Tài liệu Báo cáo Y học: The Ikaros family protein Eos associates with C-terminal-binding protein corepressors pptx

CtBP has previously been shown to bind to a PXDLS-type motif in Ikaros, and we show that another Ikaros-related protein TRPS1 also contains a PXDLS CtBP contact motif within its repressi

The Ikaros family protein Eos associates with C-terminal-binding protein corepressors

Jose´ Perdomo and Merlin Crossley

Department of Biochemistry, G08, University of Sydney, NSW, Australia

Eos is a zinc finger transcription factor of the Ikaros family

It binds typical GGGAA Ikaros recognition sites in DNA

and functions as a transcriptional repressor Here we show

that Eos associates with the corepressor C-terminal-binding

protein (CtBP) CtBP has previously been shown to bind

Pro-X-Asp-Leu-Ser (PXDLS) motifs in several

DNA-binding proteins We note that Eos contains a related motif

PEDLA, and we demonstrate that CtBP can bind this site

weakly but that it also contacts additional regions of Eos

Consistent with this finding, mutation of the PEDLA motif

does not negate CtBP binding or CtBP-mediated repression

by Eos CtBP has previously been shown to bind to a PXDLS-type motif in Ikaros, and we show that another Ikaros-related protein TRPS1 also contains a PXDLS CtBP contact motif within its repression domain We conclude that several Ikaros family proteins utilize CtBP corepressors

to inhibit gene expression

Keywords: corepressors; gene regulation; Ikaros; repression; transcription

The zinc finger transcription factor Ikaros was originally

identified as a DNA-binding protein that recognized a

critical regulatory region of the T cell-restricted CD3d gene

[1] Ikaros expression is confined to erythroid and myeloid

precursors in the early stages of differentiation and to the

lymphoid compartment in the adult [2,3] The Ikaros gene

codes for a protein with six zinc fingers that comply with the

Kru¨ppel C2-H2 consensus The N-terminal fingers are

involved in sequence-specific DNA binding [4], while the

two zinc fingers that form the C-terminal domain mediate

homodimerization [5] Alternative mRNA splicing

gener-ates at least eight isoforms (Ik-1 to Ik-8) containing subsets

of the N-terminal fingers and all sharing the C-terminal

domain Isoforms containing at least three N-terminal

fingers are able to bind to the Ikaros consensus recognition

sequence [4]

The subsequent cloning of Aiolos and Helios [6–8], and of

Eos and Pegasus [9], revealed the existence of a family of

related factors Ikaros, Aiolos and Helios are all abundantly

expressed in the haematopoietic system and are all known

or predicted to have roles in lymphoid development,

whereas Eos and Pegasus are more broadly expressed, as

mRNA is detected in several human tissues [9] Recently a

more distantly related member of the Ikaros family, the

tricho-rhino-phalangeal syndrome protein TRPS1, has been

described [10,11] This protein contains the characteristic

C-terminal domain consisting of two zinc fingers capable

of mediating dimerization, but also contains additional

Kru¨ppel-like zinc fingers and one GATA-type finger

Figure 1 shows schematic representations of Ikaros, Eos and TRPS1

Studies of murine knockouts have revealed that Ikaros is essential for the regulation of commitment of haematopoi-etic stem cells to the lymphoid lineage In Ikaros null mice (Ik–/–), B cells and their precursors are absent, and T cells are undetected in the fetus but develop (abnormally) post partum [12] Mice expressing an Ikaros protein that lacks the DNA-binding domain (dominant negative DN–/– mutation) display more extreme effects, with a complete absence of T cells and death from severe infections soon after birth [2] The severity of the DN–/–mutation suggests that this aberrant Ikaros protein, which cannot bind DNA,

is still able to dimerize with other Ikaros family proteins and, most likely, interfere with their functions Aiolos–/– mice show expanded B cell populations and autoimmunity, but are normal in their thymic and splenic T cell develop-ment [13], a phenotype consistent with the predominant expression of Aiolos in B cells No knockouts have been reported for the other family members

The molecular mechanisms by which members of the Ikaros family recognize DNA and regulate gene expression are under intense investigation [14–18] Ikaros, Aiolos, Helios and Eos all recognize the consensus Ikaros-binding site GGGAA in vitro and in cellular assays, whereas Pegasus recognizes a distinct binding sequence GNNTGTNG [9] TRPS1, by virtue of its GATA-type zinc finger, can recognize GATA sites in DNA but may also bind to additional elements through its Kru¨ppel-like fingers In transient assays, all these proteins are able to modestly influence the transcription of reporter genes driven by their cognate sites [4,8,9] Aiolos and Helios have been reported

to function as activators, Ikaros has been implicated in both activation and repression, and Eos, Pegasus and TRPS1 have so far only been implicated in transcriptional repres-sion Recently attention has focused on the role of Ikaros as

a repressor, and interactions have been reported with Sin3 [19] and Mi-2 [20], which are components of deacetylase and chromatin remodelling complexes

Correspondence to M Crossley, Department of Biochemistry,

G08, University of Sydney, NSW, Australia, 2006.

Fax: 61 29351 4726, Tel.: 61 29351 2233,

E-mail: M.Crossley@biochem.usyd.edu.au

Abbreviations: TRPS1, tricho-rhino-phalangeal syndrome protein;

CtBP, C-terminal-binding protein; GST, glutathione S-transferase.

(Received 8 August 2002, revised 19 September 2002,

accepted 15 October 2002)

Ikaros has also been shown to bind coregulatory proteins

of the C-terminal-binding protein (CtBP) family (reviewed

in [21]) CtBP was named after it was first purified as a

protein that bound to the C-terminus of the Adenovirus

E1A protein [22] CtBPs have been identified in

Caenor-habditis, Drosophila, and mammals, and data from

Drosophila and mammals have shown that CtBPs can

function as transcriptional corepressors in vivo C tBP

recognizes PXDLS motifs found in the repression domains

of a wide range of transcription factors Identified partners

include Drosophila Hairy, Snail and Kru¨ppel [23,24],

BKLF/KLF3 [25], FOG [26], KLF8 [27] and Ikaros [15]

There are two highly homologous mammalian CtBP family

members, CtBP1 and CtBP2, encoded by separate genes,

but, to date, no differences in the activity of these two

proteins have been reported In this study, we used murine

CtBP2 [25], but it is likely that the results will also apply to

CtBP1, and therefore, in some instances, we use the term

CtBP for simplicity

CtBP has been shown to associate with Ikaros through

a PEDLS motif in its N-terminus (Fig 1A,B) [15] As

Ikaros makes multiple contacts with coregulatory proteins,

experiments with full-length Ikaros can be difficult to

interpret but, by studying the N-terminal repression

domain in isolation, Koipally & Georgopoulos [15] were

able to show that CtBP contact was required for the

repression activity of this domain This repression activity

is histone deacetylase independent, and the precise

mech-anism of repression remains unknown Here, we show that

Eos also interacts with CtBP to repress transcription CtBP recognizes a PEDLA motif in the C-terminus of Eos, but the interaction does not completely depend on this motif, suggesting that the Eos–CtBP interaction involves multiple surfaces Consistent with this result, we demonstrate that CtBP recognizes several regions within Eos In addition, we show that CtBP can bind a PXDLS motif in a previously well-characterized C-terminal repres-sion domain in the TRPS1 protein [11], and we suggest that a CtBP-mediated mechanism may be common in Ikaros-like proteins

E X P E R I M E N T A L P R O C E D U R E S

Plasmids Bait plasmids used in the yeast two-hybrid experiments were generated by fusing the desired regions downstream and in-frame of the Gal4 DNA-binding domain of the yeast expression vector pGBT9 (Clontech) All numbers indicated refer to amino acids in the respective sequences The bait plasmids were pGBT9.Eos364–400, pGBT9.Eos364– 400.mut, pGBT9.Eos364–400DEtoIk (Eos 372 PED-LADGG379 changed to Ikaros site PEDLSTTS), pGBT9.TRPS1210-1281, and pGBT9.mCtBP2 [25] Prey plasmids were constructed by inserting the desired sequences in-frame and downstream of the Gal4 activation domain of the pGAD10 vector (Clontech) Full-length vectors are pGAD10.Eos and pGAD10.mCtBP2, pGAD10.Pegasus and pGAD10.Ikaros2 Other plasmids are pGA-D10.Eos101–531, pGAD10.Eos101–531.mut, pGAD10 Eos101–364, pGAD10.Eos101–331, pGAD10.Eos101–

231, pGAD10.Eos364–518, pGAD10.Eos364–518.mut, pGAD10.Ikaros1–81, pGAD10.Ikaros1–81.mut, pGA D10.TRPS1068–1186, pGAD10.TRPS1068–1186.mut, pGAD10.TRPS1210–1281 and pGAD10.Aiolos447–507 Expression vectors for mammalian systems used were constructed in the parental plasmids pcDNA3 (Invitrogen) and pMT2 The vectors include pcDNA3 Gal4DBD, pcDNA3.mCtBP2 [25], pcDNA3.Eos, pcDNA3.Gal4-DBD.Eos364–518, pcDNA3.Gal4DBD.Eos364–518.mut, pcDNA3.Gal4DBD.Ikaros1–81, pcDNA3.Gal4DBD.-Ikaros1–81.mut, pcDNA3.Gal4DBD.TRPS1068–1281, pcDNA3.Gal4DBD.TRPS1068–1281.mut, pMT2.mCTBP2, pMT2.FLAG.Eos, pMT2.FLAG.Eos.mut, pMT2.FLA-G.Eos101–389, pMT2.FLAG.Eos101–331, and pMT2 FLAG.Eos101–231 Glutathione S-transferase (GST) fu-sion mCtBP2 protein was produced by inserting mCtBP2 cDNA in-frame with GST in the pGEX-2T vector (Amer-sham Pharmacia Biotech) [25] The luciferase reporter vector contained five copies of the Gal4 DNA-binding domain upstream of the TK promoter in the vector

pGL2-TK (Promega)

Yeast two-hybrid and pull-down assays The Clontech yeast two-hybrid system was used according

to the manufacturer’s instructions The prey and bait plasmids used are described above Recombinant GST-mCtBP2 was produced in Escherichia coli strain BL-21, purified as described [28] and immobilized on glutathione beads.35S-labelled Eos and Eos.mut production and the pull-down experiments were carried out as described [9]

Ikaros 31MPVPEDLSTTS41

Eos 369GEGPEDLADGG379

TRPS1 1160NDIPLDLAIKH1170

A

B

Ikaros

Eos

TRPS1

DNA binding Dimerization

517

532

1281

Dimerization GATA finger

PXDLS motif

Intron/Exon boundary

Fig 1 Schematic representation of Ikaros, Eos and TRPS1 (A)

Diagram of Ikaros, Eos and TRPS1 The zinc fingers are represented

by unfilled semi-ellipses, the positions of the PXDLS-like motif are

indicated by filled rectangles, and the intron/exon boundaries by

arrows Amino-acid numbers are indicated in each case For Ikaros

and Eos, the N-terminal fingers involved in sequence-specific DNA

recognition are shown, and the GATA-type finger required for

binding of TRPS1 to GATA sites is shown The C-terminal fingers

that are characteristic of the Ikaros family and mediate dimerization

are also indicated (B) The sequences corresponding to the

PXDLS-like motif for the three proteins in (A) are shown Amino-acid

numbers are indicated.

In vitro transcription and translation

In vitrotranscription and translation of proteins has been

described [9]

Western blot and immunoprecipitation

Transfected COS cells were washed with cold NaCl/Piand

resuspended in 400 lL cold solution A (10 mM Hepes,

pH 7.8, 1.5 mMMgCl2, 10 mMKCl) supplemented before

use with 1 mM dithiothreitol, 50 ngÆmL)1

phenyl-methanesulfonyl fluoride, 5 lgÆmL)1 leupeptin and

5 lgÆmL)1aprotinin The tubes were incubated on ice for

10 min, vortex-mixed for 10 s, and centrifuged for 10 s at

12 000 g to pellet the nuclei The nuclei were resuspended in

30–50 lL solution C(20 mMHepes, pH 7.8, 25% glycerol,

420 mMNaCl, 1.5 mMMgCl2, 0.2 mMEDTA)

supplemen-ted as above, centrifuged for 3 min at 14 000 r.p.m at 4C

The extracts were used immediately or stored at)70 C

Proteins were separated by SDS/PAGE on 8–10%

polyacrylamide gels and transferred on to a BiotraceTM

nitrocellulose blotting membrane (Pall Gelman Sciences,

Ann Arbor, MI, USA) in a TE series TransphorTM

electrophoresis unit (Hoefer), at 50 mA overnight at 4C

For Western blotting, the membrane was washed once in

50 mM Tris/HCl, pH 7.5, containing 150 mM NaCl and

0.05% Tween-20 (Tris/NaCl/Tween), then incubated at

room temperature in skimmed milk powder solution

[5% (w/v) in Tris/NaCl/Tween] for 1 h The membrane

was rinsed in Tris/NaCl/Tween and incubated for 1 h with

gentle shaking in 10 mL Tris/NaCl/Tween containing 10 lg

primary antibody After a wash with 4· 100 mL Tris/

NaCl/Tween, the secondary antibody solution was added

and incubation was continued for 1 h The membrane was

washed for 1 h in several changes of Tris/NaCl/Tween

Detection was carried out using the Renaissance

Chemi-luminescence reagent plus (NEN Life Sciences, Boston,

MA, USA), and the signal detected on X-ray film (Eastman

Kodak Company, Rochester, NY, USA) and developed

using Kodak reagents

Covalently linked protein A/G-agarose beads

(Boehrin-ger, Mannheim, Germany) and the antibody of interest

were prepared as follows The beads plus antibody were

incubated for 1 h at room temperature in 1 mL NaCl/Piat

2 lg antibody/lL wet beads The bead–antibody complex

was washed twice with 10 vol 0.2Msodium borate, pH 9.0,

and the beads were resuspended in 10 vol 0.2M sodium

borate, pH 9.0 Solid dimethyl pimelimidate (Sigma) was

added to a final concentration of 20 mM, incubated for

30 min at room temperature, and the reaction stopped by

washing once in 10 vol 0.2Methanolamine, pH 8.0, and

then incubating for 2 h at room temperature in 10 vol

0.2M ethanolamine, pH 8.0 Coupled beads were

resus-pended in 1 vol NaCl/Piand stored at 4C

For immunoprecipitations, nuclear extracts were diluted

1 : 3 in Nonidet P40 buffer (50 mM Tris/HCl, pH 7.4,

150 mMNaCl, 0.5–1.0% Nonidet P40, 1 lgÆmL)1

leupep-tin, 1 lgÆmL)1 aprotinin, 1 mM phenylmethanesulfonyl

fluoride) Lysates were precleared with 20 lL protein A/G

beads for 30 min at 4C The cleared lysates were treated

with 5–15 lL beads–antibody complex for 1 h at 4Cwith

rocking The beads were pelleted at 14 000 r.p.m for 10 s,

the supernatant discarded, and the beads washed (4· 1 mL

cold Nonidet P40 buffer) The proteins retained on the beads were separated by SDS/PAGE and detected by Western blotting

Transfections and luciferase assay NIH-3T3 cells were transfected with 3 lg of the reporter pGL2(Gal4)5TK and different amounts (0.5–2 lg) of pcDNA3.Gal4DBD, pcDNA3.mCtBP2, pcDNA3.Gal4 DBD.Eos364–518, pcDNA3.Gal4DBD.Eos364–518.mut, pcDNA3.Gal4DBD.Ikaros1–81, pcDNA3.Gal4DBD.-Ikaros1–81.mut, pcDNA3.Gal4DBD.TRPS1068–1281 and pcDNA3.Gal4DBD.TRPS1068–1281.mut using the calcium phosphate method [29] Luciferase activity was measured as described [9] COS cells were transfected with

2 lg pMT2, pMT2.mCTBP2, pMT2.FLAG.Eos, pMT2 FLAG.Eos.mut, pMT2.FLAG.Eos101–389, pMT2.FLAG Eos101–331 and pMT2.FLAG.Eos101–231 by the DAE-Dextran method [29], and harvested 48–60 h after transfec-tion The total amount of transfected DNA was kept constant in all cases by addition of naked pcDNA3 or pMT2 vectors, as appropriate

R E S U L T S

Eos interacts with CtBPin vitro and in vivo The observation that Eos can function to repress gene expression [9] prompted us to investigate whether it associated with recognized corepressor proteins We noted that the C-terminal region of Eos contains a sequence,

372PEDLA376, that resembles the accepted consensus CtBP-binding motif PXDLS [21] The Eos motif differs from the consensus at the final residue, but a previously identified partner, Enhancer of Split md, also has alanine at the fifth position, suggesting that this change would not preclude CtBP binding [23] We first tested the ability of in vitro transcribed and translated Eos protein to interact with bacterially expressed and purified GST–CtBP Figure 2A shows that GST–CtBP but not GST alone is able to retain

35S-Eos The Eos–CtBP contact was also confirmed using the yeast two-hybrid system We cotransformed yeast with vectors encoding a Gal4 activation domain–Eos fusion, and a Gal4 DNA-binding domain–CtBP fusion and observed activation of the HIS3 reporter gene as indicated

by yeast growth in the absence of histidine (Fig 2B) Finally, we assessed the interaction using coimmunopre-cipitation experiments COS cells were transfected with vectors expressing FLAG-tagged Eos and native CtBP FLAG-Eos was recovered by immunoprecipitation with a FLAG antibody, and the presence of CtBP assessed by Western blotting using anti-CtBP serum As shown in Fig 2C, CtBP was present in the recovered material Taken together these results indicate that Eos and CtBP physically interact

The PEDLA motif of Eos is not the sole determinant

of the CtBP interaction

In most instances, such as the case of Ikaros [15], deletion or mutation of a single critical PXDLS motif results in abrogation of CtBP contact and functional consequences

of CtBP association We examined whether the binding of

CtBP to Eos required the PEDLA motif by mutating this

motif to AAALA (Eos-mut) Interestingly, we found that

this mutation did not eliminate the Eos–CtBP interaction

Figure 2D shows that GST–CtBP is also able to retain

radiolabelled Eos-mut Figure 2E shows that yeast

har-bouring an expression vector encoding a Gal4 activation

domain–Eos-mut fusion and a Gal4 DNA-binding

domain–CtBP fusion grow in the absence of histidine, and

Fig 2F indicates that CtBP can be immunoprecipitated

with FLAG-tagged Eos-mut These results suggest that

CtBP does not depend exclusively on the Eos PEDLA motif

for interaction and may make additional contacts through

other domains within the Eos protein

There are several precedents for CtBPs contacting

partners through regions outside recognizable PXDLS

motifs [30–32] To delineate additional Eos domains

involved in CtBP recruitment, a series of deletion mutants

was constructed (Fig 3A) The CtBP-interacting properties

of these mutants were tested using the yeast two-hybrid

system As seen in Fig 3A, CtBP was able to associate with

both the N-terminal and C-terminal domains of Eos To

determine whether the 372PEDLA376 motif in the

C-terminus was primarily responsible for binding to this

domain, we again mutated this motif to AAALA, but this

time in the context of the C-terminal domain Eos364–518

Again mutation of the motif did not significantly affect

CtBP binding We carried out the same experiment in the

context of a minimal Eos domain, Eos364–400 and found

that this region still bound CtBP In this construct, however,

the PEDLA to AAALA mutation reduced binding We also

made a second mutant replacing the nontypical PEDLA motif of Eos with the recognized PEDLS motif of Ikaros and observed a stronger interaction (Fig 3A) Taken together these results suggest that the PEDLA motif in Eos is suboptimal and not the major determinant of CtBP binding, but that it and other sites within the N-terminus and C-terminus of Eos contribute to CtBP contact

We also confirmed the presence of the N-terminal CtBP-binding domain using coimmunoprecipitation experiments The FLAG-tagged Eos constructs shown in Fig 3B were cotransfected with CtBP into COS cells, and immunoprecipitation experiments were carried out Figure 3Cis a Western blot showing that all three Eos constructs are expressed at comparable levels Figure 3D shows an immunoprecipitation experiment with FLAG antisera or an irrelevant antibody in the mock lane Western blotting with a CtBP antiserum shows that CtBP

is associated with the immunoprecipitated material in all cases in which the FLAG antiserum was used, although most CtBP was retained by the longest construct The results were confirmed by the reciprocal experiment, immunoprecipitating with CtBP antiserum and analyzing the material by Western blotting with anti-FLAG serum (Fig 3E) As can be seen, the three Eos deletion constructs are detected in the material immunoprecipitated by the anti-CtBP serum, and again the longest construct appears

to have been retained more efficiently No Eos fragments were detected when an irrelevant antibody was used in a similar experiment (data not shown) These findings confirm the observation that, in addition to binding the

Fig 2 Eos interacts with CtBP (A) Purified GST and GST–CtPB were used to assess the interacting activities of in vitro transcribed and translated Eos protein GST–CtBP but not GST alone retained radiolabelled Eos protein (B) The interaction was confirmed using the yeast two-hybrid system Plasmids present in the various yeast derivatives are shown Growth on this plate lacking histidine, leucine and tryptophan is indicative of a positive interaction (C) FLAG-tagged Eos and CtBP were cotransfected into C OS cells and nuclear extracts used for immunoprecipitations Lane

1 (input) indicates the migration of CtBP in the extracts, control mock transfected cells (lane 2) and detected (lane 3) CtBP after immunoprecipitation with anti-FLAG serum.

IP, Immunoprecipitation (D) Purified GST and GST–CtPB were used to assess the inter-acting activities of in vitro transcribed and translated Eos.mut protein GST–CtBP retained radiolabelled mutant os protein (E) and (F), as for (B) and (C) Mutant Eos was tested in both cases IP, Immunoprecipitation.

C-terminal domain of Eos, CtBP can also bind sites within

the N-terminal domain of Eos

TRPS1 interacts with CtBP

The TRPS1 protein is a multi-(zinc finger) protein that

contains two C-terminal fingers highly related to the Ikaros

family dimerization domain [10,11] Little is known of the

molecular roles of TRPS1 or its target genes It is known

that TRPS1 is capable of binding typical GATA sites via its GATA-type zinc finger and that TRPS1 can act to repress the expression of GATA-dependent reporter genes [11] The relevant repression domain has been localized to the C-terminal 119 residues of the protein [11] We noted that this minimal repression domain contains a potential CtBP contact motif, 1163PLDLA1167 This observation implied that the repression domain might function by recruiting the corepressor CtBP

As the yeast two-hybrid system has proved a very reliable indicator of CtBP contact in all instances previously reported [21], we used this assay to determine whether the repression domain of TRPS1 was capable of interacting with CtBP We found that there was a strong interaction in yeast (Fig 4A,B) The amount of yeast growth was comparable to that observed for the isolated CtBP-binding region of Ikaros (Fig 4A,B, construct 1) Mutation of the putative CtBP-binding motif 1163PLDLA1167 to ALAAA abolished the interaction (Fig 4A, construct 4), suggesting that it was the major determinant of CtBP binding within the repression domain

CtBP-interacting regions of Eos, Ikaros and TRPS1 function as CtBP-dependent repression domains Deletion analysis of Ikaros has indicated that it contains distinct domains that are implicated in activating or repressing transcription [4,5,19,33] One discrete domain within the N-terminus contains the motif PEDLS and has been shown to contact CtBP and function as a CtBP-dependent repression domain We investigated whether the PXDLS regions of Eos and TRPS1 also functioned as CtBP-dependent repression domains The regions were tested as Gal4 DNA-binding domain fusions for their ability to repress transcription in transient transfection experiments in mammalian cells Gal4Ikaros1–81, Gal4Eos364–518, and Gal4TRPS1068–1281 were

transfect-ed individually into NIH-3T3 cells and testtransfect-ed against a luciferase reporter gene driven by five Gal4-binding sites upstream of the TK promoter Figure 5A shows that Gal4Ikaros1–81 represses the transcription of the reporter gene A mutation in the PEDLS motif abolished the ability

of this domain to repress transcription, consistent with previous findings that these residues are required for CtBP recruitment and repression We also observed that when submaximal amounts of the Ikaros construct (0.1 lg) were

Fig 3 Eos deletion constructs interact with CtBP in yeast and in COS

cells (A) Schematic representation of Eos constructs tested against

CtBP in the yeast two-hybrid system Numbers indicate the amino

acids in the Eos sequence, the filled rectangle indicates the position of

the PEDLA motif, and the cross represents mutation of this motif.

(Rectangle) Ik indicates mutation to resemble the Ikaros motif

PED-LSTT (+) growth observed; (–) no growth For comparison, the

CtBP-interacting region of Ikaros (Ikaros1–81) is also shown Yeast

growth on plates lacking histidine is shown for selected constructs.

These plates represent growth after 4 days of 10 lL of undiluted,

1 : 10 and 1 : 100 dilutions of D 600 solutions (B) Schematic

repre-sentation of FLAG-tagged Eos constructs used for

immunoprecipi-tations Numbers indicate the amino acids in the Eos sequence (C)

Western blot of the Eos constructs in (B) expressed in COS cells (D)

Immunoprecipitation showing that the three Eos N-terminal

con-structs are able to associate with cotransfected CtBP, lanes 1–3 (E)

The reciprocal experiment with anti-CtBP serum precipitating the

cotransfected Eos constructs (arrows) shown in (B) and (C) The

prominent bands seen above the bands of interest (arrowheads)

cor-respond to the heavy chain of the antibodies used because of

incom-plete covalent coupling of the antibodies to the agarose beads IP,

Immunoprecipitation.

Fig 4 TRPS1 and Ikaros interact with CtBP through their PXDLS-like motifs (A) Schematic representation of the fragments tested Numbers indicate the amino acids of the respective protein Filled rectangles indicate the position of the PXDLS-like motif, and the cross represents mutation of this motif (B) Yeast growth in the absence of histidine is indicative of a positive interaction.

used, cotransfection with a CtBP expression vector (0.5 lg)

potentiated repression These results corroborate previous

observations on CtBP-dependent repression by this Ikaros

domain [15] Figure 5B shows a similar experiment on the

PEDLA motif-containing domain of Eos This domain also

functions to repress the reporter, but mutation of the

PEDLA motif does not abrogate repression This result is

consistent with the protein interaction data showing that

this mutation does not prevent contact with CtBP Again,

when low amounts of the Eos construct (0.1 lg) were used,

addition of a CtBP expression vector (0.5 lg) potentiated

repression, confirming the result that Eos364–518 depends

on CtBP, although the PEDLA motif is not essential for its

recruitment Finally, Fig 5Cshows results with the TRPS1

PLDLA-containing domain This portion of the protein

functioned as a potent repression domain and mutation of

the PLDLA motif abrogated repression activity, consistent

with the protein interaction result that this motif was required for CtBP contact Again addition of a CtBP expression vector potentiated repression when low amounts

of the TRPS1 construct were tested Overall these results indicate that these three Ikaros family proteins all contain repression domains that are dependent on CtBP

D I S C U S S I O N

CtBP has previously been shown to bind to repression domains in a number of transcription factors and other regulatory proteins [21] The results reported here show that CtBP binds repression domains within three mem-bers of the Ikaros family of transcription factors In each case, the domains contain a recognizable PXDLS-type motif but the PEDLA motif in Eos is not the sole determinant of CtBP contact Furthermore the PEDLA motif in Eos appears to be a relatively weak binding site,

as its replacement with the well-characterized PEDLS motif of Ikaros substantially increased the association with CtBP Although the PEDLA motif in Eos supports only weak binding of CtBP, we show that additional regions within the N-terminal and C-terminal regions of Eos also contact CtBP, and, taken together, the results of GST-pulldown, yeast two-hybrid and immunoprecipita-tion experiments demonstrate that Eos and CtBP can stably associate

Although PXDLS motifs have often been shown to be the primary determinant of CtBP binding, there are other examples where they are not essential for corepressor contact, and there are even cases where CtBP partner proteins contain no recognizable PXDLS motif The zinc finger protein Tramtrack69 contains a PPDLS motif, but this is not required for binding [31], and HDAC5 contains

a related motif (PVELR) that is dispensable for C tBP contact [32] In the case of HDAC1, no canonical CtBP recognition sequences have been found [30] We presume that generally the PXDLS motif on the DNA-binding protein slots into a putative PXDLS-accepting pocket within CtBP In the cases where the PXDLS motif is not required for binding and other contacts are made, we expect that other (nonpocket) regions of CtBP will be involved in the interaction In this way, it seems likely that proteins such as Eos may be able to bind to CtBP that is already associated with another PXDLS motif-containing protein For instance, Eos may bind to a CtBP molecule, the pocket of which is already complexed to Ikaros Indeed, we have previously shown that the conserved C-terminal dimerization domain of Eos can associate with the related domain in Ikaros [9], so Eos may in fact be able

to bind Ikaros and CtBP to form a trimeric complex We have also tested the association of Eos with other Ikaros family members and shown that it can bind the dimeriza-tion domain of TRPS1 (unpublished results), raising the possibility that Eos may also function in a similar manner

in a complex with TRPS1 and CtBP

There is good evidence that Ikaros proteins dimerize [5] and possibly form higher-order multimers in vivo [7,16], but the precise mechanisms by which various Ikaros-containing complexes operate remains under investigation In addition

to binding CtBP, Ikaros has been found in T cells as part of two discrete histone deacetylase complexes by virtue of its interaction with the ATPase Mi-2 [20] and the corepressor

X

1 81

Eos

Gal4DBD

Ikaros Gal4DBD

0.5µg

0.1µg

1.0µg

0.1µg

1.0µg

0.5µg

0.1µg

1.0µg

1.0µg

X

1068

1281 0.5µg

0.1µg

1.0 µg

0.1µg

Gal4DBD

TRPS1

A

B

C

0.1µg

CtBP

+

-+

-364 518

X

CtBP

+

-+

0 10 20 30 40 50 1.0 µg

CtBP

+

-+

-Fold repression

1 81

Fold repression

Fold repression

1 81

1 81

364 518

364 518

364 518

1068

1281

1068

1281 1068

1281

Fig 5 CtBP binding domains of Ikaros, Eos and TRPS1 act as potent

repression domains Plasmids encoding Gal4 DNA-binding domain

alone or fused to Eos364–518, Ikaros1–81 and TRPS1068–1281 were

transfected into NIH-3T3 cells and the luciferase activity determined.

The rectangle indicates the location of the PXDLS-like motif, and the

cross indicates mutation of this motif (A) Ikaros1–81 is a repressor

only when its PEDLS motif is intact Addition of a minimal amount

does not affect transcription, but repression is seen on cotransfection of

CtBP (B) Eos364–518 represses transcription even when the PEDLA

motif has been mutated This Eos domain is responsive to

cotrans-fected CtBP (C) TRPS1068–1281 is a strong repressor the activity of

which is abrogated by mutation of its PLDLA motif Transfection of a

minimal amount still represses transcription; this activity is potentiated

on CtBP cotransfection.

Sin3 [19] It has also been implicated in silencing gene

expression in B cells by targeting genes to inactive

centro-meric chromatin [3,16,18] Recently, Ikaros, Helios, Aiolos

and murine Eos were shown to be able to interact with

CtBP-interacting protein (CtIP) independently of CtBP

association [34] The Ikaros–CtIP complex was shown to be

capable of repressing transcription in the absence of histone

deacetylase activity and to perhaps function through a

mechanism that depends on interactions with components

of the basal transcriptional machinery such as TATA

binding protein and transcription factor IIB [34] Thus, the

number of possible mechanisms employed by Ikaros

complicates studies on the full-length protein, but

experi-ments on isolated domains have established a role for the

N-terminal CtBP contact region and shown that repression

was trichostatin A independent [15] This result suggests

that the CtBP–Ikaros repression domain complex does not

repress gene expression through a conventional HDAC

mechanism In our experiments we also found that the Eos–

CtBP complex was not sensitive to trichostatin A, again

consistent with a non-HDACmechanism (unpublished

results) The specific mechanisms by which other Ikaros

family members influence gene expression are still under

scrutiny, and very little is known about the overall

biological roles of these proteins Naturally occurring

mutations in the TRPS1 gene lead to faciocranial

abnor-malities and skeletal deformations [10], but precise target

genes remain to be identified The finding that CtBP

associates with a functional repression domain in TRPS1

confirms former results suggesting that TRPS1 acts as a

repressor protein and can counter GATA-mediated gene

activation [11] Figure 5Cshows strong repression by

TRPS1, which suggests that the proposed TRPS1–CtBP

interaction is stronger than that of the other constructs

investigated Ultimately determination of the relevant

association constants is likely to clarify this observation

Further work to identify target genes upregulated in the

absence of functional TRPS1 may illuminate the molecular

causes underlying the observed phenotype

A C K N O W L E D G E M E N T S

We are grateful to members of the laboratory and Margot Kearns for

reading the manuscript We thank Dr R.A Shivdasani for his gift of a

TRPS encoding plasmid This work was supported by a grant from the

Australian Health Management Group to M.C J.P is supported by an

Australian Post-graduate Award.

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