QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00194

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Trieu, M. Articles by Turner, E. E. Search for Related Content PubMed PubMed Citation Articles by Trieu, M. Articles by Turner, E. E.

Direct autoregulation and gene dosage compensation by POU-domain transcription factor Brn3a

Department of Psychiatry, University of California, San Diego and San Diego VA Medical Center, La Jolla, CA 92093-0603, USA

View larger version (59K): [in a new window]   Fig. 1. Transgene expression controlled by a Brn3a sensory enhancer is strongly autoregulated. (A) Diagram of the mouse Brn3a locus and the structure of a Brn3a/lacZ transgene. (B) The Brn3a upstream flanking sequences target ß-galactosidase expression to the cranial sensory and dorsal root ganglia and their axons in an E13.5 Brn3a heterozygote embryo. (C) Enlargement of the cervical dorsal root ganglia of three littermate embryos with different Brn3a genotypes, stained under identical conditions, in which ß-gal activity is inversely proportional to Brn3a gene dosage.

View larger version (26K): [in a new window]   Fig. 2. Brn3a autoregulation increases with developmental age. The expression of ß-gal mRNA from the Brn3a/lacZ transgene was measured in embryonic tissues by RT-PCR. Autoradiograms and the corresponding phosphorimager integration of the triplicate assays are shown. (A) E12.5 head, representing ß-gal expression in the cranial sensory ganglia. (B) E13.5 head and (C) trunk, representing expression in the dorsal root ganglia. To confirm that Brn3a autoregulation is not dependent on the transgene insertion site, the relationship between ß-gal expression and gene dosage was examined for two independent reporter lines, which gave indistinguishable results. The ß-gal staining in Fig. 1B,C, and the quantitative assays in B represent the first of these lines, and the results shown in A,C are from the second line. Assays represent the mean±s.d. of triplicate PCR assays, and all determinations were repeated in at least three experiments. Phosphorimager units are arbitrarily scaled and cannot be compared between different sets of assays.

View larger version (46K): [in a new window]   Fig. 3. Brn3a-binding sites are highly conserved in the mouse and human sensory enhancer regions. Pairwise alignment of the mouse and human Brn3a sequences reveals several regions of upstream homology, the most extensive of which encompasses a cluster of Brn3a binding sites (red), and an octamer site (blue). The y-axis represents the percent of base pair identity within a moving window of 120 bases, at intervals of 40 bases. The designated map positions are for the mouse sequence and are specified relative to the most frequently used transcription start site (red G residue in the right-hand box).

View larger version (29K): [in a new window]   Fig. 4. Mutagenesis of the autoregulatory region of the Brn3a enhancer. (A) Shows the location of the Brn3a autoregulatory region, including four consensus Brn3a-binding domains (red) and one octamer binding site (blue). Two rounds of mutagenesis were required to eliminate Brn3a binding completely, finally incorporating 19 nucleotide changes (mut2). (B) The stoichiometry and stability of Brn3a/DNA complexes were assessed in complex-stability EMSA assays. In these assays, a competitor oligonucleotide was added at the stated time prior to the start of electrophoresis. The wild-type enhancer forms a stable complex with multiple Brn3a molecules, while the Mut1 enhancer retains a single stable binding site and binding is eliminated in Mut2. NP, no protein; NC, no competitor. (C) Luciferase reporter constructs containing wild-type or mutant enhancer domains, or a minimal promoter (-36prl) were co-transfected into CV1 epithelial cells with a Brn3a expression plasmid or vector alone (pcDNA). Elimination of the identified Brn3a-binding sites in the mutant enhancer construct eliminated transactivation by Brn3a. Bars represent the mean of triplicate assays, and similar results were observed in three experiments.

View larger version (86K): [in a new window]   Fig. 5. Elimination of Brn3a binding to its own enhancer abolishes autoregulation. Embryos expressing a Brn3a-mut/lacZ transgene and varying Brn3a gene dosage were analyzed for ß-gal expression. To ensure that the loss of autoregulation by the mutant enhancer was not an artifact of transgene insertion, two independent Brn3a-mut/lacZ lines were analyzed. (A,B) ß-gal staining is similar in wild-type and knockout embryos in the first of two lines analyzed. ß-gal expression in Brn3a+/+ embryos (A) is qualitatively indistinguishable from embryos expressing the wild-type Brn3a/lacZ transgene. Abnormal features of the Brn3a-/- embryos are indicated in B, including defasciculation of axon bundles and aberrant axons (small arrows), an ectopic mass of cell bodies and fibers adjacent to the caudal hindbrain (arrowheads), and abnormal trigeminal innervation of the CNS [the normal extent of trigeminal innervation of the principal (pr5) and spinal (sp5) trigeminal nuclei is outlined]. 5g, trigeminal ganglion; 9g, 9/10 cranial ganglion complex; drg, dorsal root ganglion. (C) A second transgenic line in which ß-gal expression is independent of Brn3a gene dosage for all three Brn3a genotypes. (D) RT-PCR assays of ß-gal mRNA in E13.5 embryonic head demonstrate that transgene expression is only slightly increased in Brn3a knockout embryos.

View larger version (46K): [in a new window]   Fig. 6. Brn3a expression is normalized towards wild-type levels in Brn3a heterozygotes. Brn3a mRNA levels were assayed in E13.5 embryonic tissues by RT-PCR; the mean ±s.d. of triplicate assays are shown. (A) Brn3a expression in the intact E13.5 head, including Brn3a neurons in the midbrain, hindbrain and cranial sensory ganglia. (B) Brn3a expression in isolated trigeminal ganglia. (C) The location of tissue samples dissected from the E13.5 midbrain tectum, in which Brn3a expressing neurons are labeled by immunohistochemistry (green). aq, aqueduct; pml, post-mitotic layer; ne, neuroepithelium; tec, tectum; teg, tegmentum. (D) Brn3a expression in the isolated E13.5 tectum.

View larger version (54K): [in a new window]   Fig. 7. Transgenic Brn3a overexpression suppresses endogenous Brn3a transcription. (A) Structure of a transgene in which the 11 kb Brn3a sensory enhancer is used to drive the expression of a Brn3a cDNA transgene. The transgene product can be distinguished from the endogenous Brn3a gene by the presence of a Myc epitope sequence immediately after the transcriptional initiation site. (B-E) Expression of the Brn3a/Myc transgene in E13.5 embryos. In the wild-type embryos shown in B,D, Brn3a is detected in the dorsal root and trigeminal ganglia, and in specific groups of spinal cord and hindbrain interneurons (brackets). In the Brn3a knockout mice shown in C,E, only Brn3a expression from the sensory transgene is detected, and Brn3a is not expressed in the CNS. The arrows in E indicate signal originating from vascular artifacts. 5g, trigeminal ganglion; drg, dorsal root ganglion; HB, hindbrain; pit, pituitary. (F) Quantitative assays of the expression of the endogenous Brn3a message in isolated trigeminal ganglia from Brn3a wild-type and heterozygous E13.5 embryos in the presence and absence of the Brn3a/Myc transgene. In these assays, Brn3a mRNA encoded by the native locus was assayed selectively by the use of a 5'-PCR oligonucleotide that is interrupted by the Myc epitope sequence inserted into the transgene. Brn3a data are normalized to the expression of neuron-specific enolase (NSE). It is unlikely that the reduction in Brn3a expression observed in the presence of the Brn3a/Myc transgene is due to the presence of regulatory sequences in the transgene itself because in the transgenic line used, the transgene was present at only one or two copies, based on quantitative RT-PCR comparison with a single copy gene.