 2004) using a previously described KIS anti-body (Srinivasan et al. 2005). Control and mu-tant chromosomes were processed in paralleland photographed using identical exposuretimes.

586 I. E. McDaniel et al.

Pol II elongation, termination, and the response totranscriptional stress (Alen et al. 2002; Simic et al. 2003;Zhang et al. 2005). Drosophila CHD1 participates innucleosome assembly in vitro (Lusser et al. 2005) andwas recently found to repackage the sperm pronuclearDNA into H3.3-containing chromatin in vivo (Konev

et al. 2007). Whether CHD1 functions to facilitatechromatin disassembly or reassembly during transcrip-tion remains to be determined.

We thank John Tamkun for antibodies, discussions, and helpfulcomments on this manuscript; Kristel Dorighi, Giorgia Siriaco,Shrividhya Srinivasan, Nick Reeves, Grant Hartzog, and Joseph Schulzfor helpful discussions and advice; Robert Perry for generouslyproviding CHD1 antibody; Helen McNeill for the chd1 P-elementinsertion line; Laura Lee and Rebecca Zabinsky for fly assistance; andRancho Santa Ana Botanic Garden for use of their sequencingfacilities. This work was supported by a grant from the NationalScience Foundation (MCB-0641379).

LITERATURE CITED

Alen, C., N. A. Kent, H. S. Jones, J. O’Sullivan, A. Aranda et al.,2002 A role for chromatin remodeling in transcriptional termi-nation by RNA polymerase II. Mol. Cell 10: 1441–1452.

Armstrong, J. A., O. Papoulas, G. Daubresse, A. S. Sperling, J. T.Lis et al., 2002 The Drosophila BRM complex facilitates globaltranscription by RNA polymerase II. EMBO J. 21: 5245–5254.

Cairns, B. R., 2005 Chromatin remodeling complexes: strength indiversity, precision through specialization. Curr. Opin. Genet.Dev. 15: 185–190.

Corona, D. F., J. A. Armstrong and J. W. Tamkun, 2004 Geneticand cytological analysis of Drosophila chromatin-remodeling fac-tors. Methods Enzymol. 377: 70–85.

Daubresse, G., R. Deuring, L. Moore, O. Papoulas, I. Zakrajsek

et al., 1999 The Drosophila kismet gene is related to chromatin-remodeling factors and is required for both segmentation andsegment identity. Development 126: 1175–1187.

Deuring, R., L. Fanti, J. A. Armstrong, M. Sarte, O. Papoulas et al.,2000 The ISWI chromatin-remodeling protein is required forgene expression and the maintenance of higher order chromatinstructure in vivo. Mol. Cell 5: 355–365.

Elfring, L. K., C. Daniel, O. Papoulas, R. Deuring, M. Sarte et al.,1998 Genetic analysis of brahma: the Drosophila homolog ofthe yeast chromatin remodeling factor SWI2/SNF2. Genetics148: 251–265.

Konev, A. Y., M. Tribus, S. Y. Park, V. Podhraski, C. Y. Lim et al.,2007 CHD1 motor protein is required for deposition of histonevariant H3.3 into chromatin in vivo. Science 317: 1087–1090.

Kooistra, R., K. Vreeken, J. B. M. Zonneveld, A. de Jong, J. C. J.Eeken et al., 1997 The Drosophila melanogaster RAD54 homolog,DmRAD54, is involved in the repair of radiation damage and re-combination. Mol. Cell. Biol. 17: 6097–6104.

Lusser, A., D. L. Urwin and J. T. Kadonaga, 2005 Distinct activitiesof CHD1 and ACF in ATP-dependent chromatin assembly. Nat.Struct. Mol. Biol. 12: 160–166.

Phatnani, H. P., and A. L. Greenleaf, 2006 Phosphorylation andfunctions of the RNA polymerase II CTD. Genes Dev. 20:2922–2936.

Saha, A., J. Wittmeyer and B. R. Cairns, 2006 Chromatin remod-elling: the industrial revolution of DNA around histones. Nat.Rev. Mol. Cell Biol. 7: 437–447.

Simic, R., D. L. Lindstrom, H. G. Tran, K. L. Roinick, P. J. Costa

et al., 2003 Chromatin remodeling protein Chd1 interacts withtranscription elongation factors and localizes to transcribedgenes. EMBO J. 22: 1846–1856.

Smith, C. L., and C. L. Peterson, 2005 ATP-dependent chromatinremodeling. Curr. Top. Dev. Biol. 65: 115–148.

Srinivasan, S., J. A. Armstrong, R. Deuring, I. K. Dahlsveen, H.McNeill et al., 2005 The Drosophila trithorax group proteinKismet facilitates an early step in transcriptional elongation byRNA polymerase II. Development 132: 1623–1635.

Stokes, D. G., K. D. Tartof and R. P. Perry, 1996 CHD1 is concen-trated in interbands and puffed regions of Drosophila polytenechromosomes. Proc. Natl. Acad. Sci. USA 93: 7137–7142.

Verheyen, E., and L. Cooley, 1994 Looking at oogenesis. MethodsCell Biol. 44: 545–561.

Zhang, L., S. Schroeder, N. Fong and D. L. Bentley, 2005 Alterednucleosome occupancy and histone H3K4 methylation in re-sponse to ‘transcriptional stress.’ EMBO J. 24: 2379–2390.

Communicating editor: M.-C. Yao

Note 587