Department of Psychiatry, University of California, San Diego and San Diego VA Medical Center, La Jolla, CA 92093-0603, USA
* Author for correspondence (e-mail: eturner{at}ucsd.edu)
Accepted 4 October 2002
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SUMMARY |
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Key words: Brn3a, POU-domain, Transcription factor, Autoregulation, Mouse
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INTRODUCTION |
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Recently, we have shown that an enhancer from the mouse Brn3a
locus targets transgene expression specifically to sensory neurons that
express this factor, but not to the CNS. Using the Brn3a sensory enhancer to
target a lacZ reporter transgene, we showed that mice lacking Brn3a
exhibit marked defects in sensory axon growth, and that all of the sensory
neurons that express Brn3a require it for survival in late gestation
(Eng et al., 2001). Defective
growth of sensory axons has also been reported for Brn3a null mutants in the
development of the vestibulocochlear system
(Huang et al., 2001
). Brn3b is
required for the normal growth of retinal ganglion cell axons
(Erkman et al., 2000
;
Gan et al., 1999
), and in the
developing retina, Brn3b and Brn3c appear to have complimentary roles in
regulating axon outgrowth (Wang et al.,
2002
).
The invertebrate Brn3 homologs Unc-86 and Acj6 also regulate sensory neural
development. In nematodes, Unc-86 is required for the development of
mechanoreceptor neurons, and manipulation of Unc-86 transcriptional activity
can cause defects in neuronal migration and axon growth
(Sze et al., 1997). In the
Drosophila antenna, Acj6 is necessary for the expression of a subset
of olfactory receptors (Clyne et al.,
1999a
; Clyne et al.,
1999b
), and in the CNS Acj6 is required for the correct projection
of retinal axons to lamina of the optic lobe, while mis-expression in
motoneurons produces abnormal axon outgrowth
(Certel et al., 2000
).
In vitro studies have shown that all of the POU4 factors have very similar
DNA recognition properties, binding with high affinity to the motif ATAATTAAT
or very similar sequences (Gruber et al.,
1997; Turner,
1996
). Presumably, the Brn3 factors regulate specific genes
required for sensory neural development via such sites. In a variety of
transfected cells in culture, Brn3a enhances the transcription of reporters
linked to both synthetic and naturally occurring binding sites. Several
downstream targets of the Brn3 genes have been suggested
(Budhram-Mahadeo et al., 1995
;
Erkman et al., 2000
;
Smith et al., 1997
), but a
direct role in the regulation of these genes in vivo has not been established.
In previous work, we have shown that Brn3a can interact with specific
recognition sequences within its own sensory enhancer region, suggesting that
Brn3a may regulate its own expression
(Trieu et al., 1999
).
We demonstrate that Brn3a negatively regulates its own expression in vivo, and that this regulation is mediated by a direct interaction between Brn3a and its recognition elements within the Brn3a sensory enhancer region. Comparison of the mouse and human Brn3a loci reveals that this regulatory region is remarkably conserved across species. Assays of Brn3a mRNA levels in mice with one or two functioning copies of the Brn3a gene show that this autoregulatory mechanism leads to a compensatory increase in the transcription of the intact copy of the Brn3a gene in heterozygotes, resulting in similar mRNA levels regardless of gene dosage. These findings suggest that Brn3a may function generally as a negative regulator of transcription in the sensory neurons, and that autoregulation of the Brn3a transcription unit can compensate for the loss of one allele, potentially suppressing haploinsufficiency in Brn3a heterozygotes.
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MATERIALS AND METHODS |
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Gene expression assays
RT-PCR for the relative quantitation of mRNA levels was performed using the
ThermoScript RT-PCR system (Invitrogen) according to the manufacturers
protocol. Reverse transcriptase reactions included gene-specific primers
consisting of 1x10-11 mol of each of the 3' primers
required for the intended assays. PCR reactions were conducted with Platinum
Taq polymerase (Invitrogen) using 32P-radiolabeled
oligonucleotides. For each transcript assayed, preliminary PCR reactions were
conducted with varying cycle numbers to determine the appropriate number of
cycles for quantitative assays, at which 2-10% of the final product was
formed. Assays for quantitation were then performed in triplicate, the
products separated in polyacrylamide gels and quantitated on a phosphorimager.
Oligonucleotide primers for the RT-PCR reactions were:
Oligonucleotide primers for the 18S ribosomal RNA were obtained from Ambion.
Genomic cloning and sequencing
Human BAC genomic clones encompassing the Brn3a locus were
obtained by PCR-based screening of a human BAC library as a commercial service
of Incyte Genomics. BAC clones were mapped by Southern hybridization using
probes derived from mouse Brn3a cDNA, and subcloned into pBKS. Approximately
10 kb of the human BRN3A gene (POU4F1 Human Gene
Nomenclature Database, extending upstream 8 kb from the open reading frame,
were sequenced by the shotgun method. Plasmid insert DNA encompassing this
region was sheared by sonication to an average fragment size of 200-400 bases,
repaired with Mung Bean Nuclease and blunt end ligated into the vector pBKSII.
About 50 overlapping cloned fragments were sequenced and assembled to produce
a contiguous map. Pairwise alignment of the homologous mouse and human genomic
sequences was performed using MegAlign software (Lasergene).
DNA binding and transcription assays
Complex-stability electrophoretic mobility shift assays (EMSAs) were
performed with DNA fragments excised from plasmid vectors, which were then
dephosphorylated, and end-labeled with 32P. Each assay contained
5x10-14 moles of radiolabeled DNA and
1x10-14 moles of Brn3a-GST fusion protein. At the zero time
point of each dissociation assay, 4x10-12 moles of specific
competitor oligonucleotide containing a consensus Brn3a recognition sequence
were added. The sequence of the oligonucleotide competitor and the assay and
electrophoresis conditions have been previously described
(Trieu et al., 1999). Under
the conditions of this assay, complexes of Brn3a with DNA sites that have high
enough affinity to mediate effectively the transcriptional effects of this
factor exhibit dissociation half lives of 5-100 minutes.
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RESULTS |
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In prior experiments, we interbred this Brn3a/LacZ reporter strain with
mice carrying a Brn3a null allele to demonstrate defects in axonal growth in
Brn3a knockout mice (Eng et al.,
2001). In the subsequent examination of a large number of embryos,
we noted that when littermates were stained for ß-gal expression under
identical conditions, the signal intensity was inversely proportional to Brn3a
gene dosage, with a marked upregulation of ß-gal expression in Brn3a
knockout embryos. Fig. 1C shows
this differential ß-gal activity in the cervical dorsal root ganglia of
Brn3a wild-type, heterozygote and knockout littermate embryos carrying the
Brn3a/lacZ reporter. A similar inverse relationship between transgene
expression and Brn3a gene dosage was also observed for the trigeminal ganglion
and the other cranial sensory ganglia expressing Brn3a.
RT-PCR assays were then used to examine the developmental relationship between Brn3a genotype and Brn3a/lacZ transgene expression, and to verify that the observed changes in ß-gal activity represented altered mRNA levels, and not a difference in the stability or distribution of ß-gal protein. Because ß-gal expression from the Brn3a/lacZ transgene was restricted to the sensory ganglia, RNA isolated from whole embryonic head or trunk tissue could be used for these assays. At E10.5, ß-gal activity was not yet detectable in sensory neurons by enzymatic staining, and no consistent difference in transgene expression between genotypes could be demonstrated by RT-PCR. At E12.5, cranial expression of ß-gal mRNA was about 2.5-fold elevated in knockouts relative to wild type (Fig. 2A).
|
At E13.5, ß-gal mRNA levels were consistently increased three- to
fourfold in both the cranial sensory ganglia (head) and the dorsal root
ganglia (trunk) of Brn3a knockout embryos relative to wild type
(Fig. 2B,C), exhibiting a
greater effect of gene dosage than that observed at E12.5. The onset of cell
death in Brn3a-/- embryos prevented the examination of
relative ß-gal expression at stages later than E13.5. A likely
explanation for the increasing effect observed with advancing developmental
age is that the cellular level of Brn3a protein increases in early
development, as sensory neurons exit the cell cycle and differentiate
(Fedtsova and Turner, 1995),
and by E13.5 reaches a threshold level that effectively suppresses transgene
expression in heterozygote and wild-type embryos. To demonstrate that the gene
dosage dependence of ß-gal expression was intrinsic to the 11 kb Brn3a
sensory enhancer, and not an artifact of the transgene insertion site, we
replicated these results using ß-gal staining and quantitative mRNA
assays in two independent strains of reporter mice and obtained very similar
results.
Brn3a interacts directly with a highly conserved autoregulatory
domain
The expression pattern of Brn3a is very similar in the mouse
(Fedtsova and Turner, 1995),
rat (Turner et al., 1994
) and
chick (Fedtsova and Turner,
2001
), and is likely to be conserved throughout the higher
vertebrates. Thus, it would be reasonable to expect that the sequences that
regulate Brn3a expression would also be somewhat conserved between species. To
search for such regulatory regions, we cloned and sequenced the human Brn3a
gene, including
8 kb of upstream flanking DNA. Alignment of the mouse
Brn3a gene and the corresponding human sequence revealed regions of
partial sequence conservation, such as the G/C-rich region surrounding the
transcription initiation site (Fig.
3), alternating with regions of divergence. In addition to these
expected regions of partial sequence conservation, this comparison revealed an
`island' of highly conserved sequence residing between 5400 and 5900 bases
upstream from the mouse Brn3a transcription start site, including a region of
244 bp that displayed 100% conservation between the mouse and human sequences.
Although we considered the possibility that this conserved sequence
represented the coding region of a closely adjacent gene, analysis of stop
codons and codon usage did not reveal an open reading frame on either DNA
strand. In previous studies, we have identified a cluster of Brn3a-binding
sites that reside
5.5 kb upstream from the mouse Brn3a transcription
start site (Trieu et al.,
1999
). These sites are all included within the region of highest
conservation between the mouse and human Brn3a sequences, and each of the
Brn3a recognition elements is entirely conserved.
|
Although such an extensive region of exact sequence identity between mouse
and human regulatory sequences is unusual, enhancer sequences of similar
extent that are 80-90% conserved between these species have been reported for
several genes, particularly for regulators of neural development. For example,
the neural bHLH gene Math1 (Atoh1 Mouse Genome Informatics), which is
required for correct development of the spinal neural tube and cerebellum, is
regulated by two 3' enhancer elements of 500 bp that show 87% and
92% homology between mouse and human
(Helms et al., 2000
). The
mouse gene for another neurogenic bHLH factor, Ngn2, contains multiple
5' and 3' enhancers, with 80-90% homology to the human sequence
extending up to 800 bp (Scardigli et al.,
2001
; Simmons et al.,
2001
). Shorter functional regulatory sequences with a similar
extent of conservation have been identified in the mouse and human
PAX6 (Kammandel et al.,
1999
), PAX4 (Brink et
al., 2001
) and PDX1 (IPF1 Human Genome
Nomenclature Database) (Marshak et al.,
2000
) genes, among others. It is unclear whether the difference
between the 80-90% conservation of the mouse and human sequences of the
previously described enhancers and the 100% conservation of the Brn3a-binding
region of Brn3a sensory enhancer has any regulatory significance, and it may
be fortuitous. It is likely that all of these domains mediate conserved
regulatory functions, although their roles in human development cannot be
directly demonstrated.
Previously, we have shown that the Brn3a-binding sites in the sensory enhancer mediate transcriptional activation in co-transfection assays performed in an epithelial cell line, suggesting that Brn3a is a positive regulator of its own expression. In the transgenic experiments shown here, however, Brn3a clearly inhibits the activity of its sensory enhancer in developing sensory neurons. There are two possible explanations for this discrepancy between the cell transfection and the transgenic results. First, Brn3a might act to inhibit the activity of this enhancer through an indirect mechanism in vivo, perhaps through the induction of a downstream regulatory factor that negatively regulates Brn3a through distinct binding sites. Second, Brn3a may act directly in both cases, but with opposite effects on transcription, perhaps due to differing populations of co-activators or co-repressors in the different cell types.
To determine whether Brn3a regulates its own expression by direct
interaction with its own upstream sequences in vivo, we introduced a number of
single base-pair mutations in the Brn3a autoregulatory region. Several
mutagenesis steps were required to eliminate Brn3a binding to this region
completely, the last two of which are shown in
Fig. 4A. In the final altered
enhancer sequence, 19 bp changes were introduced in four Brn3a consensus
binding sites, a variant octamer site and two AT-rich sites that weakly
matched the Brn3a consensus. As shown in
Fig. 4B, the elimination of
significant Brn3a-binding activity to the mutant enhancer sequences was then
demonstrated using by complex stability electrophoretic mobility shift assays
(Trieu et al., 1999). In these
assays, recombinant Brn3a protein is allowed to form a complex with
radiolabeled DNA. Then, at various times prior to electrophoresis, a large
excess of unlabeled competitor oligonucleotide is added. In the presence of
the competitor, the dissociation of the complex between Brn3a and the labeled
site exhibits first-order kinetics and a constant half-time (T1/2)
of dissociation (Gruber et al.,
1997
). In prior work, we have shown that the stability of Brn3a
binding to its recognition sites is highly correlated with its ability to
mediate transcriptional effects, and under the conditions of these assays,
sites with a dissociation T1/2 of less than 5 minutes are unlikely
to be transcriptionally active.
|
The altered enhancer sequences were then tested in transfection assays in CV1 epithelial cells (Fig. 4C). As expected, the Mut1 enhancer shows significantly reduced transcriptional activation compared with wild type (eightfold compared with 90-fold), and the Mut2 enhancer showed no transcriptional effect of Brn3a. The Mut2 enhancer sequence was then used to construct a new lacZ transgene (Brn3a-mut/lacZ), which differed from the original Brn3a/lacZ transgene only by the introduced point mutations in the Brn3a recognition sites. The Brn3a-mut/lacZ transgene construct was then used to generate new lines of transgenic mice. Elimination of Brn3a binding to the sensory enhancer had no effect on the qualitative expression pattern of the transgene, with ß-gal activity detectable in the cranial sensory ganglia, the dorsal root ganglia, and their central and peripheral axons (Fig. 5A,C).
|
Mice carrying the Brn3a-mut/lacZ transgene were then interbred
with mice heterozygous for a Brn3a null allele, and the resulting
Brn3a+/-, Brn3a-mut/lacZ reporter mice were again
crossed with Brn3a+/- mice to generate littermate embryos
containing the mutant reporter and all three Brn3a genotypes
(Fig. 5A-D). These embryos were
examined by whole-mount ß-gal staining at E13.5. Those embryos lacking
Brn3a exhibited several defects consistent with previous findings
(Eng et al., 2001), including
defasciculation of axon bundles and aberrant axons, failure to correctly
innervate peripheral targets, and an ectopic mass of cell bodies and fibers
adjacent to the caudal hindbrain. Trigeminal innervation of the CNS was also
abnormal, as shown in Fig. 5B,
where the normal patterns of trigeminal innervation of the principal (pr5) and
spinal (sp5) trigeminal nuclei are outlined.
Although ß-gal expression in Brn3a-mut/lacZ embryos was qualitatively indistinguishable from that regulated by the wild-type enhancer, the relationship between ß-gal expression and Brn3a gene dosage was nearly eliminated. Staining for ß-gal activity appeared equal in Brn3a wild-type, heterozygote and knockout embryos, and this effect was consistent in two independent Brn3a-mut/lacZ reporter lines. RT-PCR assays of RNA samples from E13.5 embryonic heads revealed that ß-gal mRNA levels in knockout embryos were 120% of wild-type (Fig. 5D), compared with the 300-400% increase in expression observed for the wild-type sensory enhancer in Brn3a knockout embryos (Fig. 1). These findings demonstrate that negative autoregulation of the Brn3a sensory enhancer is mediated by a direct interaction between Brn3a and its specific upstream recognition sequences.
Autoregulation provides a mechanism for Brn3a gene dosage
compensation
Brn3a heterozygote mice are viable, fertile and exhibit no obvious
behavioral abnormalities. Detailed studies of sensory neuron survival
(Huang et al., 1999) and axon
growth (Eng et al., 2001
) in
Brn3a knockout mice also have not detected significant defects in
heterozygotes. There are several possible explanations for the lack of
haploinsufficiency in heterozygous Brn3a animals. However, Brn3a regulation of
its own sensory enhancer suggests a direct transcriptional mechanism for gene
dosage compensation in mice with only one functional Brn3a allele.
To test whether Brn3a mRNA levels were normalized toward wild-type expression levels in heterozygotes, we measured Brn3a mRNA in neural tissues from wild-type and heterozygous embryos (Fig. 6). In the E13.5 embryonic head, heterozygote Brn3a levels were 83±8% of wild-type. Brn3a-expressing neurons in the embryonic head include the cranial sensory ganglia and also developing Brn3a interneurons in the midbrain and hindbrain. Therefore, to determine whether autoregulation pertains to Brn3a-expressing neurons in both the sensory PNS and the CNS, we also assayed Brn3a mRNA in dissected E13.5 trigeminal ganglia and midbrain tecta from wild-type and heterozygous embryos. In the trigeminal ganglion three sets of triplicate assays yielded heterozygote Brn3a mRNA levels corresponding to 89%, 89% and 97% of wild type. For assays of Brn3a expression in the CNS, we harvested tissue samples from the developing tectum of E13.5 embryos as shown in Fig. 6C. The analysis of three matched pairs of midbrain samples from heterozygote and wild-type littermates showed heterozygote Brn3a expression levels that were 77%, 78% and 85% of wild type, suggesting that Brn3a is also autoregulated in CNS neurons, but possibly to a lesser extent than in sensory ganglia. Because the enhancer sequences regulating Brn3a expression in the CNS have not been identified, it is not possible to determine whether the autoregulatory sites in the Brn3a sensory enhancer also confer autoregulation in CNS neurons, or whether the regulation of Brn3a in the CNS and PNS is entirely independent.
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To further test the effect of Brn3a on the regulation of its own transcription, we used the 11 kb Brn3a sensory enhancer to overexpress Brn3a in sensory ganglia. Fig. 7 shows the structure of the transgene construct used (Brn3a/Myc), which includes the Brn3a 11 kb sensory enhancer and a Brn3a cDNA insert that has been modified by the inclusion of a Myc epitope sequence immediately after the Brn3a initiator methionine codon. Because the native Brn3a sensory enhancer used to make this transgene is negatively autoregulated by Brn3a, it was anticipated that the transgene would be weakly expressed in Brn3a+/+ animals, but show increased expression in Brn3a heterozygote and knockout mice. Negative autoregulation of the transgene in wild-type mice has the advantage of minimizing any deleterious effects of overexpressing Brn3a, and three transgenic lines carrying the Brn3a/Myc transgene were viable, fertile and showed no obvious behavioral deficits.
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Brn3a/Myc transgenic mice were then interbred with Brn3a+/- mice, and Brn3a heterozygotes from this cross that expressed the Brn3a/Myc transgene were again bred to Brn3a+/- mice to generate Brn3a-/-, Brn3a/Myc+ embryos. The expression of Brn3a protein at E13.5 in these embryos was compared with wild-type littermates by immunohistochemistry. As expected, the normal pattern of Brn3a expression in spinal cord and hindbrain interneurons (Fig. 7B,D) was entirely absent in Brn3a-/-, Brn3a/Myc+ embryos (Fig. 7C,E). However, transgenic Brn3a expression in the dorsal root ganglia and trigeminal ganglion appeared qualitatively and quantitatively similar to wild type.
The loss of the majority of sensory neurons in late gestation is probably a
sufficient cause of neonatal death in Brn3a knockout mice, as other
mutant mice with extensive sensory loss, such as Bdnf/Nt3 double
knockouts, also die at birth (Liebl et
al., 1997). We examined newborn mice (n=20) from
Brn3a+/-, Brn3a/Myc+ crosses with
Brn3a+/- mice to see if the transgenic expression of Brn3a
in the sensory ganglia would rescue neonates from the expected lethal effects
of the Brn3a null mutation. However, Brn3a-/-,
Brn3a/Myc+ pups died within 24 hours of birth, as did the
knockout neonates without the transgene. The failure of the transgenic
expression of Brn3a in the sensory system to rescue pups from neonatal
lethality suggests that the function of Brn3a in the CNS is also essential for
survival.
Next, we determined the effect of the Brn3a/Myc transgene on Brn3a expression from the endogenous Brn3a locus in trigeminal ganglia isolated from E13.5 Brn3a-/-, Brn3a/Myc+ embryos. Transcripts originating from the native Brn3a locus were distinguished from the transgenic mRNA in RT-PCR assays by the use of a 5' PCR oligonucleotide that was interrupted by the Myc epitope and thus did not amplify the transgene product (Fig. 7A). Expression of the Brn3a/Myc transgene led to a significant but modest reduction in levels of the endogenous Brn3a mRNA (Fig. 7F). The limited effect of the Brn3a transgene on the expression of the endogenous Brn3a mRNA is probably due to the fact that the transgene itself is negatively autoregulated in Brn3a+/+ embryos, lessening the extent to which Brn3a can be overexpressed, and thus limiting the repression of the native Brn3a locus.
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DISCUSSION |
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In sensory neurons, Brn3a is essential for normal axon growth and for cell
survival in late gestation (Eng et al.,
2001; Huang et al.,
1999
; Huang et al.,
2001
). The role of Brn3a is less well understood in the CNS, but
it appears to affect the migration and survival of at least some of the CNS
neurons in which it is expressed (McEvilly
et al., 1996
; Xiang et al.,
1996
). Brn3a, like all transcriptional regulators of development,
is assumed to affect gene expression by interacting with cis-acting
elements in the regulatory regions of its target genes, and the DNA-binding
properties of Brn3a and other neural POU-domain factors have been extensively
characterized in vitro and in cell transfection models
(Gruber et al., 1997
;
Rhee et al., 1998
;
Turner, 1996
). However, very
few of the direct regulatory targets of these late neural transcription
factors have been identified, and in most cases it is not known whether they
act primarily as enhancers or repressors of transcription.
In previous work, we have demonstrated that Brn3a expression in the sensory
system, but not in the CNS, is regulated by an upstream sensory enhancer
region containing multiple high-affinity Brn3a-binding sites
(Eng et al., 2001). Here, we
have shown, by manipulating the Brn3a-binding sites within this enhancer, that
Brn3a directly attenuates its own expression in vivo. In sensory ganglia
containing two intact copies of the Brn3a gene, transcriptional
activity of each copy is partly suppressed. Conversely, when one allele is
disrupted by a targeted mutation, the transcriptional activity of the intact
allele is increased
80%, nearly compensating for the inactive gene copy.
When both alleles are disrupted, a functional Brn3a mRNA cannot be
transcribed, but the activity of a reporter transgene driven by the Brn3a
sensory enhancer is about fourfold increased. Partial compensation for Brn3a
gene dosage also appears to occur in the midbrain tectum, but to a lesser
extent than in the trigeminal ganglion. Whether Brn3a autoregulation in the
tectum is controlled by the same upstream Brn3a recognition sites, or by
distinct elements, cannot presently be determined, because a specific enhancer
that regulates Brn3a expression in the CNS has not yet been identified.
Negative autoregulation by Brn3a raises the possibility that the general
function of this factor will be to repress the transcription of all of its
direct targets, at least in the developing sensory system. In the developing
spinal cord, where the molecular mechanisms of neural specification are best
understood, several homeodomain proteins that characterize progenitor cell
domains in the spinal neuroepithelium act as transcriptional repressors
(Lee and Pfaff, 2001). These
repressive activities prevent the inappropriate expression of transcription
factors that characterize adjacent domains, and refine the boundaries between
their domains. By contrast, examples of direct positive regulation of
downstream target genes by neural transcription factors are relatively few,
and it appears increasingly likely that neurons are transcriptionally defined
to a large extent by the repression of inappropriate gene expression.
The role of Brn3a as a negative regulator of its own expression in vivo is
somewhat surprising given that it strongly activates transcription when
co-transfected with reporter plasmids in cultured epithelial cells
(Gruber et al., 1997;
Trieu et al., 1999
). This
difference cannot be accounted for by the Brn3a recognition elements used or
their immediate context, because in transfection assays Brn3a strongly
activates transcription from reporters containing the same
200 base pair
autoregulatory region that confers negative regulation in vivo. Although it is
possible that this reversal of transcriptional effect depends on the broader
context of the cis-acting sequences, it seems more likely that
sensory neurons express a co-repressor that converts Brn3a from a positive to
a negative regulator of transcription, or that dividing epithelial cells
express an essential Brn3a co-activator.
One set of candidates for a Brn3a co-repressor that could act in the
sensory system are the murine Gro/TLE proteins, homologs of
Drosophila groucho (Smith and
Jaynes, 1996), which have recently been shown to mediate
transcriptional repression by many of the progenitor zone homeodomain proteins
in the developing spinal cord (Muhr et
al., 2001
). The Gro/TLE repressors interact with a variety of
homeodomain proteins through a conserved Engrailed homology (eh1) domain. The
locus for one factor containing an eh1 domain, the motoneuron transcription
factor HB9, exhibits autoregulatory behavior very similar to Brn3a, in that
reporter genes linked to this locus are markedly upregulated in HB9 knockout
mice (Arber et al., 1999
).
However, it is not known whether HB9 autoregulation occurs by a direct or
indirect mechanism. The Gro/TLE protein Grg4 is expressed in the developing
cranial and dorsal root ganglia where it might be available to interact with
Brn3a (Koop et al., 1996
), but
Brn3a does not have a readily apparent eh1 domain to mediate this
interaction.
Autoregulation and haploinsufficiency
In humans, a majority of the known transcription factor diseases exhibit
dominant inheritance (Veitia,
2002). This may reflect in part an ascertainment bias, because
homozygous mutations in many transcription factors result in embryonic or
neonatal lethality and thus would not be represented in human populations. In
some cases, dominant inheritance has been attributed to the dominant negative
effect of toxic proteins, or to allele-specific regulation. However, the
default explanation for dominance is haploinsufficiency caused by reduced
protein levels (Nutt et al.,
1999
).
In mice, null mutants have been generated for a large number of
transcriptional regulators of neurodevelopment. For a majority of the members
of the LIM, POU, variant homeodomain and bHLH families, these mutations have
no known phenotype in heterozygous animals. By contrast, several members of
the Pax gene family exhibit haploinsufficiency in both mice and humans
(Chi and Epstein, 2002).
Among the members of the POU family expressed in the nervous and endocrine
systems, loss-of-function mutations appear to be consistently recessive.
Mutations in Pit1, a regulator of pituitary development, cause recessive
dwarfism in mice and in humans. Autosomal dominant Pit1 mutations are also
known, but these result from mutations outside the DNA-binding domain of the
molecule, and most probably cause dominant-negative transcriptional effects
(Parks et al., 1999). Pit1
exhibits positive autoregulation that maps to a conserved distal enhancer, and
is necessary for the sustained but not the embryonic expression of the gene
(DiMattia et al., 1997
;
Rhodes et al., 1993
).
Within the POU4 transcription factor class, the effects of Brn3a
loss-of-function on sensory neuron survival and axon growth and fasciculation
observed in Brn3a knockout mice do not appear in heterozygotes
(Eng et al., 2001;
Huang et al., 1999
).
Similarly, the excessive retinal ganglion cell death and aberrant axon growth
observed in Brn3b-null mutants does not occur in heterozygotes
(Gan et al., 1999
), and the
auditory defects observed in Brn3c-null mice are also recessive
(Keithley et al., 1999
;
Xiang et al., 1997
). Mutations
in Brn3c have also been identified as a causes of progressive heritable
hearing loss in humans (Vahava et al.,
1998
). This syndrome exhibits autosomal dominant inheritance,
which has been attributed to the dominant-negative effects of the mutant
protein. No enhancers have been characterized that accurately reproduce the in
vivo expression patterns of Brn3b and Brn3c, so it is not known if
autoregulatory elements are present in these genes.
Haploinsufficiency can be suppressed by several mechanisms, including the expression of functionally redundant genes, expression of sufficient protein from a single functional allele to saturate binding sites, post-translational modulation of transcription factor activity or by regulatory feedback from downstream targets. In the case of Brn3a, the loss of one allele partly relieves direct transcriptional suppression, leading to an increase in expression of the intact gene copy. This is a particularly economical, self-contained homeostatic mechanism for maintaining the expression level of a crucial developmental regulator. Given that a large number of transcription factors regulating neurodevelopment appear to act as transcriptional repressors, and that the null mutations for a majority of these do not exhibit heterozygote phenotypes, it will be interesting to see if such a mechanism is widely employed.
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ACKNOWLEDGMENTS |
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