PLZF is a negative regulator of retinoic acid receptor transcriptional activity

Martin, Delmotte Marie-Hélène, Formstecher Pierre, Lefebvre, Philippe Lefebvre

Description

Retinoic acid receptors (RARs) are ligand-regulated transcription factors controlling cellular proliferation and differentiation. Receptor-interacting proteins such as corepressors and coactivators play a crucial role in specifying the overall transcriptional activity of the receptor in response to ligand treatment. Little is known however on how receptor activity is controlled by intermediary factors which interact with RARs in a ligand-independent manner. Results We have identified the promyelocytic leukemia zinc finger protein (PLZF), a transcriptional corepressor, to be a RAR-interacting protein using the yeast two-hybrid assay. We confirmed this interaction by GST-pull down assays and show that the PLZF N-terminal zinc finger domain is necessary and sufficient for PLZF to bind RAR. The RAR ligand binding domain displayed the highest affinity for PLZF, but corepressor and coactivator binding interfaces did not contribute to PLZF recruitment. The interaction was ligand-independent and correlated to a decreased transcriptional activity of the RXR-RAR heterodimer upon overexpression of PLZF. A similar transcriptional interference could be observed with the estrogen receptor alpha and the glucocorticoid receptor. We further show that PLZF is likely to act by preventing RXR-RAR heterodimerization, both in-vitro and in intact cells. Conclusion Thus RAR and PLZF interact physically and functionally. Intriguingly, these two transcription factors play a determining role in hematopoiesis and regionalization of the hindbrain and may, upon chromosomal translocation, form fusion proteins. Our observations therefore define a novel mechanism by which RARs activity may be controlled.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2003
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

BioMed CentralNuclear Receptor
Open AccessResearch
PLZF is a negative regulator of retinoic acid receptor
transcriptional activity
1 2 1Perrine J Martin , Marie-Hélène Delmotte , Pierre Formstecher and
1Philippe Lefebvre*
1Address: INSERM U 459 and Ligue Nationale Contre le Cancer, Faculté de Médecine Henri Warembourg, 1 place de Verdun, 59045 Lille cedex,
2France and Department of Biochemistry and Molecular Biology, Howard Hughes Medical Institute, University of Massachusetts Medical School,
Worcester MA 01605, USA
Email: Perrine J Martin - [e-mail]; Marie-Hélène Delmotte - [e-mail];
Pierre Formstecher - [e-mail]; Philippe Lefebvre* - [e-mail]
* Corresponding author
Published: 06 September 2003 Received: 16 May 2003
Accepted: 06 September 2003
Nuclear Receptor 2003, 1:6
This article is available from: http://www.nuclear-receptor.com/content/1/1/6
© 2003 Martin et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all
media for any purpose, provided this notice is preserved along with the article's original URL.
Abstract
Background: Retinoic acid receptors (RARs) are ligand-regulated transcription factors controlling
cellular proliferation and differentiation. Receptor-interacting proteins such as corepressors and
coactivators play a crucial role in specifying the overall transcriptional activity of the receptor in
response to ligand treatment. Little is known however on how receptor activity is controlled by
intermediary factors which interact with RARs in a ligand-independent manner.
Results: We have identified the promyelocytic leukemia zinc finger protein (PLZF), a
transcriptional corepressor, to be a RAR-interacting protein using the yeast two-hybrid assay. We
confirmed this interaction by GST-pull down assays and show that the PLZF N-terminal zinc finger
domain is necessary and sufficient for PLZF to bind RAR. The RAR ligand binding domain displayed
the highest affinity for PLZF, but corepressor and coactivator binding interfaces did not contribute
to PLZF recruitment. The interaction was ligand-independent and correlated to a decreased
transcriptional activity of the RXR-RAR heterodimer upon overexpression of PLZF. A similarional interference could be observed with the estrogen receptor alpha and the
glucocorticoid receptor. We further show that PLZF is likely to act by preventing RXR-RAR
heterodimerization, both in-vitro and in intact cells.
Conclusion: Thus RAR and PLZF interact physically and functionally. Intriguingly, these two
transcription factors play a determining role in hematopoiesis and regionalization of the hindbrain
and may, upon chromosomal translocation, form fusion proteins. Our observations therefore
define a novel mechanism by which RARs activity may be controlled.
Background factors in the form of RAR/RXR heterodimers. RAR is acti-
atRA receptors (RARs) α, β and γ and 9-cis retinoic acid vated by atRA and binding of this ligand induces receptor
receptors α, β and γ (RXRs) are encoded by three different conformational changes that switch on transcription of
genes and are members of the nuclear receptor super- genes containing RA Response Elements (RAREs) by
family. They function as ligand-inducible transcription favoring coactivator tethering to regulated promoters.
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This protein complex assembly at regulated promoters PLZF protein to retinoic acid receptor " (RAR α, [15–17]).
induces chromatin remodeling and increased binding of The PLZF-RAR α fusion protein maintains most of the
RNA polymerase II to these promoters, thereby inducing DNA and dimerization properties of both moieties, and
a variety of biological effects (reviewed in [1,2]). While a PLZF-RAR binds to retinoic acid response elements
detailed understanding of the ligand-dependent activa- (RAREs) as a heterodimeric partner of RXR, interfering
tion of RARs has been achieved by structural and func- with RAR α functions by exerting a dominant negative
tional studies, little is known about factors regulating the effect [16,18]. The resistance of t(11;17) APL to pharma-
activity of the unliganded receptor. We therefore under- cological doses of atRA contrasts with the sensitivity of the
took a 2-hybrid screen in yeast using an AF2-inactivated more common t(15;17) APL, which is characterized by a
hRAR α as a bait, thus unable to respond transcriptionally fusion between the promyelocytic leukemia transcription
to ligand, to identify proteins potentially able to regulate factor PML and the RAR α proteins [19]. Thus the highly
RAR functions in a ligand-independent manner. Among stable, targeted recruitment of NCoRs and HDACs to
the identified proteins, PLZF was found to physically PLZF-RAR, mostly through the BTB/POZ domain, is likely
interact with RAR α through its zinc finger domain. to underlie the pathogenesis of the t(11;17) APL and
renders it refractory to atRA chemotherapy, although
The human promyelocytic leukemia zinc finger (PLZF) additional factors are involved in the t(11;17)-induced
protein is a 673 amino acid (AA) transcriptional repressor leukemogenesis [20].
belonging to a large protein family characterized by a 120
AA N-terminal bric-à-brac, tramtrack, brad complex Interestingly, the PML protein acts either as a corepressor
(BTB)/poxvirus zinc finger (POZ) domain. Proteins con- or a coactivator in a DNA-binding independent manner.
taining this BTB/POZ domain are associated to multiple PML gene inactivation leads to a strongly decreased tran-
functions such as development, embryogenesis and chro- scriptional activation of the p21 gene and to impaired
matin remodeling. The BTB/POZ domain allows protein myeloid differentiation in response to retinoid stimula-
homodimerization [3] and is involved in the recruitment tion [21]. Consistent with its role of coactivator, it has
of transcriptional corepressor complexes (NCoR) harbor- been shown to be integrated in the DRIP complex [22]
ing histone deacetylases (HDAC) activity [4,5]. In addi- and to interact with CBP [23].
tion, this multimeric NCoR complex has been shown to
provide a docking site for eight-twenty one (ETO), a non- Thus, quite intriguingly, PML and RAR have a functional
DNA binding transcriptional repressor fused to the tran- relationship during transcriptional regulatory processes,
scriptional activator AML1 in acute myelogenous leuke- and are chromosomal translocation partners. In this
mia [6,7]. Another structural feature of PLZF is its C- paper, we describe the physical interaction of PLZF with
terminal DNA binding domain made of nine C H Krup- RAR α and explore the functional consequences of this2 2
pel-like zinc fingers that binds the consensus sequence interaction on retinoid-regulated transcription.
GTACAGTTSCAU [8]. The first two zinc fingers are dispen-
sable for DNA binding [9,10], although other domains of Results and Discussion
the protein seem to contribute to the DNA binding specif- PLZF interacts with RAR α in-vitro
icity by restricting the DNA binding repertoire of PLZF [8]. In a search for proteins that could interact with the unlig-
Finally, a proline-rich and an acidic domains are found in anded, transcriptionally inactive RAR α, we set up a yeast
the central part of the molecule (see also Figure 1 for more two hybrid screen using a mutated receptor (Figure 1A).
details). Mutations were designed on the basis of the three-dimen-
sional structure of the RAR α ligand binding domain
The exact biological role of PLZF remains to be estab- (LBD). It defines K262 as establishing salt bridges with
lished. However, its localization to nuclear bodies [11], E412 and E415 of the RAR α activating function 2 (AF2)
which are nuclear structures associated to a central, tran- activating domain (AD) upon agonist binding [24,25].
scriptional regulatory role [12], as well as its down regula- Mutation of K262 and of the neighboring K244 into
tion upon myeloid cell differentiation hint at a crucial role alanine residues (RAR α 2 K) prevents the ligand-induced
in cell growth control [13]. Indeed, genetic ablation of the folding of RAR α AF2, impedes coactivator recruitment,
PLZF gene in mice led to aberrant limb modeling resulting weakens corepressor interaction (Figure 1A) and inacti-
from deregulated cell proliferation and apoptosis, and vates the transcriptional activity of RAR α [26].
also suggested that PLZF is, like all trans retinoic acid
(atRA), a critical regulator of the linear expression of the A human ovary cDNA library was screened for interaction
Hox gene cluster [14]. Another strong argument for the with RAR α 2 K and twelve positive clones were isolated
biological importance of PLZF is the association of the and further characterized by DNA sequencing. A BLAST
chromosomal translocation t(11;17) to a rare variant of search indicated that we isolated, among these clones, a
acute promyelocytic leukemia (APL), which fuses the cDNA encoding amino acids 389 to 658 of human
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A)
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