Download LHX3 transcription factor mutations associated with combined

Gene 265 (2001) 61±69
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LHX3 transcription factor mutations associated with combined pituitary
hormone de®ciency impair the activation of pituitary target genes
Kyle W. Sloop 1, Gretchen E. Parker 1, Kimberly R. Hanna, Heather A. Wright, Simon J. Rhodes*
Department of Biology, Indiana University-Purdue University Indianapolis, 723 West Michigan Street, Indianapolis, IN 46202-5132, USA
Received 15 December 2000; received in revised form 9 January 2001; accepted 24 January 2001
Received by A.J. van Wijnen
Abstract
The Lhx3 LIM homeodomain transcription factor is critical for pituitary gland formation and speci®cation of the anterior pituitary
hormone-secreting cell types. Two mutations in LHX3, a missense mutation changing a tyrosine to a cysteine and an intragenic deletion
that results in a truncated protein lacking the DNA-binding homeodomain, have been identi®ed in humans. These mutations were identi®ed
in patients with retarded growth and combined pituitary hormone de®ciency and also abnormal neck and cervical spine development. For
both the LHX3a and LHX3b isoforms, we compared the ability of wild type and mutant LHX3 proteins to trans-activate pituitary genes, bind
DNA recognition elements, and interact with partner proteins. The tyrosine missense mutation inhibits the ability of LHX3 to induce
transcription from selected target genes but does not prevent DNA binding and interaction with partner proteins such as NLI and Pit-1.
Mutant LHX3 proteins lacking a homeodomain do not bind DNA and do not induce transcription from pituitary genes. These studies
demonstrate that mutations in the LHX3 isoforms impair their gene regulatory functions and support the hypothesis that defects in the LHX3
gene cause complex pituitary disease in humans. q 2001 Elsevier Science B.V. All rights reserved.
Keywords: Alpha glycoprotein subunit; Growth; Homeodomain; LIM; Prolactin
1. Introduction
The anterior pituitary gland is the central endocrine regulator of growth, metabolism, lactation, reproduction, and the
response to stress. These processes are controlled by the
actions of hormones released from the distinct cell types
that comprise this organ. These cell types are somatotropes
that secrete GH, thyrotropes that release TSH, lactotropes
that produce PRL, gonadotropes that synthesize LH and
FSH, and corticotropes that secrete ACTH. Following
early inductive signals, these cells differentiate from a
common origin in a spatial and temporal manner that is
controlled by the actions of pituitary-speci®c and pituitary-enriched transcription factors (reviewed in Burrows et
Abbreviations: ACTH, adrenocorticotropin; aGSU, alpha glycoprotein
subunit; CPHD, combined pituitary hormone de®ciency; EMSA, electrophoretic mobility shift analysis; FSH, follicle-stimulating hormone; GH,
growth hormone; GST, glutathione-S-transferase; LH, luteinizing
hormone; LHX3, LIM homeobox gene 3; LIM-HD, LIM homeodomain;
NLI, nuclear LIM interactor; PCR, polymerase chain reaction; PRL, prolactin; TSH, thyroid-stimulating hormone
* Corresponding author. Tel.: 11-317-278-1797; fax: 11-317-274-2846.
E-mail address: [email protected] (S.J. Rhodes).
1
These authors contributed equally to this study.
al., 2000). These regulatory factors include the Lhx3, Lhx4,
Hesx1, Pitx-1, Pitx-2, Prop-1, and Pit-1 homeodomain
proteins (Burrows et al., 2000).
Mutations in the genes encoding several of these factors
have been demonstrated to cause pituitary disease. Recessive and dominant mutations of the PIT1 gene lead to the
development of a hypoplastic anterior pituitary gland and
CPHD (reviewed in Parks and Brown, 1999). These patients
display loss of circulating GH and PRL and often have low
or absent levels of TSH. Several types of mutations in PIT-1
have been shown to impair its function. For example, the
A158P (PfaÈf¯e et al., 1992) and P239S (Pernasetti et al.,
1998) mutations reduce the ability of PIT-1 to induce transcription of target genes. Nonsense mutations such as
R172X (Tatsumi et al., 1992) and E250X (Irie et al.,
1995) result in shortened proteins that do not contain the
DNA-binding homeodomain. The R271W defect may exert
a dominant negative effect by enabling this mutant of PIT-1
to bind certain DNA elements better than wild type PIT-1
(e.g. Cohen et al., 1995). This mutation also may prevent
dimerization of the molecule (Jacobson et al., 1997). To
date, all mutations described in the PROP-1 gene are recessive (reviewed in Parks and Brown, 1999). PROP-1 defects
cause a similar form of CPHD to that observed in PIT-1
0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0378-111 9(01)00369-9
62
K.W. Sloop et al. / Gene 265 (2001) 61±69
patients, but individuals with this disease also typically
display low or absent levels of LH and FSH (e.g. Duquesnoy
et al., 1998; Wu et al., 1998; Pernasetti et al., 2000) and may
exhibit late-onset ACTH de®ciency (e.g. Pernasetti et al.,
2000). PROP-1 mutations include missense mutations that
affect DNA interactions and frameshift mutations that result
in truncated, inactive proteins. Panhypopituitarism and
septo-optic dysplasia have been shown to result from an
autosomal recessive mutation in the HESX1 gene (Dattani
et al., 1998). This mutation causes an amino acid substitution that inhibits the DNA binding ability of this factor
(Dattani et al., 1998). Humans with pituitary disease of
unknown etiologic origin, such as patients with hypopituitarism and ectopically located posterior pituitary glands,
have been hypothesized to have defects in pituitary regulatory genes such as PIT-1, PROP-1, and LHX3 (Hamilton et
al., 1998; Pinto et al., 1999; Sloop et al., 2000a; Schmitt et
al., 2000), but the molecular basis for these diseases remains
unknown.
Lhx3 (also known as P-Lim/LIM-3) is a LIM-HD transcription factor (Seidah et al., 1994; Bach et al., 1995;
Zhadanov et al., 1995). This factor contains a DNA-binding
homeodomain and two LIM domains that mediate protein/
protein interaction and trans-activation functions. Lhx3 is
expressed in the embryonic rodent brain and spinal cord
and later is restricted to the developing and adult pituitary
gland (Seidah et al., 1994; Bach et al., 1995; Zhadanov et
al., 1995). Studies in mice have demonstrated that it is
essential for early pituitary structural development, for the
speci®cation of motor neuron subtypes, and later for the
differentiation of anterior and intermediate pituitary cell
types (Sheng et al., 1996, 1997; Sharma et al., 1998).
Lhx3 can activate transcription from anterior pituitary
hormone gene promoters, either alone or in synergy with
other pituitary regulatory proteins (e.g. Bach et al., 1995;
Meier et al., 1999; Sloop et al., 1999). In humans, two
LHX3 isoforms, LHX3a and LHX3b, are generated from
the LHX3 gene (Sloop et al., 1999, 2000b; Schmitt et al.,
2000). These isoforms possess identical LIM domains,
homeodomains, and carboxyl termini but possess different
amino termini that confer distinct functions upon the molecules (Sloop et al., 1999). Recently, mutations in the LHX3
gene have been shown to be associated with a novel, severe
form of CPHD (Netchine et al., 2000). Two mutations in
LHX3, a missense mutation of a tyrosine to a cysteine in the
second LIM domain (LHX3aY111C, LHX3bY116C) and
an intragenic deletion that results in a truncated protein
lacking the DNA-binding homeodomain, were identi®ed.
Patients with these mutations display retarded growth, pituitary hormone de®ciency, and abnormal neck and cervical
spine development. The underlying mechanism of pituitary
disease caused by mutations in the LHX3 gene has not been
explored. In this study, we investigated the molecular basis
of the LHX3 defects that cause human pituitary disease by
comparing the gene regulatory properties of wild type and
mutant LHX3 protein isoforms.
2. Materials and methods
2.1. Plasmid construction/mutagenesis
The myc epitope-tagged LHX3a and LHX3b expression
plasmids in the vector pcDNA3.1 have been described
(Sloop et al., 1999). Y111/116C and Y111/116F mutations
were introduced as described (Parker et al., 2000) using the
following primers: 5 0 -cgcgcccaggacttcgtgtgccacctgcactgctttgcc-3 0 and 5 0 -ggcaaagcagtgcaggtggcacacgaagtcctgggcgcg-3 0 (Y111/116C); 5 0 -cgcgcccaggacttcgtgttccacctgcactgctttgcc-3 0 and 5 0 -ggcaaagcagtgcaggtggaacacgaagtcctgggcgcg-3 0 (Y111/116F). The LHX3 a/b DHD cDNA was
generated using Expand High Fidelity DNA polymerase
(Roche Biochemical) and the following primers: 5 0 -cgggatccatgctgctggaaacggggct-3 0 (LHX3a), 5 0 -cgggatccatggaggcgcgcggggagct-3 0 (LHX3b), and 5 0 -gcgaagcttggaccaggaaaggtgggagctgcttggcggtttcgtagtccgc-3 0 (LHX3 a/b DHD).
Bacterial recombinant protein expression vectors for GSTLHX3 fusion proteins were constructed by cloning LHX3
cDNAs into pGEX-KT as described (Sloop et al., 1999).
The integrity of all plasmids was con®rmed by DNA
sequencing (Biochemistry and Molecular Biology, Indiana
University School of Medicine).
2.2. In vitro transcription/translation
Radiolabeled LHX3 proteins were synthesized in vitro
from pcDNA3.1 expression vector substrates containing
either wild type or mutant LHX3 cDNAs as described
(Sloop et al., 1999).
2.3. Cell culture, transfection, and luciferase assays
Mouse pituitary GHFT1-5 cells (Lew et al., 1993; kind
gift of Dr. Pamela Mellon, University of California San
Diego) and human embryonic kidney 293T cells were
cultured as described (Sloop et al., 1999). Using the CalPhos
system (Clontech), 1:5 £ 105 293T cells or 2:5 £ 105
GHFT1 cells/60 mm dish were transfected with calcium
phosphate/DNA precipitates. Reporter plasmid 0.5 mg and
0.1±1.0 mg of expression vector were added per 60 mm dish,
and all groups received equal ®nal DNA concentrations.
Control cultures received empty expression vector DNA.
The murine aGSU promoter luciferase plasmid (Roberson
et al., 1994) was a kind gift of Dr. Richard Maurer (Oregon
Health Sciences University). The Lhx3 consensus binding
site reporter gene was previously described (Sloop et al.,
1999). The rat Pit-1 and rat PRL promoter plasmids also
have been described (Meier et al., 1999). Luciferase activity
was measured 48 h after transfection as described (Meier et
al., 1999). All assay points were performed in triplicate.
Total cell protein was determined by the Bradford method
(BioRad), and luciferase activity was normalized to protein
concentration.
K.W. Sloop et al. / Gene 265 (2001) 61±69
2.4. Statistical analysis
Data points were compared using a one-tailed Student's ttest for paired samples using Sigma Plot 5.0 (Jandel Corp.).
Values were considered signi®cantly different when
P , 0:01.
2.5. Western analysis
Western analyses of cells transfected with LHX3 expression vectors were performed as described (Meier et al.,
1999). Mouse anti-myc 9E10 ascites ¯uid (Developmental
Studies Hybridoma Bank, University of Iowa) was used at
1:5000. The secondary antibody was a goat anti-mouse/
horseradish peroxidase (Sigma) at 1:15000. Results were
visualized using Lumi-Light PLUS chemiluminescence
reagents (Roche) and Biomax MR ®lm (Kodak).
2.6. Recombinant protein preparation/electrophoretic
mobility shift analysis
Recombinant GST-LHX3 proteins were expressed in E.
coli BL21 (DE3) pLysS and af®nity-puri®ed as previously
described (Meier et al., 1999). Proteins were analyzed on
12% SDS-PAGE gels followed by staining with Coomassie
brilliant blue. EMSAs were performed as described (Meier
et al., 1999). Oligonucleotides representing the Lhx3
consensus binding site and murine aGSU promoter 2350
to 2323 bp element have been described (Sloop et al.,
1999).
2.7. Protein/protein interaction assays
Expression vectors containing mouse NLI and rat Pit-1
cDNA have been described (Meier et al., 1999). Labeled
NLI and Pit-1 proteins were synthesized in vitro using
TNT rabbit reticulocyte lysate reagents (Promega) and
35
S-methionine. Protein/protein interaction assays using
labeled NLI or Pit-1 incubated with wild type and mutant
GST-LHX3 fusion proteins were performed as previously
described (Bach et al., 1995; Meier et al., 1999).
3. Results
3.1. Transcriptional properties of mutant LHX3 proteins
Expression vectors encoding wild type LHX3a and
LHX3b and LHX3aY111C, LHX3bY116C, LHX3aDHD,
and LHX3bDHD mutant protein isoforms were constructed
(Fig. 1A). In addition, because the amino acid residue at
position 111/116 often is a phenylalanine in Lhx-class
LIM-HD factors, expression vectors encoding LHX3aY111F and LHX3bY116F were generated to assess the
general importance of this position (Fig. 1A). In vitro transcription/translation analysis demonstrated that the expression vectors produced proteins of the predicted relative
molecular masses (Fig. 1B). We previously have demon-
63
strated that LHX3a isoform activates the promoter of the
aGSU gene (Sloop et al., 1999). This gene encodes the
common subunit of the LH, FSH, and TSH anterior pituitary
hormones. By contrast, the LHX3b isoform has little or no
effect on the induction of transcription from this promoter
(Sloop et al., 1999). LHX3 expression vectors were cotransfected with an aGSU luciferase gene into either heterologous
human embryonic kidney cells (293T) or mouse pituitary
cells (GHFT1) and gene activity was recorded (Fig. 2A,B).
In both cell types, LHX3a strongly induced the aGSU
promoter, while LHX3b and all mutant derivatives of both
LHX3 isoforms did not (P # 4 £ 1025 ). In 293T cells, the
data points for LHX3bY116C and LHX3bY116F were
slightly more than that for LHX3b (Fig. 2A), but this activity
was not signi®cantly different from controls (P . 0:01), and
this observation was not made in experiments using the
GHFT1 pituitary cells (Fig. 2B). These data demonstrate
that the LHX3 mutations associated with pituitary disease
impair the ability of LHX3a to activate the aGSU promoter
and do not inappropriately confer aGSU gene activation
function to LHX3b.
Experiments also were performed to test the ability of the
mutant LHX3 proteins to activate complex pituitary promoters acting in combination with the Pit-1 transcription factor.
Transient transfection experiments were performed using
the PRL promoter in 293T and GHFT1 cells as described
above. In these experiments, the LHX3aY111C and
LHX3aY111F proteins were able to synergistically activate
the PRL promoter with Pit-1 in 293T cells (Fig. 3A). In
comparison to wild type LHX3a, the LHX3aY111C mutant
exhibited signi®cantly reduced capacity to activate this gene
in 293T cells (P ˆ 8 £ 1024 ) but LHX3aY111F did not
(P ˆ 0:05). Similar observations were made in experiments
using GHFT1 cells (Fig. 3B). The LHX3aDHD and
LHX3bDHD mutants did not demonstrate synergy in these
experiments (Fig. 3A,B). LHX3b did synergize with Pit-1 in
these experiments, but the induction of the gene was low in
comparison to assays using LHX3a (Fig. 3A,B). As
observed with the aGSU promoter in 293T cells, luciferase
activities in cotransfection experiments with LHX3bY116C
or LHX3bY116F and Pit-1 were slightly higher than
observed for LHX3b and Pit-1 (Fig. 3A) but, again, this
observation was not made in experiments using GHFT1
cells (Fig. 3B). These results demonstrate that the Y116C
mutation does not confer an increased capacity upon
LHX3b to synergize with Pit-1.
The ability of the mutant LHX3 proteins to activate a
synthetic minimal reporter gene containing three copies of
a Lhx3 binding site also was examined. Both LHX3a and
LHX3b, with LHX3a having the greater activity, activate
this reporter gene (Sloop et al., 1999). The Y111C and
Y111F mutants of LHX3a both signi®cantly activated the
synthetic reporter gene (Fig. 4A). The Y111C mutant
displayed reduced activity compared to wild type
(P ˆ 8 £ 1024 ), but LHX3aY111F did not (P ˆ 0:1). Similar effects were observed when the corresponding LHX3b
64
K.W. Sloop et al. / Gene 265 (2001) 61±69
Fig. 1. Wild type and mutant LHX3 proteins. (A) Schematic depiction of the LHX3a and LHX3b isoforms and of mutant molecules used in this study. Hatched
regions, isoform-speci®c amino terminal domains; X, location of mutation; solid box, additional amino acids resulting from a frameshift caused by an
intragenic deletion. (B) Radiolabeled wild type and mutant LHX3 proteins were generated from cDNA expression vectors by in vitro transcription/translation,
separated by SDS electrophoresis, and dried gels were visualized by ¯uorography. The migration positions of protein standards (in kilodaltons) are shown.
derivatives were tested (Fig. 4A). The mutant LHX3
proteins lacking the homeodomain were inactive. Western
analysis was performed as a control to examine the levels of
mutant and wild type proteins produced in transfected cells
(Fig. 4B). In these experiments, it often was observed that
LHX3aY111F was expressed at slightly reduced levels
compared to wild type LHX3a. Together, these data indicate
that the Y111/116C mutant LHX3 proteins have a reduced
capacity to activate pituitary target genes and that the truncated mutants lacking the homeodomain are inactive.
3.2. DNA binding properties
Recombinant derivatives of the wild type and mutant
LHX3 proteins were generated as GST fusion proteins by
expression in E. coli followed by af®nity puri®cation.
EMSA analysis was performed using equivalent amounts
of puri®ed proteins and radiolabeled Lhx3 DNA binding
sites. LHX3a and LHX3aY111C displayed similar interaction with a consensus high-af®nity Lhx3 binding site (Fig.
5). By comparison to LHX3a, LHX3b has a reduced af®nity
K.W. Sloop et al. / Gene 265 (2001) 61±69
65
family of factors (Bach, 2000), Pit-1 (Bach et al., 1995),
MRG1 (Glenn and Maurer, 1999), and SLB (Howard and
Maurer, 2000). To test the hypothesis that the Y111/116C
mutation disrupts the LIM2 domain structure of LHX3
proteins and their binding to the partner proteins, we
performed in vitro binding assays using puri®ed LHX3
proteins and NLI or Pit-1. Af®nity resins containing equivalent amounts of wild type and Y111/116C LHX3 isoforms
were synthesized (Fig. 6E) and used as substrates in binding
assays with radiolabeled NLI or Pit-1 ligands. Both the wild
type and Y111/116C LHX3 proteins demonstrated signi®cant binding to NLI or Pit-1 (Fig. 6A/C,B/D, respectively).
LHX3a, LHX3b, and the Y111/116C mutants displayed
similar binding to NLI (Fig. 6A,C). However, the interac-
Fig. 2. Mutations in LHX3 inhibit activation of the aGSU gene promoter.
Human 293T cells (A) and mouse pituitary GHFT1 cells (B) were transiently transfected with an aGSU luciferase reporter gene and the indicated
expression vectors. Promoter activity was assayed by measurement of luciferase activity after 48 h. Activities are mean [light units/10 s/mg total
protein] of triplicate assays ^SEM. A representative experiment of at
least ®ve experiments is depicted.
for this class of DNA element (Sloop et al., 1999). As
observed for LHX3a, the Y116C mutation did not affect
the DNA binding capacity of LHX3b (Fig. 5). As expected,
LHX3aDHD and LHX3bDHD proteins did not bind the
tested DNA probes (Fig. 5). Similar data were obtained
using the 2323 bp aGSU promoter Lhx3 binding element
(data not shown).
3.3. Interaction of mutant LHX3 proteins with regulatory
protein partners
The two LIM domains of LIM-HD proteins such as Lhx3
allow interactions with partner proteins (reviewed in Bach,
2000). These proteins include the NLI/Ldb1/CLIM/Chip
Fig. 3. Mutations in LHX3 impair synergistic induction of the PRL promoter. 293T (A) and GHFT1 (B) cells were transiently transfected with a PRL
promoter reporter gene plasmid and wild type or mutant LHX3 and/or Pit-1
expression vectors. Luciferase activity was assayed after 48 h. Values are
mean [light units/10 s/mg total protein] of triplicate assays ^SEM. A
representative experiment of at least three experiments is shown.
66
K.W. Sloop et al. / Gene 265 (2001) 61±69
to date only a few mutations in this class of regulatory genes
have been correlated with human diseases. For example,
mutations in the LMX1B gene cause Nail-Patella syndrome
(Dreyer et al., 1998) and Zhao et al. (1999) have suggested
LHX8 as a candidate gene for a form of cleft palate in
humans. Loss-of-function mutations of mouse LIM-HD
genes often are lethal (e.g. reviewed in Hobert and Westphal, 2000), suggesting that severe mutations in the orthologous human genes might have similar effects. However, in
contrast to the observation that mice lacking the Lhx3 gene
die as neonates (Sheng et al., 1996), patients with LHX3
gene mutations survive and display a new form of CPHD
(Netchine et al., 2000).
In this study, we have demonstrated that the mutant
LHX3 proteins have a reduced gene activation capacity.
The Y111/116C mutant and the truncated LHX3 proteins
are incapable of inducing the aGSU gene, which encodes an
essential component of the three anterior pituitary glycopro-
Fig. 4. Mutations in LHX3 reduce activation of a synthetic luciferase
reporter gene containing Lhx3 binding sites. (A) Human 293T cells were
transiently transfected with a Lhx3 reporter gene and the indicated expression vectors. Promoter activity was assayed by measurement of luciferase
activity after 48 h. Activities are mean [light units/10 s/mg total protein] of
triplicate assays ^SEM. A representative experiment of at least three
experiments is depicted. (B) Western analysis using an anti-myc monoclonal antibody of cells transfected with myc epitope-tagged LHX3 expression
vectors con®rmed expression of proteins. The migration positions of
protein standards (in kilodaltons) are shown.
tion of the Y111/116C proteins to Pit-1 was reduced
compared to wild type LHX3 (Fig. 6B,D). These data indicate that the mutant LHX3 molecules retain their ability to
interact with NLI and Pit-1, but the binding to Pit1 is
reduced.
4. Discussion
Although critical roles for LIM-HD genes in guiding the
development of both vertebrate and invertebrate species
have been demonstrated (reviewed in Dawid et al., 1998),
Fig. 5. DNA binding of wild type and mutant LHX3 proteins. An EMSA
using a Lhx3 binding site probe was performed. Radiolabeled probe was
incubated with the indicated proteins and the resulting complexes were
separated from free probe (F) by electrophoresis. LHX3 protein/DNA
complexes are indicated by an arrow. Lane 1, probe alone; lane 2, GST
as a negative control; lanes 3±8, recombinant LHX3 proteins expressed in
E. coli.
K.W. Sloop et al. / Gene 265 (2001) 61±69
67
Fig. 6. Interaction of wild type and mutant LHX3 proteins with protein partners. In vitro binding assays demonstrate interaction of wild type and mutant LHX3
proteins with NLI (A) and Pit-1 (B) proteins. Radiolabeled proteins were generated by translation in the presence of [ 35S]-methionine and incubated with the
indicated GST fusion proteins or with GST alone as a control. After washing, equivalent samples of bound proteins were separated by electrophoresis and
visualized by ¯uorography. The migration positions of molecular weight standards (in kilodaltons) are shown. The binding of NLI (C) and Pit-1 (D) proteins
also was quanti®ed by scintillation counting. Bars indicate binding relative to that of LHX3a and represent the means of three independent experiments ^SEM.
(E) Coomassie brilliant blue stain of a SDS-polyacrylamide gel analysis of GST-LHX3 fusion proteins used in interaction studies. The migration of molecular
weight standards (in kilodaltons) is indicated.
tein hormones and is an early marker for pituitary development. The truncated isoforms also are unable to activate the
PRL gene. By contrast, the LHX3aY111C protein retains
some ability to synergize with Pit-1 in activating PRL,
although at a reduced level compared to wild type
LHX3a. However, the fact that the Y111/116C mutation is
associated with a similar disease to the more severe truncation mutation (Netchine et al., 2000) indicates that the
Y111/116 residue is critical to overall LHX3 function.
Indeed, a reduced capacity to activate certain target genes,
rather than complete loss of activity, may be suf®cient to
dramatically perturb pituitary development, especially
target genes that are involved in critical regulatory steps
of early pituitary ontogeny: a role that is predicted for
LHX3 (Sheng et al., 1996, 1997). Modest changes in gene
expression levels can have drastic consequences, especially
68
K.W. Sloop et al. / Gene 265 (2001) 61±69
if the affected gene is critical for the function of a cell or
tissue. For example, a modest change in collagen type I gene
expression is a key feature of ®brotic diseases (Jimenez et
al., 1986).
Maurer and colleagues have mapped a trans-activation
function within the LIM domains of mouse Lhx3 (Glenn
and Maurer, 1999). The reduced activity of the Y111/
116C proteins, therefore, is consistent with a reduction in
the ability of this domain to positively contribute to gene
activation. It also is possible that Y111/116 is post-translationally modi®ed by phosphorylation and that such modi®cation is required for activity of the protein. The LIM
domain is characterized by the presence of coordinated
zinc ions at the base of each of the two `®ngers' (PerezAlvarado et al., 1994). In the Y111/116C mutant, the introduced cysteine may affect the structure of the LHX3 LIM2
motif by providing an inappropriate coordination position
for the zinc ions that are integral to the LIM domain structure. Alternately, the introduced cysteine might form novel
disul®de bridges that disrupt LHX3 structure. It is clear that
the LIM domains are essential for Lhx-class protein functions. For example, Cheah and colleagues (2000) recently
have demonstrated that mice homozygous for a Lhx1 allele
encoding a protein with point mutations of the LIM domains
are a phenocopy for Lhx1 null mice that display dramatic
defects in head development.
The LIM domains of LIM-HD proteins mediate protein/
protein interactions (reviewed in Bach, 2000). In our experiments, we demonstrated that LHX3 interactions with the NLI
partner protein are maintained in the presence of the diseasecausing Y111/116C mutation. However, because LHX3
contains two LIM domains, both of which possess some
independent ability to interact with known partners such as
the NLI/CLIM proteins (e.g. Bach et al., 1997), disruption of
LIM2 by the Y111/116C mutation might not affect overall
recognition of such proteins by LHX3. It is possible, therefore, that the Y111/116C mutation prevents LIM2-dependent
LHX3 interaction with other factors that are required for its
activities in vivo. Binding of the Y111/116C mutant forms of
LHX3 to Pit-1 is reduced. This observation may, in part,
explain the reduction in ability of LHX3aY111C to activate
the PRL promoter in the presence of Pit-1.
We conclude that the mutant forms of the LHX3a and
LHX3b neuroendocrine transcription factor proteins display
impaired abilities to activate pituitary hormone gene promoters. Whereas the Y111/116C mutant LHX3 proteins exhibit
similar DNA binding properties to the wild type proteins on
known LHX3 DNA-binding elements, the truncated LHX3a
and LHX3b proteins lacking the homeodomain do not bind
DNA. The Y111/116C mutant LHX3 proteins retain some
ability to interact with the NLI and Pit-1 partner proteins. To
fully understand the mechanism by which these mutations
cause complex disease, a more complete identi®cation of
the target genes and partner proteins for the LHX3 proteins
during pituitary and motor neuron development will be
required.
Acknowledgements
We are grateful to Drs R. Maurer and P. Mellon for
materials and J. Bridwell and A. Hartman for advice.
Supported by grants to SJR from the National Science Foundation and the NRICGP/USDA.
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