1

γTub23C interacts genetically with Brahma chromatin-remodeling complexes in

Drosophila melanogaster.

Martha Vázquez1, Monica T. Cooper2, Mario Zurita1, and James A. Kennison2.

1Departamento de Fisiología Molecular

y Genética del Desarrollo,

Instituto de Biotecnología, UNAM.

Cuernavaca, Morelos 62250, México.

2Laboratory of Molecular Genetics,

Eunice Kennedy Shriver National Institute of Child Health and Human Development,

National Institutes of Health

Bethesda, MD 20892, USA

Genetics: Published Articles Ahead of Print, published on September 9, 2008 as 10.1534/genetics.108.093492

2

Running head: γTub23C interacts genetically with Brahma

Keywords: . γ-tubulin, Brahma, Grip91

Corresponding author: Martha Vázquez

Av. Universidad 2001

Departamento de Fisiología Molecular

y Genética del Desarrollo,

Instituto de Biotecnología, UNAM

Cuernavaca, Morelos 62250, México

**mvazquez@ibt.unam.mx

Phone: (52)(55)56227631

Fax: (52)(777)3172388

3

ABSTRACT

The brahma gene encodes the catalytic subunit of the Drosophila melanogaster BRM

chromatin-remodeling complexes. Screening for mutations that interact with brahma, we

isolated the dominant-negative Pearl-2 allele of γTub23C. γTub23C encodes one of the

two γ-tubulin isoforms in Drosophila and is essential for zygotic viability and normal

adult patterning. γ-tubulin is a subunit of microtubule organizer complexes. We show that

mutations in lethal (1) discs degenerate 4, which encodes the Grip91 subunit of

microtubule organizer complexes, suppress the recessive lethality and the imaginal

phenotypes caused by γTub23C mutations. The genetic interactions between γTub23C

and chromatin remodeling mutations suggest that γ-tubulin might have a role in

regulating gene expression.

4

The trithorax and Polycomb group genes encode positive and negative factors required

for the proper function of homeotic genes. Kennison and Tamkun (KENNISON and

TAMKUN 1988) identified brahma (brm) as a trithorax group gene required for the

maintenance of homeotic gene expression, but brm also regulates the expression of many

developmental regulators and facilitates global transcription from RNA polymerase II

(ARMSTRONG et al. 2002). The Brm protein is a SWI2/SNF2 family-ATPase and is the

catalytic subunit of BRM chromatin remodeling complexes. These complexes modify

nucleosome structure; they can also act to generate Z-DNA structures (reviewed in

FLAUS and OWEN-HUGHES 2004).

Drosophila BRM complexes and related mouse and human SWI/SNF complexes have

roles in a variety of processes, including cell proliferation, differentiation, viral infection,

and cancer (reviewed by (ROBERTS and ORKIN 2004). Targeting of the BRM complexes

for transcriptional regulation involves contact with members of the basal transcription

machinery and gene-specific transcriptional activators (for examples (ARMSTRONG et al.

2005; SHARMA et al. 2003). To identify proteins that are required for proper function of

homeotic genes, we screened for mutations that showed genetic interactions with brm

mutations to cause a held-out wings phenotype. This approach allowed us to isolate

mutations in the trithorax group genes osa, tonalli, and taranis (GUTIÉRREZ et al. 2003;

VÁZQUEZ et al. 1999). In this work, we describe the characterization of another mutation

isolated in this genetic screen, the Pearl-2 allele of γTub23C. Some γTub23C mutant

phenotypes are modified (enhanced or suppressed) by mutations in genes encoding

subunits of the BRM complexes, and by mutations in Grip91, a γ -tubulin ring complex

subunit. These data suggest a role for γ-tubulin in transcription.

5

MATERIALS AND METHODS

Fly strains: Flies were raised at 25oC on a yeast-sucrose-agar medium with either

Nipagin or propionic acid or on a cornmeal-molasses-yeast-agar medium with Tegosept.

Unless otherwise noted, all mutations and chromosome aberrations are described in

Lindsley and Zimm (LINDSLEY and ZIMM 1992). Mutant stocks carrying l(1)dd4G0122,

l(1)dd42, l(2)23CeA6-2, l(2)23CeA14-9, l(2)23CeA15-2, and γTub23Cbmps1 were provided by

the Bloomington stock center.

Mutant phenotypes: The viability (in percentage) of homozygous or heteroallelic

combinations of alleles was determined by dividing the observed number of flies by the

expected number and multiplying by 100%. The expected numbers were calculated by

counting the numbers of progeny in the crosses that received the balancer chromosomes

and dividing by half.

Genetic mapping: The γTub23CPl-2 mutant was first mapped meiotically between the

visible markers al and dp.

The dd4su(Pl) mutation was first mapped by meiotic recombination using visible

markers. Individual recombinant sons from females heterozygous for dd4su(Pl) and a y2

wa ct6 g2 f mutant chromosome were recovered and tested for the survival of

γTub23Cbmps1/γTub23CA6-2 transheterozygotes. After the initial mapping, 28

recombinants between ct6 and g2 and 13 recombinants between g2 and f were recovered

and tested. None of these recombinants separated the suppressor from g+.

6

P-induced male recombination mapping of γTub23CPl-2: Females with the P-element

insertions shown in Table 1 were crossed to males of the genotype al γTub23CPl-2 KrIf/+;

TMS, P{ry+t7.2=Delta2-3}99B/+. Sons that were P{X}/ al γTub23CPl-2 KrIf; TMS/+ were

crossed to al dp b pr c px sp females and the progeny scored for recombinants between al

and KrIf. Recombinants were recovered and balanced for further testing. To determine

which recombinants carried flanking deletions that removed essential genes, each

recombinant chromosome was crossed to deletions in 23CD and to known mutants in

23C (lilli, l(2)23Cb, l(2)23Cd, γTub23CA6-2, and okra). Although l(2)23Cb has been

renamed l(2)23Dd by Flybase, our deletion mapping places the gene between Rpb9 and

γTub23C, consistent with the original mapping to 23C and the original gene name.

Molecular analyses: After γTub23CPl-2 was mapped between P{EP}Rrp1020 and

P{EPgy2}CG9643EY07345, the DNA sequences of the open reading frames of all four

predicted genes in the region (CG9641, CG3165, CG9643, and γTub23C) were

determined from DNA isolated from homozygotes of γTub23CPl-2 (and the parental

w;red e strain in which it was induced) and γTub23CA6-2 (and the parental cn bw

chromosome in which it was induced). As the only non-synonomous changes found

between the two mutants and their parental chromosomes were in the γTub23C open

reading frame (Figure 2C), we then determined the DNA sequence of γTub23C from

γTub23CA14-9 and γTub23CA15-2 homozygotes. Sequencing was done from PCR amplified

genomic fragments. Mutant homozygotes were identified using a GFP-expressing

balancer chromosome [CyO, P{w+mC=ActGFP}JMR1].

7

RESULTS

The γTub23CPl-2 mutation enhances brahma mutants: Flies heterozygous for some

combinations of mutations in trithorax group genes have held-out wings (VÁZQUEZ et al.

1999). On the basis of this phenotype we isolated several dominant enhancers of brm,

including alleles of the trithorax group genes osa (osa), tonalli (tna), and taranis (tara)

(GUTIÉRREZ et al. 2003; VÁZQUEZ et al. 1999). From that same genetic screen, we also

isolated the γTub23CPl-2 mutation. In addition to its dominant enhancement of brm (Table

2 and Figure 1C), γTub23CPl-2 has additional dominant phenotypes in the wing blade

(Figure 1B), including pearl-like structures [predominantly in the second (L2) and/or

third (L3) wing vein(s)], blisters in the wing blade, and notches or gaps in the ventral and

dorsal margins in one or both wings (Figure 1B). γTub23CPl-2 heterozygotes also have

small round eyes. We mapped γTub23CPl-2 to the same chromosomal region as Pearl (Pl)

(ROSIN 1951; ROSIN 1952), a dominant mutation that had the same unique combination

of phenotypes. The original Pl mutant is no longer extant. We originally called our

mutation, Pl2, but since it is allelic to γTub23C (see below), we have renamed it

γTub23CPl-2.

Mapping of γTub23CPl-2: We first mapped γTub23CPl-2 by meiotic recombination, and

then by complementation with available chromosome deletions, to polytene chromosome

subdivisions 23CD (Figure 2A). We next used the P-element insertion lines in the 23CD

region to map γTub23CPl-2 by P-induced male-recombination (CHEN et al. 1998). Of 63

recombinants recovered, all except one appear to have resulted from recombination at the

P-element insertion site. These 62 recombinants all place γTub23CPl-2 in the 7kbp region

8

between P{EP}Rrp11020 and P{EPgy2} CG9643EY07345 (Figure 2B). Inverse PCR with

recombinants that still retained the P insertion, and genetic complementation with

mutants in the region, were both used to identify recombinants with flanking deletions

and duplications in the 23BD region. The flanking deletions were useful for determining

the order of the essential genes in the region. One of the flanking deletions recovered by

P-induced male recombination from P{EPgy2}CG9643EY07345 is Df(2L)3G, which

behaves as a deletion of γTub23CPl-2 (Table 3), but does not delete any of the other

essential genes in 23C that have been identified [lilli, Rpb9, or l(2)23Cb].

l(2)23Ce is allelic to γTub23CPl-2: We mapped all of the phenotypes associated with

γTub23CPl-2 (the held-out wing phenotype in the presence of brm alleles and the

dominant phenotypes in the wing blade) to polytene chromosome bands 23CD. We then

tested mutants previously mapped to 23CD. We found that all alleles of l(2)23Ce that

we tested [l(2)23CeA6-2, l(2)23CeA14-9, and l(2)23CeA15-2] failed to complement

γTub23CPl-2 for viability (Table 3). We have renamed the l(2)23Ce alleles as γTub23C A6-

2, γTub23CA14-9, and γTub23CA15-2. When transheterozygous to other alleles, γTub23CA15-

2 is similar to the two deficiencies tested [Df(2L)JS17 and Df(2L)3G], and is probably a

null allele. The γTub23CA14-9 allele behaves as a hypomorph, often eclosing when

heterozygous to the deficiencies and most other alleles. All of the eclosing mutant flies

(regardless of which combination of alleles) have the same phenotypes observed for

γTub23CPl-2 heterozygotes (small round eyes and held-out and blistered wings with

pearl-like structures), but with far fewer notches in the wing margins. While flies

hemizygous for γTub23CA14-9 eclosed at 70 % to 83% of the expected numbers, no

9

γTub23CA14-9/γTub23CPl-2 flies eclosed. γTub23CPl-2 is an antimorphic, or dominant-

negative, allele and the phenotypes observed in γTub23CPl-2 heterozygotes are loss-of-

function phenotypes for γTub23C caused by interference of the γTub23CPl-2 mutant

protein with the wild-type γTub23C protein. Consistent with this interpretation is the

suppression of the dominant phenotypes of γTub23CPl-2 by an additional wild-type copy

of γTub23C (observed with both Dp(2;1)JS13 and with tandem duplications recovered

from the P-induced male recombination). These tests allowed us to establish a γTub23C

allelic series, being in order of decreasing activity: γTub23CA14-9>γTub23CA6-2>

γTub23CA15-2=Df(2L)JS17=Df(2L)3G>γTub23CPl-2.

Molecular characterizations: We mapped the γTub23CPl-2 mutation to a 7 kbp

genomic region that includes four predicted genes, γTub23C, CG9641, CG3165, and

CG9643 (Figure 2B). Our analyses showed changes in the γTub23C open-reading frame

for all four alleles that we sequenced (Figure 2C). The γTub23C gene encodes a protein

of 475 residues. The γTub23CA15-2 mutation changes tryptophan 104 to a stop codon,

predicting the formation of a truncated protein. This is in agreement with our

complementation analyses that suggested γTub23CA15-2 behaves as a null mutation.

γTub23CA14-9 changes arginine 217 to histidine (R217H), γTub23CA6-2 changes serine 233

to phenylalanine (S233F), and γTub23CPl-2 changes methionine 382 to isoleucine

(M382I).

10

Differential suppression of γTub23C lethality by an X-linked suppressor:

Subsequent to our molecular analyses, we obtained the γTub23Cbmps1 mutant (MAHONEY

et al. 2006). While γTub23Cbmps1 and γTub23CPl-2 both change the same methionine to

isoleucine (M382I), the description of γTub23Cbmps1 differed from our observations for

γTub23CPl-2 in two important aspects. The first difference was the much lower

penetrance of the dominant phenotypes for γTub23Cbmps1. The second important

difference was the eclosion of many γTub23Cbmps1 hemizygotes. We observed these

same differences when we began experiments with γTub23Cbmps1, but noticed striking

differences in the results of the reciprocal crosses with the γTub23Cbmps1 mutant flies.

For example, when γTub23CA6-2 heterozygous females were mated to γTub23Cbmps1

heterozygous males, no γTub23Cbmps1/γTub23CA6-2 mutant progeny eclosed. In the

reciprocal cross in which γTub23Cbmps1 heterozygous females were mated to γTub23CA6-2

heterozygous males, 73% of the expected γTub23Cbmps1/γTub23CA6-2 sons eclosed (Tables

3 and 4), while less than 2% of the expected γTub23Cbmps1/γTub23CA6-2 daughters eclosed

(Table 4). The eclosion of mutant sons only when the mothers were from the

γTub23Cbmps1 stock suggested the presence of an X-linked suppressor in this stock. We

replaced the X chromosome in the γTub23Cbmps1 stock with an X chromosome marked

with w1 and repeated the crosses. The penetrance of the dominant phenotypes in

γTub23Cbmps1 heterozygotes was much greater and the differential eclosion of mutant

sons in reciprocal crosses disappeared. When we replaced the X chromosomes in the

other γTub23C mutant and deficiency stocks with the X chromosome from the original

γTub23Cbmps1 stock, we found that more mutant flies eclosed. The γTub23Cbmps1 stock

11

contains a recessive X-linked suppressor that we named l(1)dd4su(Pl) (see the following

section for the allelism to l(1)dd4). We will refer to the suppressor as dd4su(Pl) for the

remainder of this work. The survival of γTub23C mutant genotypes with the suppressor

[dd4su(Pl)] and without [dd4+] are given in Table 3.

While most heteroallelic γTub23C combinations were lethal, dd4su(Pl) rescued some

genotypes to eclosion (Table 3). Based on the ability to be rescued by dd4su(Pl), we

separated heteroallelic γTub23C combinations into two classes (Class I and Class II,

Table 3). For example, for the Class I genotype γTub23Cbmps1/γTub23CA6-2 no flies

eclosed when they were dd4+ but 73% eclosed when they were dd4su(Pl). The same effect

was observed for γTub23CPl-2/γTub23CA6-2 and γTub23CA14-9/γTub23CA15-2 flies, where

6% and 25% eclosed, respectively, with dd4+, but 74% and 94% eclosed, respectively,

with dd4su(Pl). For Class II genotypes, there were almost no differences in the eclosion

rates between dd4+ and dd4su(Pl).

The Class I genotypes include one, and only one, of the following alleles: γTub23CA14-9,

γTub23CPl-2, and γTub23Cbmps1. Genotypes that do not include one of these three

suppressible alleles (or which include two of the suppressible alleles) are Class II. The

failure of suppression in flies with two suppressible alleles is interesting. For example,

dd4+ flies heterozygous for one of the suppressible alleles, γTub23CPl-2 or γTub23Cbmps1,

do not eclose when also heterozygous for a deficiency or for the weak hypomorphic allele

γTub23CA14-9. The dd4su(Pl) flies that are heterozygous for γTub23CPl-2 or γTub23Cbmps1,

however, eclose at 16-24% of the expected frequencies if also heterozygous for one of

the deficiencies, but at only 1-2% of the expected frequency if also heterozygous for the

hypomorphic (and suppressible) γTub23CA14-9 allele.

12

The X-linked suppressor of γTub23C lethality is in the gene encoding the Grip91 γ-

tubulin-interacting protein: We used meiotic recombination to map the X-linked

suppressor in the γTub23Cbmps1 stock to a region that includes the garnet gene. Because

we recovered no recombinants between the suppressor and garnet in our mapping

experiments, the suppressor is very close to garnet. The gene next to garnet in the

genome is lethal (1) discs degenerate 4 [l(1)dd4], which we will refer to as dd4. dd4

encodes the Grip91 protein, a subunit of the γ-tubulin γTuSC and γTuRC complexes

(BARBOSA et al. 2000). Some dd4 mutant alleles cause zygotic lethality, while flies with

other mutant alleles survive in dry media and have held-up wings, absence of some

bristles, defects in abdominal segments, and male sterility (BARBOSA et al. 2000).

Although the suppressor had no mutant phenotype in either sex, we decided to test for

allelism to dd4 because of the biochemical data. We used two lethal alleles, dd42 and

dd4G0122, and the test genotype γTub23Cbmps1/γTub23CA6-2. The data are in Table 4.

Rescue of γTub23Cbmps1/γTub23CA6-2 lethality by the suppressor is almost completely

recessive (only 2% of the expected flies eclosed if heterozygous for the suppressor, while

77% survived if homozygous for the suppressor). Both lethal alleles of dd4 (dd42 and

dd4G0122) complemented the suppressor (dd4su(Pl)) for viability, with the mutant females

eclosing at the expected numbers. However, neither of the lethal alleles complemented

dd4su(Pl) for the suppression of γTub23Cbmps1/γTub23CA6-2 lethality, i.e., both lethal alleles

of dd4 also suppressed the γTub23C lethality. We conclude that the suppressor, dd4su(Pl),

is an allele of dd4. The suppression is a loss of function phenotype, but the suppressor

must retain some functions necessary for zygotic viability and fertility.

13

γTub23CPl-2 genetically interacts with BRM complex mutations, and with tna and

tara: γTub23CPl-2 was isolated because it interacts with brm2 to cause a held-out wings

phenotype (Table 2). Since several trithorax group mutants were isolated in this same

genetic screen, we tested γTub23CPl-2 for genetic interactions with a collection of

trithorax group mutants and with mutants in some general transcription factors. From the

general transcription factors, we chose to test Taf1, Taf4, and Taf6, and the Mediator

complex subunits Med12 and Med13 encoded by the trithorax-group genes kohtalo (kto)

and skuld (skd), respectively. We tested mutants in subunits of nucleosome-remodeling or

histone-modification complexes, including brm, mor, snr1, osa, kismet, ash1, ash2, and

trithorax. We also tested the trithorax group genes Trithorax-like (Trl), tna, tara,

verthandi (vtd), sallimus (sls), devenir (dev), Vha55, and urdur (urd). The mutations

tested and the results are summarized in Table 2. We found that only alleles of brm, osa,

and tna showed strong genetic interactions with γTub23CPl-2 to cause the held-out wings

phenotype. The genetic interactions between osa1 and γTub23CPl-2 (Table 2 and Figure

1F-G) are even stronger than the genetic interactions between brm2 and γTub23CPl-2

(Table 2 and Figure 1C). Flies heterozygous for all three mutations (γTub23CPl-2, brm2,

and osa1) have poor survival and twisted and blistered wings even more severe than the

phenotypes of the γTub23CPl-2/+; osa1/+ double heterozygotes (Figure 1C-D). Other

mutations that showed weaker but significant interactions with γTub23CPl-2 included

brm1, osa2, mor1 , mor2, mor6, tna1, tara2, and tara20 (Table 2). We did not find

significant interactions with any of the other mutations tested. We considered the

possibility that the interactions between the BRM complex mutations and γTub23CPl-2

could be explained by reduced transcription of γTub23C. If heterozygous brm mutants

14

have reduced transcription of γTub23C, then all loss-of-function γTub23C phenotypes

should be enhanced in all mutant genotypes. To test this prediction, we chose several

combinations of hypomorphic γTub23C alleles to examine in brm2 heterozygotes.

CyO/γTub23CA14-9 ; TM6C/brm2 females were crossed to males with mutations or

deletions of γTub23C. A reduction in transcription would be expected to reduce viability

and enhance all of the γTub23C mutant phenotypes in progeny that receive the brm2

mutant when compared to their TM6C siblings. While γTub23CA14-9/γTub23CA6-2,

γTub23CA14-9/γTub23CA15-2, and γTub23CA14-9/ Df(2L)3G transheterozygous flies all have

reduced viability, the viability was not further reduced in brm2 heterozygous flies. As

expected, the held-out wings phenotype of γTub23CA14-9/ Df(2L)3G flies was enhanced

in brm2 heterozygotes, but the expressivity of pearl-like structures in the wings was not

enhanced. Finally, we examined interactions between brm2, dd4su(Pl) , and γTub23C.

The rescue of γTub23CPl-2/γTub23CA6-2 by dd4su(Pl) was reduced by about half in brm2

heterozygotes. The enhancement of the held out wings phenotype in γTub23CPl-2/+;

brm2/+ double heterozygotes was reduced from 76% to 14% in dd4su(Pl) males.

DISCUSSION

Proteins identified as part of the eukaryotic cytoskeleton may have more direct roles in

transcriptional regulation than originally thought (reviewed in OLAVE et al. 2002). Actin

and actin-related proteins (ARPs) are found in BRM complexes from yeast to humans,

including the BRM complexes in Drosophila (PAPOULAS et al. 1998). The function of

actin and ARPs in these complexes is not well understood. Some ARPs interact with

DNA-bending proteins and with histones and it was proposed that they facilitate

15

chromatin achitecture and interactions between complexes or function as histone

chaperones (SHEN et al. 2003; SZERLONG et al. 2003). Actin is also part of pre-initiation

complexes and is necessary for transcription by RNA polymerases I, II, and III

(HOFMANN et al. 2004; HU et al. 2004; PHILIMONENKO et al. 2004). The α- and/or β-

tubulins are also found with a subset of trithorax-group proteins in the mammalian

ASCOM complex (Activating signal cointegrator 2, Asc2-complex), which is required

for transactivation by nuclear receptors (GOO et al. 2003; LEE et al. 2006), and in a

histone H2A deubiquitinase complex (ZHU et al. 2007). γ-tubulin is essential for

microtubule function, but unlike α- and β-tubulin, it is not a component of microtubules.

Rather, it is located at microtubule-organizing centres (MTOCs) and functions in the

initiation of microtubule nucleation and in the establishment of microtubule polarity

(LUDERS and STEARNS 2007; OAKLEY 1992). γ-tubulin contributes to the proper

formation of mitotic spindles and cytoplasmic microtubular arrays. There are critical

cytoskeletal and nuclear envelope connections, linking for example MTOCs to the

nuclear lamina (reviewed in (TADDEI et al. 2004). In addtion, γ-tubulin has been proposed

to have microtubule- and/or centrosome-independent function(s) in mitosis (PRIGOZHINA

et al. 2004) or spindle assembly checkpoints (MULLER et al. 2006).

Drosophila embryonic γ-tubulin exists in two related complexes: a large complex

similar to the Xenopus γTuRC (γ-Tubulin Ring Complex) (36.9S, ~2000 kDa) and a

small soluble complex called γTuSC (γ-tubulin Small Complex) (8.5S ~240kDa)

(OEGEMA et al. 1999). The Drosophila γTuRC consists of ~8 polypeptides, including γ-

tubulin, Grip163, Grip128, Grip91, Grip84, Grip75, and GP71WD (GUNAWARDANE et al.

2000; GUNAWARDANE et al. 2003; OEGEMA et al. 1999). The γTuRC has a lockwasher-

16

like structure and a cap at one of the ends of the complex. The Drosophila γTuSC is a

tetramer of two γ-tubulin molecules and one molecule each of Grip91 and Grip84.

Several γTuSCs form the γTuRC lockwasher region. The other Grips (Grip163, 128, and

75) form the cap (MORITZ et al. 2000).

Drosophila is the only metazoan in which the genes encoding subunits of the γTuSC

and γTuRC complexes have been functionally studied using genetic approaches (for

recent examples BARBOSA et al. 2003; COLOMBIÉ et al. 2006; GUNAWARDANE et al.

2003; VOGT et al. 2006). Null mutations in dd4 (which encodes Grip91) and Grip84 are

lethal and display defects in spindle assembly (BARBOSA et al. 2003; COLOMBIÉ et al.

2006), while null mutations in Grip128 and Grip75 are viable, but sterile (SCHNORRER et

al. 2002; VOGT et al. 2006).

In Drosophila there are two γ-tubulin genes, γTub23C and γTub37C. They encode very

similar (but not identical) proteins, but they have different expression patterns and mutant

phenotypes. γTub37C is largely restricted to the female germ line and early stages of

embryogenesis. It is required for bicoid (bcd) mRNA localization at mid-oogenesis

(SCHNORRER et al. 2002), female meiosis, and nuclear proliferation (TAVOSANIS et al.

1997; WILSON et al. 1997). In syncytial embryos γTub23C is in the soluble small γTuSC

fraction (RAYNAUD et al. 2001) and is absent at the centrosome (WILSON et al. 1997). At

this stage, γTub37C is found in both the γTuSC and γTuRC fractions (RAYNAUD et al.

2001). It is localized at the centrosome (TAVOSANIS et al. 1997; WILSON et al. 1997) and

over the spindle regions (TAVOSANIS et al. 1997). γTub37C mutants are female sterile

(TAVOSANIS and GONZÁLEZ 2003; TAVOSANIS et al. 1997).

17

The γTub23C isoform is expressed in a variety of tissues in both sexes (WILSON et al.

1997), including larval brains and imaginal discs, and it is required for somatic mitotic

divisions. It is also expressed in ovaries and is the only isoform expressed in testes.

γTub23C is required for meiosis in males and for spermatogenesis (SAMPAIO et al. 2001).

γTub23C is essential for zygotic viability and for development of imaginal tissues:

We isolated the γTub23CPl-2 mutation in a mutant screen designed to identify genes that

interact with brm in wing development. In addition to showing genetic interactions with

brm, γTub23CPl-2 mutants are homozygous lethal, while the heterozygotes have defects in

imaginal eye and wing development. We showed that γTub23CPl-2 is a dominant-

negative mutation and that l(2)23Ce alleles are loss-of-function mutations in γTub23C

with recessive phenotypes similar to the dominant phenotypes of γTub23CPl-2. γTub23C

has 30% identity to α and β tubulins, which are structural components of microtubules. It

is known which parts of the β tubulin protein are involved in auto-regulation for

translation and for binding and hydrolisis of GTP. The γ-tubulin protein shares some of

these regions with β-tubulin. The γTub23C mutations characterized in this work do not

map to any of these known regions, with the exception of the truncated form in the

γTub23CA15-2 allele (Figure 2C). This suggests that the proteins synthesized from the

γTub23CA14-9, γTub23CA6-2, γTub23CPl-2, and γTub23Cbmps1 alleles might be affecting

other γ-tubulin functions (see below).

We were surprised during the course of this work to identify a dd4 allele with no

discernable phenotype except the suppression of some γTub23C mutant phenotypes

(including zygotic lethality). Since dd4 encodes Grip91, a protein that physically

18

interacts with γ-tubulin, we believe that the genetic interactions have important

implications.

Implications of the genetic interactions between γTub23C and dd4 (Grip91)

mutations on current structural models of γTuRC and γTuSC complexes: Grip91,

Grip84, and γ-tubulin form the lockwasher region of γTuRC and γTuSC complexes

(MORITZ et al. 2000). Grip91 and Grip84 (or their orthologues in yeast and human)

interact with each other and with γ-tubulin (ALDAZ et al. 2005; MORITZ et al. 2000;

WIESE 2008). The interactions between Grip91 and γ-tubulin facilitates binding of GTP

binding to γ-tubulin (GUNAWARDANE et al. 2003). Grip91 is required for correct bipolar

spindle assembly during mitosis and male meiosis and it helps to locate γ-tubulin to the

centrosome (BARBOSA et al. 2003; BARBOSA et al. 2000).

Grip91 is an essential protein encoded by the dd4 gene (BARBOSA et al. 2003;

BARBOSA et al. 2000). Semi-lethal alleles have held-up wings and other imaginal defects

and are male sterile (BARBOSA et al. 2003; BARBOSA et al. 2000). The dd4su(Pl) allele that

we identified is unusual in that it has no defects in viability, fertility, or developmental

patterning. Its only phenotype is the suppression of Class I (but not Class II) genotypes

of γTub23C.

What is the significance of the two types of γTub23C alleles from the functional point

of view? The defects produced by suppressible alleles may involve γTuSC and/or γTuRC

functions, while the defects produced by non-suppressible alleles may involve γTub23C

functions independent of the γTuSC and γTuRC complexes. It is also possible that

different mutant proteins, although in some cases retaining partial activity, may affect

19

other different functions of γTub23C. Some of these other functions may require Grip91

(and possibly the integrity of γTuRC and/or γTuSC complexes) and some may not. Such

functions could affect the assembly of the γTuSC and/or γTuRC complexes, the transport

of the complex(es) to subcellular compartments, and/or the relationships of γTub23C

with other proteins involved in microtubule-independent processes. We believe that the

new alleles of γTub23C and dd4 that we have characterized can help to test the current

structural models of γTuRC and γTuSC complexes proposed in the biochemical and

crystallographic studies (ERICKSON 2000; MORITZ et al. 2000).

Recent work shows that γ-tubulin has a microtubule-independent role in establishing or

maintaining a mitotic checkpoint block (PRIGOZHINA et al. 2004) and that γTuRCs

proteins may have a centrosome-independent role in the spindle assembly checkpoint.

For this last function γ-tubulin is probably in a complex associated with Cdc20 and

BubR1 kinases (MULLER et al. 2006). We have found that the genetic interactions

between γTub23C and Brm are caused not by reduced γTub23C transcription, but more

probably by the presence of defective γ-tubulin proteins. This suggests roles for γ-tubulin

in transcription and/or chromatin remodeling. This is further supported by the recent

description of interactions between Pericentrin (an integral centrosomal component) and

CHD3, a Brm-related protein in the NuRD chromatin-remodeling complex (SILLIBOURNE

et al. 2007).

This work was supported by grants from CONACyT (M.V.) and PAPPyT-UNAM (M.V.

and M. Z.), Howard Hughes Medical Institute (M. Z.). This research was supported in

part by the Intramural Research Program of the National Institutes of Health, NICHD.

20

LITERATURE CITED

ALDAZ, H., L. M. RICE, T. STEARNS and D. A. AGARD, 2005 Insights into microtubule

nucleation from the crystal structure of human γ-tubulin. Nature 435: 523-527.

ARMSTRONG, J. A., O. PAPOULAS, G. DAUBRESSE, A. S. SPERLING, J. T. LIS et al., 2002

The Drosophila BRM complex facilitates global transcription by RNA

polymerase II. EMBO J. 21: 5245-5254.

ARMSTRONG, J. A., A. S. SPERLING, R. DEURING, L. MANNING, S. L. MOSELEY et al.,

2005 Genetic screens for enhancers of brahma reveal functional interactions

between the BRM chromatin-remodeling complex and the Delta-Notch signal

transduction pathway in Drosophila. Genetics 170: 1761-1774.

BARBOSA, V., M. GATT, E. REBOLLO, C. GONZÁLEZ and D. GLOVER, 2003 Drosophila

dd4 mutants reveal that γTuRC is required to maintain juxtaposed half spindles in

spermatocytes. J. Cell Sci. 116: 929-941.

BARBOSA, V., R. R. YAMAMOTO, D. S. HENDERSON and D. GLOVER, 2000 Mutation of

a Drosophila γ-tubulin ring complex subunit encoded by discs degenerate-4

differentially disrupts centrosomal protein localization. Genes Dev. 14: 3126-

3139.

BURNS, R. G., 1995 Analysis of the γ-tubulin sequences: implications for the functional

properties of γ-tubulin. J. Cell Sci. 108: 2123-2130.

CHEN, B., T. CHU, E. HARMS, J. P. GERGEN and S. STRICKLAND, 1998 Mapping of

Drosophila mutations using site-specific male recombination. Genetics 149: 157-

163.

21

COLOMBIÉ, N., C. VÉROLLET, P. SAMPAIO, A. MOISAND, C. E. SUNKEL et al., 2006 The

Drosophila γ-tubulin small complex subunit Dgrip84 is required for structural and

functional integrity of the spindle apparatus. Mol. Biol. Cell 17: 272-282.

ERICKSON, H. P., 2000 γ-tubulin nucleation: template or protofilament? Nat. Cell Biol.

2: E93-E96.

FLAUS, A., and T. OWEN-HUGHES, 2004 Mechanisms for ATP-dependent chromatin

remodelling: farewell to the tuna-can octamer? Curr. Opin. Genet. Dev. 14: 165-

173.

GOO, Y. H., Y. C. SOHN, D. H. KIM, S. W. KIM, M. J. KANG et al., 2003 Activating

signal cointegrator 2 belongs to a novel steady-state complex that contains a

subset of trithorax group proteins. Mol. Cell. Biol. 23: 140-149.

GUNAWARDANE, R. N., O. C. MARTIN, K. CAO, L. ZHANG, K. DEJ et al., 2000

Characterization and reconstitution of Drosophila γ-tubulin ring complex

subunits. J. Cell Biol. 151: 1513-1523.

GUNAWARDANE, R. N., O. C. MARTIN and Y. ZHENG, 2003 Characterization of a new

γTuRC subunit with WD repeats. Mol. Biol. Cell 14: 1017-1026.

GUTIÉRREZ, L., M. ZURITA, J. A. KENNISON and M. VÁZQUEZ, 2003 The Drosophila

trithorax group gene tonalli (tna) interacts genetically with the Brahma

remodeling complex and encodes an SP-RING finger protein. Development 130:

343-354.

HOFMANN, W. A., L. STOJILJKOVIC, B. FUCHSOVA, G. M. VARGAS, E. MAVROMMATIS et

al., 2004 Actin is part of pre-initiation complexes and is necessary for

transcription by RNA polymerase II. Nat. Cell Biol. 6: 1094-1101.

22

HU, P., S. WU and N. HERNÁNDEZ, 2004 A role for β-actin in RNA polymerase III

transcription. Genes Dev. 18: 3010-3015.

KENNISON, J. A., and J. W. TAMKUN, 1988 Dosage-dependent modifiers of Polycomb

and Antennapedia mutations in Drosophila. Proc. Natl. Acad. Sci. USA 85: 8136-

8140.

LEE, S., D. K. LEE, Y. DOU, J. LEE, B. LEE et al., 2006 Coactivator as a target gene

specificity determinant for histone H3 lysine 4 methyltransferases. Proc. Natl.

Acad. Sci .U S A 103: 15392-15397.

LINDSLEY, D. L., and G. G. ZIMM, 1992 The genome of Drosophila melanogaster.

Academic Press, San Diego, Calif.

LUDERS, J., and T. STEARNS, 2007 Microtubule-organizing centers: a re-evaluation. Nat.

Rev. Mol. Cell Biol. 8: 161-167.

MAHONEY, M. B., A. L. PARKS, D. A. RUDDY, S. Y. K. TIONG, H. ESENGIL et al., 2006

Presenilin-based genetic screens in Drosophila melanogaster identify novel Notch

pathway modifiers. Genetics 172: 2309-2324.

MORITZ, M., M. B. BRAUNFELD, V. GUÉNEBAUT, J. HEUSER and D. A. AGARD, 2000

Structure of the γ-tubulin ring complex: a template for microtubule nucleation.

Nat. Cell Biol. 2: 365-370.

MULLER, H., M. L. FOGERON, V. LEHMANN, H. LEHRACH and B. M. H. LANGE, 2006 A

centrosome-independent role for γ-TuRC proteins in the spindle assembly

checkpoint. Science 314: 654-657.

OAKLEY, B. R., 1992 γ-tubulin: the microtubule organizer? Trends Cell Biol. 2: 1-5.

23

OEGEMA, K., C. WIESE, O. C. MARTIN, R. A. MILLIGAN, A. IWAMATSU et al., 1999

Characterization of two related Drosophila γ-tubulin complexes that differ in their

ability to nucleate microtubules. J. Cell Biol. 144: 721-733.

OLAVE, I. A., G. R. RECK-PETERSON and G. R. CRABTREE, 2002 Nuclear actin and

actin-related proteins in chromatin remodeling. Annu. Rev. Biochem. 71: 755-

781.

PAPOULAS, O., S. J. BEEK, S. L. MOSELEY, C. M. MCCALLUM, M. SARTE et al., 1998

The Drosophila trithorax group proteins BRM, ASH1 and ASH2 are subunits of

distinct protein complexes. Development 125: 3955-3966.

PHILIMONENKO, V. V., J. ZHAO, S. IBEN, H. DINGOVA, K. KYSELA et al., 2004 Nuclear

actin and myosin I are required for RNA polymerase I transcription. Nat. Cell

Biol. 6: 1165-1172.

PRIGOZHINA, N. L., C. E. OAKLEY, A. M. LEWIS, T. NAYAK, S. A. OSMANI et al., 2004

γ-tubulin plays an essential role in the coordination of mitotic events. Mol. Biol.

Cell 15: 1374-1386.

RAYNAUD, M., B., A. DEBEC, Y. TOLLON, M. GARES and M. WRIGHT, 2001 Differential

properties of the two Drosophila γ-tubulin isotypes. Eur. J. Cell Biol. 80: 643-649.

ROBERTS, C. W., and S. H. ORKIN, 2004 The SWI/SNF complex-chromatin and cancer.

Nat. Rev. Cancer 4: 133-142.

ROSIN, S., 1951 Zur Entwicklungsphysiologie der mutante Pearl (Pl) von Drosophila

melanogaster. Developmental physiology of the mutant Pearl (Pl) of Drosophila

melanogaster. Rev. suisse Zool. 58: 398-403.

24

ROSIN, S., 1952 Veränderungen des borstenmusters bei der mutante Pearl von

Drosophila melanogaster. Alterations of bristle patterns in the Pearl mutant of

Drosophila melanogaster. Rev. suisse Zool. 59: 261-268.

SAMPAIO, P., E. REBOLLO, H. VARMARK, C. E. SUNKEL and C. GONZÁLEZ, 2001

Organized microtubule arrays in γ-tubulin-depleted Drosophila spermatocytes.

Curr. Biol. 11: 1788-1793.

SCHNORRER, F., S. LUSCHNIG, I. KOCH and C. NÜSSLEIN-VOLHARD, 2002 γ-tubulin37C

and γ-tubulin ring complex protein 75 are essential for bicoid RNA localization

during Drosophila oogenesis. Dev. Cell 3: 685-696.

SHARMA, V. M., B. LI and J. C. REESE, 2003 SWI/SNF-dependent chromatin

remodeling of RNR3 requires TAF(II) and the general transcription machinery.

Genes Dev. 17: 502-515.

SHEN, X., H. XIAO, R. RANALLO, W. H. WU and C. WU, 2003 Modulation of ATP-

dependent chromatin-remodeling complexes by inositol polyphosphates. Science

299: 112-114.

SILLIBOURNE, J. E., B. DELAVAL, S. REDICK, M. SINHA and S. J. DOXSEY, 2007

Chromatin remodeling proteins interact with pericentrin to regulate centrosome

integrity. Mol. Biol. Cell 18: 3667-3680.

SZERLONG, H., A. SAHA and B. R. CAIRNS, 2003 The nuclear actin-related proteins

Arp7 and Arp9: a dimeric module that cooperates with architectural proteins for

chromatin remodeling. EMBO J. 22: 3175-3187.

TADDEI, A., F. HEDIGER, F. R. NEUMANN and S. M. GASSER, 2004 The function of

nuclear architecture: a genetic approach. Annu. Rev. Genet. 38: 305-345.

25

TAVOSANIS, G., and C. GONZÁLEZ, 2003 γ-tubulin function during female germ cell

development and oogenesis in Drosophila. Proc. Natl. Acad. Sci. U S A 100:

10263-10268.

TAVOSANIS, G., S. LLAMAZARES, G. GOULIELMOS and C. GONZÁLEZ, 1997 Essential

role for γ-tubulin in the acentriolar female meiotic spindle of Drosophila. EMBO

J. 16: 1809-1819.

VÁZQUEZ, M., L. MOORE and J. A. KENNISON, 1999 The trithorax-group gene osa

encodes an ARID-domain protein that interacts with the Brahma chromatin-

remodeling factor to regulate transcription. Development 126: 733-742.

VOGT, N., I. KOCH, H. SCHWARZ, F. SCHNORRER and C. NUSSLEIN-VOLHARD, 2006 The

γTuRC components Grip75 and Grip128 have an essential microtubule-anchoring

function in the Drosophila germline. Development 133: 3963-3972.

WIESE, C., 2008 Distinct Dgrip84 isoforms correlate with distinct γ-tubulins in

Drosophila. Mol. Biol. Cell 19: 368-377.

WILSON, P. G., Y. ZHENG, C. E. OAKLEY, B. R. OAKLEY, G. G. BORISY et al., 1997

Differential expression of two γ-tubulin isoforms during gametogenesis and

development in Drosophila. Dev. Biol. 184: 207-221.

ZHU, P., W. ZHOU, J. WANG, J. PUC, K. A. OHGI et al., 2007 A histone H2A

deubiquitinase complex coordinating histone acetylation and H1 dissociation in

transcriptional regulation. Mol. Cell 27: 609-621.

26

Table 1. P-induced male recombination.

____________________________________________________

P-transposon Polytene No. of

insertion Location recombinants

____________________________________________________

P{lacW}lilli05431 23C1-3 4

P{SUPor-P}Rrp1KG01159 23C3 9

P{EP}Rrp11020 23C3-4 3

P{EPgy2} CG9643EY07345 23C4 31

P{SUPor-P}CG3558KG02323 23C4 7

P{lacW}v(2)k05816 23C5 3

P{PZ}toc01361 23D2 3

P{lacW}Madk00237 23D3 3

27

Table 2. γTub23CPl-2 interactions with general transcription machinery and

trithorax-group mutants.

____________________________________________________________

Genotype Number of flies with Penetrance

held-out wings/Total (%)

____________________________________________________________

brm2/+ 9/498 2

mor1/+ 0/120 0

osa1/+ 6/208 3

osa2/+ 0/41 0

tna1/+ 19/115 17

tara2/+ 0/129 0

tara20/+ 0/151 0

γTub23CPl-2/+ 5/727 1

γTub23CPl-2/+; brm1/+ 16/46 35

γTub23CPl-2/+; brm2/+ 71/94 76

γTub23CPl-2/+; mor1/+ 10/105 10

γTub23CPl-2/+; mor2/+ 18/43 42

γTub23CPl-2/+; mor6/+ 20/107 19

γTub23CPl-2/+; osa1/+ 307/314 98

28

γTub23CPl-2/+; osa2/+ 17/44 39

γTub23CPl-2/+; tna1/+ 111/148 75

γTub23CPl-2/+; tara2/+ 19/88 22

γTub23CPl-2/+; tara20/+ 19/91 21

When trans-heterozygous with γTub23CPl-2, the following mutations gave no more than

3% penetrance for the held-out wings phenotype: Taf11, Taf41, Taf4S466, Taf61, Taf62,

trxE2, trxB11, ash16, ash1B1, ash21, ash22, kis1, kis2, kto1, kto3, vtd3, vtd14, sls1, dev1, dev2,

Vha5512, Vha5516, urd2, Trl3, Trl62, and skd3.

29

Table 3. Survival to eclosion (%) of γTub23C mutant transheterozygotes with dd4+ or dd4su(Pl)

_________________________________________________________________________________

dd4su(Pl) suppressible (Class I) dd4su(Pl) non-suppressible (Class II)

__________________________________________________________________________________

γTub23C genotype dd4+ dd4su(Pl) γTub23C genotype dd4+ dd4su(Pl)

__________________________________________________________________________________

γTub23CA14-9/Df(2L)3G 83 100 γTub23CA6-2/γTub23CA15-2 3* 0

γTub23CA14-9/Df(2L)JS17 70 100 γTub23CA6-2/Df(2L)3G 2* 0

γTub23CA14-9/γTub23C A6-2 65 74 γTub23CA6-2/Df(2L)JS17 0 0

γTub23CA14-9/γTub23CA15-2 25 94 γTub23CA14-9/γTub23Cbmps1 0 2

γTub23CPl-2/γTub23CA6-2 6* 74 γTub23CA14-9/γTub23CPl-2 0 1

γTub23Cbmps1/γTub23CA6-2 0 73 γTub23CA15-2/Df(2L)JS17 0 2

γTub23CPl-2/Df(2L)JS17 0 24 γTub23CA15-2/Df(2L)3G 0 0

γTub23CPl-2/Df(2L)3G 0 19 Df(2L)3G/Df(2L)JS17 0 0

γTub23Cbmps1/Df(2L)3G 0 19 γTub23CPl-2/γTub23Cbmps1 0 0

30

γTub23Cbmps1/Df(2L)JS17 0 16

γTub23CA15-2/γTub23Cbmps1 0 13

γTub23CA15-2/γTub23CPl-2 0 7

*eclosed, but all quickly became stuck in the medium.

31

Table 4. Suppression of γTub23Cbmps1/γTub23CA6-2 lethality by dd4 mutations.

__________________________________________________________

dd4 genotype Survival of γTub23Cbmps1/γTub23CA6-2 flies

# observed/expected %

_________________________________________________________

+/Y 0/351 0

+/+ 0/218 0

+/dd4su(Pl) 8/478 2

+/dd42 0/38 0

+/dd4G0122 2/109 2

dd4su(Pl)/Y 186/255.5 73

dd4su(Pl)/dd4su(Pl) 58/75.5 77

dd4su(Pl)/dd42 102/101 101

dd4su(Pl)/dd4G0122 105/105 100

The expected numbers of γTub23Cbmps1/γTub23CA6-2 flies is half the number of CyO

individuals in the same crosses. For +/Y or dd4su(Pl)/Y genotypes the expected frequency

was calculated with the CyO sons and for the rest of the genotypes it was calculated with

the CyO daughters.

32

Figure legends.

Figure 1.- γTub23CPl-2-dependent phenotypes. (A) Wild-type fly with the wings held

back parallel to the body axis. (B) γTub23CPl-2/+ flies, note the notches and the pearl-like

structures in the wings (indicated by arrows, one of them shown in the inset). (C)

γTub23CPl-2/+; brm2/+ (shown in picture) and γTub23CPl-2/+; osa1/+ double heterozygous

flies have held-out wings in addition to the wing notches and pearl-like structures. (D)

γTub23CPl-2/+; brm2 /osa1 triple heterozygous fly with twisted wings (in addition to held-

out and notched wings with pearl-like structures). (E) Wild-type wing from Oregon R

stock. (F) Wing from γTub23CPl-2/+; osa1 /+ double heterozygous fly. The arrow indicate

a pearl-like structure along the third wing vein shown in the inset (G).

Figure 2.- Genetic and molecular characterization of the Pearl region in 23C. (A)

Deficiency mapping of the Pearl region. (B) Genomic map of region 23C. Predicted

transcriptional units in the region are indicated by arrows according their transcriptional

direction. P-element transposons in the region are indicated by inverted triangles. EP1020

is P{EP}Rrp11020, KG01159 is P{SUPor-P}Rrp1KG01159, and EY07345 is

P{EPgy2}CG9643EY07345. The wide line marks the 7 kbp region where γTub23CPl-2 was

localized by P-induced male recombination. (C) The amino acid sequences for the two γ-

tubulin proteins (labeled 23C and 37C) in Drosophila (WILSON et al. 1997) and the

changes found in the γTub23C mutant alleles. The arrows indicate the changes in the

γTub23C mutant alleles. At the top of each arrow are the allele name and the aminoacid

substitution. The numbered blocks indicate peptides with known or presumed functions

(taken from BURNS 1995) and references therein): 1 is a peptide implicated in the

33

autoregulation of β tubulin translation; peptides 2-10 are implicated in the binding or the

hydrolysis of GTP by β tubulin. Peptide 10 is also implicated in the release of α, β, and γ

tubulin from the TCP1α chaperonine. Note that none of the γTub23C mutations are in

any of the peptides with known or presumed functions and that all the changed residues

in the γTub23C mutants are identical in both γTub23C and γTub37C wild-type genes.