Trl-GAGA directly interacts with lola like and both are part of the

repressive complex of Polycomb group of genes

Krishnaveni Mishra, Vivek S. Chopra, Arumugam Srinivasan, Rakesh K. Mishra*

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India

Received 11 December 2002; received in revised form 1 April 2003; accepted 1 April 2003

Abstract

Epigenetic inheritance to maintain the expression state of the genome is essential during development. In Drosophila, the cis regulatory

elements, called the Polycomb Response Elements (PREs) function to mark the epigenetic cellular memory of the corresponding genomic

region with the help of PcG and trxG proteins. While the PcG genes code for the repressor proteins, the trxG genes encode activator proteins.

The observations that some proteins may function both as PcG and trxG member and that both these group of proteins act upon common cis

elements indicate at least a partial functional overlap among these proteins. Trl-GAGA was initially identified as a trxG member but later was

shown to be essential for PcG function on several PREs. In order to understand how Trl-GAGA functions in PcG context, we have looked for

the interactors of this protein. We identified lola like, aka batman, as a strong interactor of GAGA factor in a yeast two-hybrid screen. lolal

also interacts with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7 PRE mediated pairing dependent silencing of mini-white

transgene. These observations suggest a possible mechanism of how Trl-GAGA plays a role in maintaining the repressed state of target genes

involving lolal, which may function as a mediator to recruit PcG complexes.

q 2003 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Polycomb group of genes; Epigenetic inheritance; Polycomb response element; Trl-GAGA; lola like

1. Introduction

During development, differential expression patterns of

genes that define cell types once established, need to be

epigenetically maintained. Specific DNA sequences and the

protein complexes working in combination confer this

cellular memory. In Drosophila, early regulatory network of

genes, referred to as segmentation genes, setup the

expression pattern of homeotic genes during the first 2–3 h

of development. After this initial period, segmentation

proteins disappear but the expression pattern of homeotic

genes set up early on is maintained by a mechanism that

involves the protein product of a number of genes grouped

into Polycomb group (PcG) and trithorax group (trxG) of

genes. Generally, these two groups of genes function in

opposite directions. Mutations in the PcG genes lead to

ectopic activation of homeotic genes, which suggests that

their normal function is to repress genes. The trxG genes, on

the other hand, have been shown to be positive regulators

(Kennison, 1993; Pirrotta, 1998; Simon, 1995).

Protein products of PcG and trxG genes are recruited to

the target loci through common DNA sequences referred

to as Polycomb Response Elements, PRE (Chan et al., 1994;

Muller and Bienz, 1991; Simon et al., 1993). Although there

are no significant sequence similarities among different

PREs that have been functionally characterized so far, there

is at least one sequence motif that is conserved (Mihaly

et al., 1998) which turned out to be the binding site for a PcG

member, pleiohomeotic, pho (Brown et al., 1998). Among

the other sequence motifs that are present in most PREs is

the GAGA factor binding site. We have shown recently that

GAGA motifs are necessary for iab-7 PRE (Mishra et al.,

2001). The significance of GAGA binding sites in PRE

function has been demonstrated by us and others in case of

different PRE by showing that GAGA protein is a part of the

protein complex that is assembled at these cis elements

(Busturia et al., 2001; Hagstrom et al., 1997; Hodgson et al.,

2001; Horard et al., 2000; Mishra et al., 2001). These

observations suggest that multiple sequence motifs, in

certain combinations, contribute to PRE function, probably,

0925-4773/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved.

doi:10.1016/S0925-4773(03)00046-7

Mechanisms of Development 120 (2003) 681–689

www.elsevier.com/locate/modo

* Corresponding author. Tel.: þ91-40-2719-2658; fax: þ91-40-2716-

0311.

E-mail address: mishra@ccmb.res.in (R.K. Mishra).

by recruiting different subsets of proteins that form the

chromatin complex at different loci or same locus in

different cell type.

Involvement of Trl-GAGA in repressive function of PcG

proteins, however, raises a paradox as Trl-GAGA has been

identified as an activator protein, an enhancer of PEV and a

member of trxG of genes (Biggins and Tjian, 1988; Soeller

et al., 1993; Farkas et al., 1994). How does Trl-GAGA

carryout these apparently opposing functions? It is now

emerging that PcG and trxG proteins function on the same

cis elements and therefore PRE and TRE may be closely

linked or overlapping (Orlando et al., 1998; Strutt et al.,

1997; Tillib et al., 1999). The mechanism of this assembly

of chromatin structure that is capable of maintaining

expression of linked genes, is not clear yet. The GAGA

factor has been shown to be involved in ATP dependent

nucleosome remodeling (Tsukiyama et al., 1994). Recent

studies provide a direct link between chromatin remodeling

and PcG mediated maintenance of the repressive chromatin

(Czermin et al., 2002; Muller et al., 2002). Any direct link

between this remodeling and Trl-GAGA remains to be

established. On the contrary, GAGA protein was not

detected in the ESC-E(Z) protein complex capable of

histone H3 lysine 27 methylation which may be one of the

steps in remodeling process that ensures nucleosomes stay

in repressive chromatin conformation (Muller et al., 2002).

There still may, however, be a more dynamic role of

Trl-GAGA in the assembly of the PcG protein complex

(Poux et al., 2001a).

We decided to investigate the mechanism behind the

function of Trl-GAGA as a component of the repressive

Polycomb system by identifying the direct interacting

partners of this protein. We used the yeast two-hybrid

system to screen a cDNA library made from Drosophila

embryos. During this screen we identified lola like (lolal)

aka batman as one of the strong interactors of GAGA factor.

Since lolal has previously been shown to interact with

polyhomeotic, ph-p, a known PcG gene, it is likely that PRE

function of Trl-GAGA is mediated by lolal. Our genetic and

biochemical interaction studies presented here suggest that

lolal may be the effecter molecule for the Trl-GAGA

mediated PcG function.

2. Results

2.1. Identification of lolal as a potential partner for GAGA

GAGA factor contains three distinct domains, Fig. 1A

(Farkas et al., 1994). The BTB/POZ domains of several

proteins have been reported to mediate homo- and

heterodimerizations (Bardwell and Treisman, 1994) and

can be functionally swapped between two proteins (Read

et al., 2000). We reasoned that GAGA may carry out its

diverse roles by recruiting different proteins to the target

DNA sequences and the BTB domain may mediate this. We,

therefore, performed a yeast two-hybrid screen using the

BTB domain of GAGA, amino acids 1–245 (Fig. 1A) as the

bait. We screened the 0–16 h embryo cDNA library to

identify potential partners for Trl-GAGA protein. Out of the

46 adenine auxotrophs, 23 strongly activated the second

reporter gene, b-galactosidase. All the 23 positives inter-

acted specifically with the bait upon retransformation and 14

turned out to be one single gene lola like (lolal) (Fig. 1A).

All the clones had complete coding region of the gene.

LOLAL also interacts with the full length GAGA protein

(Fig. 1B). Quantitative assays showed that the interaction of

LOLAL with BTB and GAGA full-length protein was

comparable (data not shown). Indeed, in an independent

screen using full length GAGA protein, LOLAL appeared

eight times (Srinivasan, A., Mishra, K., Mishra, R.K.,

unpublished). We also isolated other BTB domain contain-

ing proteins multiple times and one already reported

interactor of Trl-GAGA, Sin3A (Espinas et al., 2000). We,

however, did not isolate GAGA factor itself in these screens,

which may suggest that BTB domain of this protein might

not form a homodimer. When tested in directed two hybrid

assay, though, we did see BTB–BTB interaction. It has

been reported that Trl-GAGA exists in several isoforms

(Benyajati et al., 1997). All the experiments reported here

have been done with the major isoform, GAGA 519. We do

not know if other isoforms can interact differently.

Furthermore, pipsqueak that binds to GAGA sequences

(Lehmann et al., 1998) and contains BTB domain has also

been shown to be essential for sequence specific targeting of

PcG protein complex (Huan et al., 2002). It is unclear,

however, how Trl-GAGA is placed in this multi-component

recruitment system working on GAGA motif of PREs.

Further studies will be needed to answer these questions.

lolal was originally identified in the Drosophila gene

disruption project (Spradling et al., 1999). Later on this

mutation was also found to enhance the homeotic phenotype

of polyhomeotic, ph, and renamed as batman (Faucheux

et al., 2001). lolal encodes a protein of 127 amino acids that

contains a BTB domain of about 90 amino acids, leaving

Fig. 1. (A) Molecular map showing the domains of GAGA factor and

LOLAL. The N-terminal region containing BTB domain of GAGA factor

was used in the yeast two hybrid screen. (B) Yeast transformants containing

the constructs indicated in the middle panel were patched on to synthetic

media lacking leucine and uracil (to select for the plasmids) and synthetic

media lacking leucine, uracil and adenine (to select for the interactors).

K. Mishra et al. / Mechanisms of Development 120 (2003) 681–689682

only few residues at both ends of the protein for any other

functional motif/domain. Another PcG protein, ESC,

consists almost entirely of six WD40 repeat motifs (Sathe

and Harte, 1995; Simon et al., 1995). Unlike multi-domain

proteins, the ones made of a single domain alone may

function as adaptor modules to bring together two different

molecules/complexes. Trl-GAGA is known to activate

transcription of several genes. In this context, lolal may

function to inhibit this activation role of Trl-GAGA, in a

way similar to MyoD–Id interaction (Sun et al., 1991),

further studies will be required to differentiate between

these mechanisms.

2.2. LOLAL interacts with GAGA factor in vitro and exists

in a complex that also contains PC

In order to further verify the GAGA–LOLAL inter-

action, we performed a GST pull-down assay. Full length

LOLAL was expressed as a GST fusion protein in

Escherichia coli. Nuclear extracts were prepared from

0–16 h wild-type Drosophila embryos and incubated with

either GST or GST–LOLAL. The complexes were pulled

down using glutathione Sepharose and tested for the

presence of GAGA protein by Western blotting. As shown

in Fig. 2A, GAGA protein comes down specifically with

GST–LOLAL. In the light of yeast two-hybrid interaction

studies, this assay further suggests that GAGA and LOLAL

probably interact directly. It has been shown that GAGA

factor associates with PC, PH and PSC (Poux et al., 2001a).

Since our results show a direct interaction between LOLAL

and GAGA factor, we tested if PC is also pulled down by

GST–LOLAL. As shown in Fig. 2A lower panel, GST–

LOLAL does enrich PC. We used antibodies raised against

two nuclear proteins, lamin B1 and F7BP1, to probe the

same blot to rule out any nonspecific protein–protein

interaction under these conditions. The two pull-down

experiments suggest that LOLAL is a part of the PcG

protein complex. LOLAL could be the adaptor between

GAGA and PC as the latter two coimmunoprecipitate

(Horard et al., 2000)

2.3. PH and GAGA factor interact in nuclear extracts

Earlier reports suggested that lolal enhances the sex

comb phenotype of ph (Faucheux et al., 2001). Also our

results described above suggest that, at least in vitro,

LOLAL can be associated with the GAGA containing

complex that includes PC and other PcG proteins. We

decided to test if GAGA and PH interact. The nuclear

extracts were made from 0–16 h embryo and larvae of

transgenic flies carrying FLAG-tagged PH (Shao et al.,

1999). Nuclei were isolated from the embryo or larvae and

extracts were prepared using standard protocols. To the

extracts as such or after incubation with bacterially

produced GST–LOLAL, M2 coupled agarose (Sigma)

was added. FLAG-PH bound complex isolated in this

manner was analyzed by western analysis for the presence

of GAGA. As shown in Fig. 2B, GAGA association with PH

in embryos was dependent on externally provided GST–

LOLAL, while in the larval extracts, PH was found to be

associated with GAGA factor, irrespective of whether or not

external GST–LOLAL was added.

2.4. Genetic interactions of lolal

2.4.1. Pairing dependent silencing caused by iab-7PRE is

dependent on lolal and ph

The pairing-dependent silencing or pairing sensitivity,

PS, assay is widely used for checking the PRE nature of a

given sequence and previous studies have shown that

mutations in PcG genes relieve this pairing-dependent

silencing (Chan et al., 1994; Gindhart and Kaufman, 1995;

Hagstrom et al., 1997; Kassis, 1994). We have already

shown that transgenic lines carrying iab-7 PRE next to

mini-white reporter, PRE-mw, show pairing dependent

silencing of the mini-white and that like several PcG

mutations, this silencing was affected by Trl mutations

(Hagstrom et al., 1997; Mishra et al., 2001). We used

these PRE-mw lines to test if lolal is required for the

PS function. Since lolal was shown to interact with ph

(Faucheux et al., 2001), we also checked ph in this PS

assay.

Fig. 2. (A) GST pull-down assay. Total nuclear extract (NE), equivalent to

one fourth of the input, are loaded as control for the antibody detection.

G-LOLAL is pull-down pellet loaded from the GST–LOLAL fusion

protein coupled to glutathione Sepharose, GST indicates the lane

where pull-down pellet is loaded from the GST protein coupled to

glutathione Sepharose as negative control. Western blotting with anti-

bodies against GAGA factor, POLYCOMB, LAMIN B and F7BP1 are

shown. (B) FLAG-PH coimmunoprecipitation: FLAG-PH nuclear extracts

prepared from embryos (EE) or larvae (LE) were incubated with GST-lolal

fusion protein or GST alone. The complex was then pulled down using M2

agarose. Western blot was probed with anti GAGA antibody.

K. Mishra et al. / Mechanisms of Development 120 (2003) 681–689 683

In several transgenic lines, PRE-mw/PRE-mw flies show

lighter eye color as compared to the PRE-mw/ þ flies

(Mishra et al., 2001). All the lines tested showed clear

requirement of both lolal and ph for the PS function, as the

eye color of PRE-mw/PRE-mw flies showed clear enhance-

ment when brought in lolal (ban Df(2R)311a) or ph ( ph-p)

background, Fig. 3. We got similar results with P element

insertion allele of lolal (lolal k02512), data not shown. In this

case, although there was a clear enhancement of pigmenta-

tion in PRE-mw/PRE-mw flies when tested in lolal back-

ground, we have also measured the red pigment (Ashburner,

1989), of these mutant combinations to distinguish the

increase in pigmentation from mere additive effect of the

light eye color of P element of the lolal allele. The results

show that value for PRE-mw/PRE-mw in lolal/CyO context

is 50% more than added values of PRE-mw/PRE-mw and

lolal/CyO flies (data not shown).

2.4.2. Interaction of lolal with other PcG and trxG genes

The sex comb phenotype of ph is known to be enhanced

by lolal and, by this criterion, lolal can be classified as a

PcG member. Often members of the PcG interact with one

another and also with the trxG members. We tested lolal in

different PcG and trxG mutant backgrounds, Table 1. Since

most of these mutations are recessive lethal, we could test

only the heterozygote combinations, except for ph which is

homozygous viable. As expected, we found sex combs on

the middle leg and often on the third leg in ph/y; lolal/ þ

males, Fig. 4A. We found similar interaction of lolal with

Pc and Pcl (Table 1). Interestingly, ph in combination with

lolal showed partial homeotic transformation of A4 ! A5,

A5 ! A6 and A6 ! A7, Fig. 5B. In contrast, Asx showed

partial A6 ! A5 transformation in the abdomen, Fig. 5C.

Pc in combination with lolal also gave a wing phenotype,

Fig. 4B. All lolal/ þ ; Pc/ þ flies had crumbled wings

with defective margin. Among the trxG mutations

tested, mor had phenotype of rough eye (Fig. 4C) and

trg showed homeotic transformation in the abdomen

(Fig. 5D). While trg/ þ flies have several bristles on

the 6th sternite in males, in the combination of lolal, we

noticed additional segment formed (shown by arrow

Fig. 3. Effect of lolal (A) and ph (B) on the pairing dependent silencing

iab-7 PRE–mini white (PRE-mw) transgenic lines. Eye color of female flies

of similar age is compared. The eye color of PRE-mw homozygous flies in

wild-type background is always lighter than those in lolal/CyO or ph/ph

background.

Fig. 4. Interaction of lolal with PcG and trxG genes. Panel (A) shows the

extra sex comb phenotype, indicated by arrows, in lolal/Pcl and lolal/ þ ;

ph males. I, II, III indicate anterior, middle and posterior legs. Similar

phenotype is seen in Pc mutation background also. Panel (B) shows the

wing phenotype of lolal/ þ ;Pc/ þ flies. All flies of this genotype with two

different alleles of Pc showed this defect. Panel (C) shows the rough eye

phenotype with irregular margin as found in lolal/ þ ;mor/ þ flies. This is

a variable phenotype with flies of this genotype showing normal to rough

eye defect to varying degree.

K. Mishra et al. / Mechanisms of Development 120 (2003) 681–689684

mark in Fig. 5D) indicating partial anteriorization,

transformation of A7 to A6.

Contrary to the expectations based on the biochemical

studies discussed above, we did not notice any apparent

interaction of lolal with Trl 13C in heterozygous condition.

Similarly, among the mutations that we tested, osa, trx,

ash1, Psc, Scm did not show any interaction with lolal.

Interestingly, it has been shown recently that Trl also

enhances ph phenotype in a fashion similar to what we see

in case of lolal and ph (Hodgson et al., 2001). This raises the

possibility that there may be a networking of Trl-lolal-ph

and, perhaps, other PcG proteins in the PRE mediated

repression mechanisms. These interactions of lolal with ph

and Pc suggest that association of lolal may be the key step

of PcG function of Trl-GAGA.

3. Discussion

Chromatin organization is a vital component of the

mechanism regulating gene expression in eukaryotes. The

molecular mechanisms of how these regulatory processes

take place – how active and inactive regions of chromatin

are established during development and maintained through

a number of cell divisions – is still far from being

understood. In Drosophila, extensive genetic and biochemi-

cal studies on the homeotic gene regulation has implicated

Polycomb and trithorax group of genes in the maintenance

of expressed state at the level of higher order chromatin

organization (Pirrotta, 1997; Simon, 1995). It is emerging

that high molecular weight complexes consisting of few

‘core’ PcG proteins and other variable components exist

(Hodgson et al., 2001; Horard et al., 2000; Poux et al.,

2001b; Saurin et al., 2001; Simon et al., 1993). The variable

members may determine stage specific or locus specific

function. For example, pho has been shown to be more

crucial for late larval and pupal stages as compared to the

early embryonic development (Poux et al., 2001b).

One of the key steps in the PcG/trxG mediated

maintenance is the recruitment of the multi-protein complex

of correct composition onto the PRE. Recent studies have

Table 1

Interaction of lolal with PcG and trxG genesa

Mutation Phenotypeb Comments

Pcl Sex comb on the 2nd leg All male flies, sex comb teeth per leg 4 ^ 1

Sex comb on the 3rd leg All male flies, sex comb teeth per leg 3 ^ 1

Pc1 Sex comb on the 2nd leg All male flies, sex comb teeth per leg 5 ^ 1

Sex comb on the 3rd leg All male flies, sex comb teeth per leg 2 ^ 1

Curved wings with posterior margin defect All flies show the phenotype

ph410 Sex comb on the 2nd leg All male flies, sex comb teeth per leg 3 ^ 2

Sex comb on the 3rd leg 12.5% male flies, sex comb teeth per leg 3 ^ 1

Segmental transformation All male flies show reduction in 6th segment

Asx[XF23] Bristle on the 6th male sternite 26.3% male flies

mor1 Rough eye (In females) This phenotype is quite variable with flies showing very mild to strong

defects

trg Segmental transformation 69.2% males show partial A7 to A6 transformation (47% males show this

phenotype when trg is brought paternally)

a Psc1, ScmR5.13B, ash1 B1, trxred, osa00090 phoCV and Trl13C did not show any interaction in double heterozygous conditions based on such clear visible

phenotypic criteria.b Phenotype shown is the most prominent one seen with the corresponding allele in lolal/ þ background. Phenotype reported for Asx[XF23], Pcl and ph

were seen when the PcG mutation was brought maternally. In case of Pc1, the Pc1/ þ ; þ /CyO siblings also showed 5 ^ 1 sex comb teeth per 2nd leg, while

this genotype did not show any sex comb teeth on the 3rd leg.

Fig. 5. Cuticular structures of the adult male abdomen. Anterior is top and

posterior is bottom. The abdomen was split mid-dorsally and flattened.

Cuticle preparation of differential mutant combinations with lolal is shown.

(A) is wild-type fly; (B) is lolal/ þ ;ph showing strong reduction in the size

of 6th tergite (indicated by black arrowhead) and additional pigmentation of

the 4th tergite (indicated by hatched arrow). (C) is lolal/ þ ;Asx/ þ

abdomen with few additional bristles on the 6th sternite (indicated by

arrow), wild-type male cuticle is devoid of bristles in this region. We almost

always found two bristles in these flies. The rest of the cuticle appears

normal. (D) is lolal/ þ ;trg/ þ cuticle preparation. These flies show

development of a small tergite (indicated by arrowheads). These

preparations have several bristles in the 6th sternite, which is not due to

lolal as these bristles are present in similar numbers in trg/ þ flies.

K. Mishra et al. / Mechanisms of Development 120 (2003) 681–689 685

shown that more than one or perhaps several recruiting

processes take place in concert. It is likely that different

recruitment possibilities provide the necessary variation that

is needed for the establishment and maintenance of varying

transcriptional states at hundreds of different loci. Our

studies identify a new member in this process. Trl-GAGA

bound to specific sites on the PREs recruits LOLAL, which

in turn, through direct or indirect means, incorporates PH

into the complex. This raises a question whether the other

recruiting agents like pho, zeste, etc. (Americo et al., 2002;

Brown et al., 1998) function in cooperation or competition

with each other. Also, it is not clear if the complex is

assembled de novo on the PREs, a pre-assembled complex

is recruited or partly assembled sub-complexes are

recruited. Since large complexes of PcG proteins can be

isolated, we can conclude that such structures, once

assembled are stable. It is not clear though if these

complexes are stable during cell division or they assemble

each time a cell divides.

As most of the PREs contain Trl-GAGA binding sites, it

is likely that at least some PcG complexes are recruited by

GAGA factor through its direct interaction with LOLAL.

These findings establish a molecular link between Trl-

GAGA and the PcG complex. Since Trl-GAGA functions in

several other processes that do not seem to be directly linked

to the PRE function, it is likely that there are several

interactors of Trl-GAGA. We are studying such interactors

and initial observations suggest that a large number of

proteins can interact with Trl-GAGA with a potential to

target a repressive or activator function to different loci. It is

not clear though how the selection of appropriate partner is

made. Is it in the context in which Trl-GAGA is bound or

different heterodimers preexist in the nucleus and these are

then recruited to appropriate loci? New assays will have to

be designed to appropriately answer these questions.

Our genetic interaction studies (Table 1) show that lolal

interacts with a variety of PcG and trxG mutations, Figs. 4

and 5. This underscores the important role of this protein in

the regulation of developmental genes. Interestingly, we

find that lolal interactions with ph mutation leads to

transformation of 2nd (and some times 3rd) leg to 1st leg,

an apparent anteriorization type of homeotic transformation

in thoracic but in the abdominal region same combination

leads to posteriorization type of homeotic transformation,

pigmentation of A4 (A4 ! A5) reduction in the size of A6

(A6 ! A7). However, appearance of sex comb in 2nd and

3rd legs is also known to be due to derepression of Scr in

posterior segments thereby explaining this phenotype as due

to loss of the repression function of the PcG proteins.

Furthermore, trxG and PcG mutations upon interaction with

lolal can give a similar phenotype. In lolal context, Asx and

trg both show partial A6 ! A5 transformation in abdominal

region. Pc is involved in pairing dependent silencing

complex recruited by iab-7 PRE. In this study we show

that ph is also involved in the PS function of iab-7 PRE.

While it was known that lolal enhances the homeotic

phenotype of ph, we demonstrate that both ph and lolal are

involved in establishing the repressive complex at the

iab-7 PRE. This indicates that lolal and ph function in

coordination to set up a repressive complex. Taken together,

these results suggest that lolal may be acting along with

Trl-GAGA or with other partners in different complexes in a

locus or stage specific manner. Depending on the context it

could be an activator or repressor function. Since not only

‘ON’ or ‘OFF’ but also several ‘levels of expression states’

for a given hox gene or indeed other regulated loci are

maintained, it is likely that a unique combination of trxG

and PcG proteins may be needed for each varying levels of

expression state of a given locus.

Affinity pull-down experiments, Fig. 2, show that

GAGA, LOLAL, PH and PC proteins coexist in a complex.

This is also in agreement with the genetic interaction

studies, where we found interaction of lolal with ph and Pc.

Genetic and biochemical studies also suggest that, like Trl,

lolal could also be not specifying the kind of complex to be

assembled. It is likely, therefore, that specificity of the

GAGA partner, PcG or trxG member, does not come from

the lolal. It is even possible that lolal could also be a

multifunctional adapter of Trl-GAGA in assembling multi

protein complexes. The specificity may come from yet

another factor or even from the transcriptional activity

around the locus. Recent observations that transcription

process itself may contribute to the cellular memory may

support this view (Bender and Fitzgerald, 2002; Hogga and

Karch, 2002; Rank et al., 2002). This might bring together

the ability of GAGA factor to support transcription and

recruitment of multi-protein complexes and nucleosome

remodeling activity in one mechanistic context.

Trl-GAGA has been suggested to be involved in creating

a nucleosome free region (Hagstrom et al., 1997). The first

step in establishing a PcG complex may be this Trl-GAGA

mediated nucleosome remodeling of the chromatin on the

PRE region to create a more accessible (DNase I

hypersensitive site). The recruitment of a protein complex

to the accessible region may take place through GAGA

factor or by other factors that can anchor the complex on to

DNA. As the next step LOLAL could mediate recruitment

of initial complex, for example, ESC-E(Z) protein complex,

which modifies nearby histone tails to covalently mark the

region for the recruitment of another complex, like PRC1.

Consistent histone modifications and remodeling may be

needed to maintain the chromatin conformation. Our studies

would place LOLAL as the factor binding to the DNA

bound GAGA even when the rest of the complex is not

recruited and therefore help in subsequent recruitment steps.

In this context, the exact function of proteins like LOLAL

becomes very important. Further studies will be required to

clarify these issues.

3.1. Note

While this manuscript was under reviewing process, lolal

K. Mishra et al. / Mechanisms of Development 120 (2003) 681–689686

(batman) was reported to interact with PcG and trxG genes

and also was found in a complex containing GAGA factor

(Faucheux et al., 2003).

4. Experimental procedures

4.1. Strains and plasmids

The full-length and BTB-containing GAGA fragments

were amplified using polymerase chain reaction (PCR)

primers and cloned in frame with the bait vector,

pGBDCU2. GST fusion constructs were made by sub

cloning full-length lolal from the pACT2-LOLAL obtained

from the screen into pGEX5x-2. E. coli strain XL1 blue was

used routinely. HB101 was used for amplifying yeast

plasmids and BL21 for expression of protein. All yeast

media were prepared according to Rose et al. (1990). PJ69-

4A (Mata trp1-901 leu2-3,112 ura3-52 his3-200 gal4 gal80

LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) was

used for the screen. All yeast transformations were done by

the high efficiency LiAc method (Gietz and Schiestl, 1995).

Library transformations were scaled up 10-fold.

4.2. Yeast two-hybrid screen

The system developed by James et al. (1996) was used

for the screen. The bait, BTB containing region of GAGA

factor (amino acids 1–245), was cloned in frame with the

Gal4 DNA binding domain of pGBDUC2. The strain

PJ69-4a was transformed with pGBDUC2-BTB and

checked to see if it turned on any of the reporters. The

bait activated the HIS3 reporter weakly and therefore the

screen was carried out using the ADE2 reporter. The strain

containing the bait was transformed with the Drosophila

embryonic cDNA library (embryo MATCH MAKER

cDNA library, Clonetech) and plated on to SC-Leu-Ura-

Ade selective plates. Out of about 50 000 transformants

screened, 46 adenine auxotrophs were obtained. Out of

these, 23 also activated the b-galactosidase reporter. The

GAL4 AD plasmid was extracted from yeast, amplified in

E. coli (HB101) and retransformed into PJ69 24A either

with the bait or with full length GAGA factor in the bait

vector or empty bait vector. All the 23 interacted

specifically with GAGA factor but did not activate the

reporter genes with the vector alone. Interactions were also

quantified using liquid b-galactosidase assays.

4.3. GST pull-down assay and coimmunoprecipitation

The pull-down assay was done essentially as described

(Sambrook and Russell, 2001). Briefly, 0–16 h embryos

were collected and nuclear extract was prepared as

described (Han et al., 1993). GST and GST–LOLAL fusion

protein were expressed in bacteria, BL21 and bound to

glutathione Sepharose. The Sepharose gel was washed

several times with PBS, resuspended as a 50% suspension

and stored at 4 8C. A small aliquot was checked on SDS

polyacrylamide gel for size and concentration of proteins.

Gel containing equal amounts of protein (30 ml) was added

to nuclear extract (100 mg of protein per reaction);

incubated for 2 h at 4 8C, washed several times with PBS

and finally resuspended in 30 ml of Laemmli buffer. The

samples were run on SDS polyacrylamide gel, transferred

onto Immobilon membrane and blotted with anti-GAGA

antibodies (a gift from Carl Wu) or anti-PC antibodies (a gift

from Renato Paro). The blots were developed using the

Chemiluminescence kit from NEN Life Science Products.

As negative controls, we also probed these blots with anti-

Lamin B (a gift from Veena Parnaik) and anti-F7BP1

antibodies. The anti-Lamin B is polyclonal rat lamin B1

antibody that recognizes DmO, the Drosophila lamin B

homolog (Jagatheesan et al., 1999). The anti-F7BP1 is

polyclonal antibody was raised against F7BP1, a protein

identified in a screen to isolate Fab-7 boundary binding

protein (R.K. Mishra, F. Karch, in preparation).

Overnight collection of FLAG-PH embryos was done

and nuclear extract was prepared as described above.

Similarly, 2nd and 3rd instar larvae were collected,

homogenized to isolate nuclei and nuclear extract was

prepared. These extracts were incubated with M2 affinity

agarose for 4–6 h at 4 8C, spun down and washed several

times in PBS. Finally, the agarose matrix was boiled in

loading buffer and separated on SDS–polyacrylamide gel

electrophoresis for Western analysis.

4.4. Fly strains and mutants

FLAG-PH expressing fly was supplied by Robert Kingston,

Trl mutations and iab-7 PRE lines were from Francois Karch,

lolal deletion mutation (ban Df(2R)311a) was from Laurent

Theodore and trg was supplied by Ruth Lehmann. Following

mutations used in this study were supplied by Bloomington

Drosophila stock center: Psc 1/CyO, Scm R5-13B/TM3,

Asx[XF23]/CyO, esc 2CyO/DpGYL(esc 2), Pcl/CyO,

Pc 1/TM6, Sb, Tb, In(1)ph410, ph-p[410] w[1],

ash1 B1/TM6C, cu 1 Sb 1 ca 1, brm 2 e s ca 1/TM6B, Sb 1

Tb 1, trx/TM6, ry 506 P{ry þt7.2 ¼ PZ}osa 00090/TM3,

mor 1/TM6B, Tb 1, lolal (y1 w67c23; P {w þ mC ¼ lacW}

lolalk02512/CyO).

4.5. Pairing sensitivity lines in lolal (batman) and ph

background

To examine the role of lolal in pairing dependent

silencing we tested lolal/ þ in the context of homozygous

and heterozygous PRE-mw, transgenic lines carrying P

element with iab-7 PRE juxtaposed to mini-white reporter.

Briefly, lolal/CyO males were crossed to w;CyO,TM3,Sb/

Xasta virgins. From the progeny of this cross, we selected

lolal/CyO; þ /TM3 males and crossed them to w;pin/CyO,

TM6 virgins. Another cross consisting of PRE-mw

K. Mishra et al. / Mechanisms of Development 120 (2003) 681–689 687

insertions on the third chromosome and w;CyO,

TM3,Sb/Xasta virgins was set up. From these crosses we

collected lolal/CyO;TM3/TM6 males and virgin females and

crossed to w 2 ; þ /CyO; PRE/TM3 flies. From the progeny

of these crosses we selected lolal/CyO;PRE-mw/TM3 males

and virgin females for checking the pairing sensitivity of the

PRE in lolal background. For examining the PRE-mw in

polyhomeotic ( ph) background we took In (1) ph 410,

ph-p 410 w 1. From this stock, virgins were crossed to

w;CyO, TM3, Sb/Xasta males. From the progeny ph/Y; TM3,

Sb/Xasta males were crossed to ph-p homozygous virgins.

Another cross was set up where ph-p homozygous virgins

were crossed to PRE-mw (II and III chromosome lines)

males. From these crosses ph/ph;CyO/ þ virgins were

crossed to ph/Y;PRE-mw/ þ males, in the progeny

homozygous and heterozygous PRE-mw were observed in

the ph background. Six different PRE-mw lines were used

for these experiments. We also used colorimetric estimation

to quantitate the intensity of the eye color. This was

particularly useful in the case of those lolal experiments

where lolal allele was a P-element insertion mutation with a

light eye color of its own.

Acknowledgements

We are thankful to Robert Kingston for the FLAG-PH fly

strain, Laurent Theodore for lolal (ban) deletion mutations,

Vincenzo Pirrotta and Carl Wu for the Trl-GAGA

antibodies, Renato Paro for the Pc antibody, Veena Parnaik

for Lamin B antibody and David Shore for yeast strains and

plasmids. We thank Mohammad Idris, Ch. Sabarinadh, R.U.

Pathak, T. Jayashree and S. Subramanian for help. We also

thank Vincenzo Pirrotta and Francois Karch for critical

reading of the manuscript. This work was supported by a

‘Young Investigator Grant’ from Human Frontier Science

Program to R.K.M. (RGY0316/2001-M) and a research

fellowship to V.S.C. by the Council of Scientific and

Industrial Research, India.

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