Molecular codes for cell type specification in Brn3 retinal ganglion cells.

Visual information is conveyed from the eye to the brain by distinct types of retinal ganglion cells (RGCs). It is largely unknown how RGCs acquire their defining morphological and physiological features and connect to upstream and downstream synaptic partners. The three Brn3/Pou4f transcription factors (TFs) participate in a combinatorial code for RGC type specification, but their exact molecular roles are still unclear. We use deep sequencing to define (i) transcriptomes of Brn3a- and/or Brn3b-positive RGCs, (ii) Brn3a- and/or Brn3b-dependent RGC transcripts, and (iii) transcriptomes of retinorecipient areas of the brain at developmental stages relevant for axon guidance, dendrite formation, and synaptogenesis. We reveal a combinatorial code of TFs, cell surface molecules, and determinants of neuronal morphology that is differentially expressed in specific RGC populations and selectively regulated by Brn3a and/or Brn3b. This comprehensive molecular code provides a basis for understanding neuronal cell type specification in RGCs.

Systemic ocular antigen immunization leads only to a minor secondary immune response.

The immunization with optic nerve homogenate antigen (ONA) or S100 induced retinal degeneration. Since many neurological diseases are reinforced or initiated by immune cells, leucocytes were analyzed. CD3(+) T-cells in the retina increased slightly in ONA rats, but not in S100 treated retinas. No CD45R(+) B-cells and granulocytes could be detected in the retinas. At early stages, CD3(+) cells, Iba1(+) macrophages and granulocytes of the secondary lymphoid organs were not affected. Yet, the sole injection of pertussis toxin led to a shift to fewer CD45R(+) cells and more granulocytes in spleens. Later, splenic Iba1(+) macrophages were increased in both groups. We conclude that the retinal infiltration of lymphocytes is not crucial for the degeneration process and rather an epiphenomenon.

The influence of activity on axon pathfinding in the optic tectum.

The relative importance of neural activity versus activity-independent cues in shaping the initial wiring of the brain is still largely an open question. While activity is clearly critical for circuit rearrangements after initial connections have been made, whether it also plays a role in initial axon pathfinding remains to be determined. Here, we investigated this question using the guidance of zebrafish retinal ganglion cell axons to their targets in the tectum as a model. Recent results have implicated biased branching as a key feature of pathfinding in the zebrafish tectum. Using tetrodotoxin to silence neural activity globally, we found a decrease in the area covered by axon branches during pathfinding. After reaching the target, there were dynamic differences in axon length, area and the number of branches between conditions. However, other aspects of pathfinding were unaffected by silencing, including the ratio of branches directed toward the target, length, and number of branches, as well as turning angle, velocity, and number of growth cones per axon. These results challenge the hypothesis that neural connections develop in sequential stages of molecularly guided pathfinding and activity-based refinement. Despite a maintenance of overall guidance, axon pathfinding dynamics can nevertheless be altered by activity loss.

Dre - Cre sequential recombination provides new tools for retinal ganglion cell labeling and manipulation in mice.

BACKGROUND: Genetic targeting methods have greatly advanced our understanding of many of the 20 Retinal Ganglion Cell (RGC) types conveying visual information from the eyes to the brain. However, the complexity and partial overlap of gene expression patterns in RGCs call for genetic intersectional or sparse labeling strategies. Loci carrying the Cre recombinase in conjunction with conditional knock-out, reporter or other genetic tools can be used for targeted cell type ablation and functional manipulation of specific cell populations. The three members of the Pou4f family of transcription factors, Brn3a, Brn3b and Brn3c, expressed early during RGC development and in combinatorial pattern amongst RGC types are excellent candidates for such gene manipulations. METHODS AND FINDINGS: We generated conditional Cre knock-in alleles at the Brn3a and Brn3b loci, Brn3a(CKOCre) and Brn3b(CKOCre). When crossed to mice expressing the Dre recombinase, the endogenous Brn3 gene expressed by Brn3a(CKOCre) or Brn3b(CKOCre) is removed and replaced with a Cre recombinase, generating Brn3a(Cre) and Brn3b(Cre) knock-in alleles. Surprisingly both Brn3a(Cre) and Brn3b(Cre) knock-in alleles induce early ubiquitous recombination, consistent with germline expression. However in later stages of development, their expression is limited to the expected endogenous pattern of the Brn3a and Brn3b genes. We use the Brn3a(Cre) and Brn3b(Cre) alleles to target a Cre dependent Adeno Associated Virus (AAV) reporter to RGCs and demonstrate its use in morphological characterization, early postnatal gene delivery and tracing the expression of Brn3 genes in RGCs. CONCLUSIONS: Dre recombinase effectively recombines the Brn3a(CKOCre) and Brn3b(CKOCre) alleles containing its roxP target sites. Sequential Dre to Cre recombination reveals Brn3a and Brn3b expression in early mouse development. The generated Brn3a(Cre) and Brn3b(Cre) alleles are useful tools that can target exogenously delivered Cre dependent reagents to RGCs in early postnatal development, opening up a large range of potential applications.

Whole number, distribution and co-expression of brn3 transcription factors in retinal ganglion cells of adult albino and pigmented rats.

The three members of the Pou4f family of transcription factors: Pou4f1, Pou4f2, Pou4f3 (Brn3a, Brn3b and Brn3c, respectively) play, during development, essential roles in the differentiation and survival of sensory neurons. The purpose of this work is to study the expression of the three Brn3 factors in the albino and pigmented adult rat. Animals were divided into these groups: i) untouched; ii) fluorogold (FG) tracing from both superior colliculli; iii) FG-tracing from one superior colliculus; iv) intraorbital optic nerve transection or crush. All retinas were dissected as flat-mounts and subjected to single, double or triple immunohistofluorescence The total number of FG-traced, Brn3a, Brn3b, Brn3c or Brn3 expressing RGCs was automatically quantified and their spatial distribution assessed using specific routines. Brn3 factors were studied in the general RGC population, and in the intrinsically photosensitive (ip-RGCs) and ipsilateral RGC sub-populations. Our results show that: i) 70% of RGCs co- express two or three Brn3s and the remaining 30% express only Brn3a (26%) or Brn3b; ii) the most abundant Brn3 member is Brn3a followed by Brn3b and finally Brn3c; iii) Brn3 a-, b- or c- expressing RGCs are similarly distributed in the retina; iv) The vast majority of ip-RGCs do not express Brn3; v) The main difference between both rat strains was found in the population of ipsilateral-RGCs, which accounts for 4.2% and 2.5% of the total RGC population in the pigmented and albino strain, respectively. However, more ipsilateral-RGCs express Brn3 factors in the albino than in the pigmented rat; vi) RGCs that express only Brn3b and RGCs that co-express the three Brn3 members have the biggest nuclei; vii) After axonal injury the level of Brn3a expression in the surviving RGCs decreases compared to control retinas. Finally, this work strengthens the validity of Brn3a as a marker to identify and quantify rat RGCs.

Combinatorial expression of Brn3 transcription factors in somatosensory neurons: genetic and morphologic analysis.

The three members of the Brn3 family of POU-domain transcription factors (Brn3a/Pou4f1, Brn3b/Pou4f2, and Brn3c/Pou4f3) are expressed in overlapping subsets of visual, auditory/vestibular, and somatosensory neurons. Using unmarked Brn3-null alleles and Brn3 conditional alleles in which gene loss is coupled to expression of an alkaline phosphatase reporter, together with sparse Cre-mediated recombination, we describe the following: (1) the overlapping patterns of Brn3 gene expression in somatosensory neurons; (2) the manner in which these patterns correlate with molecular markers, peripheral afferent arbor morphologies, and dorsal horn projections; and (3) the consequences for these neurons of deleting individual Brn3 genes in the mouse. We observe broad expression of Brn3a among DRG neurons, but subtype-restricted expression of Brn3b and Brn3c. We also observe a nearly complete loss of hair follicle-associated sensory endings among Brn3a(-/-) neurons. Together with earlier analyses of Brn3 gene expression patterns in the retina and inner ear, these experiments suggest a deep functional similarity among primary somatosensory neurons, spiral and vestibular ganglion neurons, and retinal ganglion cells. This work also demonstrates the utility of sparse genetically directed labeling for visualizing individual somatosensory afferent arbors and for defining cell-autonomous mutant phenotypes.

Electroporation-mediated gene delivery of cleavage-resistant pro-nerve growth factor causes retinal neuro- and vascular degeneration.

PURPOSE: Neurotrophins, including nerve growth factor (NGF), are secreted by glia as a pro-form (proNGF) that is normally cleaved into the mature ligand. Increases of proNGF has been well documented in retinal neurodegenerative diseases. Since systemic overexpression of proNGF exhibits embryonic lethality, we aimed to establish a model that specifically and stably overexpresses a cleavage-resistant mutant of proNGF (proNGF123) plasmid in the retina using electroporation. METHODS: Male Sprague-Dawley rats were injected intravitreally with pGFP or pGFP-proNGF123 plasmids, then electroporated with various settings for optimization. Retinal cell death and ganglion cell count were assessed by TUNEL and immunostaining with anti-Brn3. Expression of proNGF, NGF, and their receptors was examined by western blot. Retinal vascular permeability was assessed by extravasation of bovine serum albumin-fluorescein. Development of acellular capillaries was assessed by periodic acid-Schiff and hematoxylin staining. RESULTS: Successful pGFP-proNGF123 gene delivery and expression of proNGF was demonstrated by western blot and extensive proNGF immunostaining in retina sections. Overexpression of proNGF reduced NGF expression while inducing the expression of neurotrophin receptors, including p75(NTR) and tyrosine receptor kinase A, but not sortilin. Overexpression of proNGF resulted in ~50% reduction in ganglion cell count and fivefold increase in TUNEL-positive cells in rat retina. In addition, overexpression of proNGF induced breakdown of the blood-retina barrier evident by twofold increase in extravasation of bovine serum albumin-fluorescein after 1 week and induced the development of acellular capillaries after 4 weeks. CONCLUSIONS: Electroporation can successfully incorporate and express biologically active cleavage-resistant proNGF locally in rat retinas. Overexpression of cleavage-resistant proNGF can be a useful tool to investigate specific molecular mechanisms by which proNGF causes neurodegeneration and vascular injury in the retina.

Differential expression of Brn3 transcription factors in intrinsically photosensitive retinal ganglion cells in mouse.

Several subtypes of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) have been reported. The M1 type of ipRGCs exhibit distinct properties compared with the remaining (non-M1) cells. They differ not only in their soma size and dendritic arbor, but also in their physiological properties, projection patterns, and functions. However, it is not known how these differences arise. We tested the hypothesis that M1 and non-M1 cells express Brn3 transcription factors differentially. The Brn3 family of class IV POU-domain transcription factors (Brn3a, Brn3b, and Brn3c) is involved in the regulation of differentiation, dendritic stratification, and axonal projection of retinal ganglion cells during development. By using double immunofluorescence for Brn3 transcription factors and melanopsin, and with elaborate morphometric analyses, we show in mouse retina that neither Brn3a nor Brn3c are expressed in ipRGCs. However, Brn3b is expressed in a subset of ipRGCs, particularly those with larger somas and lower melanopsin levels, suggesting that Brn3b is expressed preferentially in the non-M1 cells. By using dendritic stratification to distinguish M1 from non-M1 cells, we found that whereas nearly all non-M1 cells expressed Brn3b, a vast majority of the M1 cells were negative for Brn3b. Interestingly, in the small proportion of the M1 cells that did express Brn3b, the expression level of Brn3b was significantly lower than in the non-M1 cells. These results provide insights about how expression of specific molecules in a ganglion cell could be linked to its role in visual function.

Primary culture of neurospheres obtained from fetal mouse central nervous system and generation of inner ear hair cell immunophenotypes in vitro.

OBJECTIVE: To investigate whether hair cell immunophenotypes can be derived from the central nervous system. DESIGN: We established in vitro cell cultures from embryonic day 14.5 fetal rat brain tissue, and analysed changes in the immunohistochemical features of these cell cultures following differentiation. RESULT: The immature neural progenitors obtained from the fetal mouse central nervous system generated cell immunophenotypes which expressed epitopes of the hair cell marker proteins myosin VIIa and Brn-3c and the supporting cell marker pan-cytokeratin. CONCLUSION: Neural progenitors have the potential to differentiate into inner ear hair cell and supporting cell phenotypes, and thus may be a useful material for cell transplantation therapy aiming to replace damaged inner ear hair cells.

Evolutionary origin of rhopalia: insights from cellular-level analyses of Otx and POU expression patterns in the developing rhopalial nervous system.

In Cnidaria, the medusae of Scyphozoa and its sister-group Cubozoa uniquely possess rhopalia at their bell margin. These sensory centers coordinate behavior and development. We used fluorescent in situ hybridization and confocal microscopy to examine mRNA expression patterns in Aurelia sp.1 (Cnidaria, Scyphozoa) during early medusa formation, while simultaneously visualizing the developing nervous system by immunofluorescence. The genes investigated include AurOtx1, and the POU genes, AurPit1, and AurBrn3, homologs of genes known to function in cephalar neural organization and sensory cell differentiation across Bilateria. Our results show that AurOtx1 expression defines the major part of the oral neuroectodermal domain of the rhopalium, within which distinct populations of AurBrn3- and AurPit1-expressing sensory cells develop. Thus, despite the unique attributes of rhopalial evolution, we suggest that the rhopalial nervous system of scyphozoan medusae involves similar patterns of differential expression of genes that function in bilaterian cephalic structure and neuroendocrine system development. We propose that rhopalia evolved from preexisting sensory structures that developed distinct populations of sensory cells differentially expressing POU genes within Otx oral-neuroectodermal domains. This implies some commonality of developmental genetic functions involving these genes in the still poorly constrained common ancestor of bilaterians and cnidarians.

Direct inhibition of the DNA-binding activity of POU transcription factors Pit-1 and Brn-3 by selective binding of a phenyl-furan-benzimidazole dication.

The development of small molecules to control gene expression could be the spearhead of future-targeted therapeutic approaches in multiple pathologies. Among heterocyclic dications developed with this aim, a phenyl-furan-benzimidazole dication DB293 binds AT-rich sites as a monomer and 5'-ATGA sequence as a stacked dimer, both in the minor groove. Here, we used a protein/DNA array approach to evaluate the ability of DB293 to specifically inhibit transcription factors DNA-binding in a single-step, competitive mode. DB293 inhibits two POU-domain transcription factors Pit-1 and Brn-3 but not IRF-1, despite the presence of an ATGA and AT-rich sites within all three consensus sequences. EMSA, DNase I footprinting and surface-plasmon-resonance experiments determined the precise binding site, affinity and stoichiometry of DB293 interaction to the consensus targets. Binding of DB293 occurred as a cooperative dimer on the ATGA part of Brn-3 site but as two monomers on AT-rich sites of IRF-1 sequence. For Pit-1 site, ATGA or AT-rich mutated sequences identified the contribution of both sites for DB293 recognition. In conclusion, DB293 is a strong inhibitor of two POU-domain transcription factors through a cooperative binding to ATGA. These findings are the first to show that heterocyclic dications can inhibit major groove transcription factors and they open the door to the control of transcription factors activity by those compounds.

Expression of osteopontin in the rat retina: effects of excitotoxic and ischemic injuries.

PURPOSE: The cytokine osteopontin (OPN) has been localized to the retinal ganglion cell layer in the normal rodent retina, prompting the suggestion that it could serve as a useful marker for identifying and quantifying such neurons in models of retinal and optic nerve neurodegeneration. In the present study, we characterized the time course and cellular localization of OPN expression in the rat retina after excitotoxic and ischemic injuries. METHODS: Excitotoxicity and ischemia-reperfusion experiments were performed by using standard techniques. Rats were killed at various time points, and the retinas were removed either for mRNA analysis or to be processed for immunohistochemistry. RESULTS: In the normal retina, double-labeling immunofluorescence indicated that OPN is expressed by the majority of, if not all, RGCs, since OPN was associated with more cells than Brn-3, but was colocalized with Thy1.1. NMDA, kainic acid, and ischemia-reperfusion all caused decreases in the total retinal levels of Thy1 and Brn-3 mRNAs, reflecting injury to RGCs, but a dramatic, short-lived upregulation in OPN mRNA. The source of the increased OPN signal after excitotoxic-ischemic insults is unlikely to be injured RGCs, as no alteration in the intensity of OPN immunostaining in RGCs was apparent. Instead, additional cells, mostly contained within the IPL, were identified as positive for OPN. Double-labeling immunofluorescence showed that ED1 always colocalized with OPN in these cells, indicating their status as activated microglia. CONCLUSIONS: OPN is exclusively expressed by RGCs in the physiological retina, but in response to retinal neurodegeneration is synthesized de novo by endogenous, activated microglia.

Differentiation of an auditory neuronal cell line suitable for cell transplantation.

The auditory neuroblast cell line US/VOT-N33 (N33), which is conditionally immortal, was studied as an in vitro model for the differentiation of spiral ganglion neurons (SGNs) and as a candidate for cell transplantation in rodents. It expresses numerous molecular markers characteristic of auditory neuroblasts, including the transcription factors GATA3, NeuroD, Brn3a and Islet1, as well as the neuronal cytoskeletal protein beta3-tubulin. It displays active migratory behaviour in vitro and in vivo. In the presence of the fibroblast growth factors FGF1 or FGF2 it differentiates bipolar morphologies similar to those of native SGNs. In coculture with neonatal cochlear tissue it is repelled from epithelial surfaces but not from native SGNs, alongside which it extends parallel neuronal processes. When injected into the retina in vivo, EGFP-labelled N33 cells were traced for 1-2 weeks and migrated rapidly within the subretinal space. Cells that found their way into the retinal ganglion cell layer extended multiple processes but did not express beta3-tubulin. The ability of N33 to migrate, to differentiate, to localize with native SGNs in vitro and to survive in vivo suggests that they provide an effective model for SGN differentiation and for cell transplantation into the ear.

Brn-3a is required for the generation of proprioceptors in the mesencephalic trigeminal tract nucleus.

The distribution of motor and proprioceptive neurons was investigated in the trigeminal nervous system of wild-type and Brn-3a knockout mice at embryonic day 18.5 and postnatal day 0. We found that the trigeminal motor nucleus (Mo5) contained abundant motoneurons in wild-type (mean number +/- SD per section = 128 +/- 22, range = 93-167) and knockout (mean number +/- SD per section = 121 +/- 23, range = 75-158) mice and that the cell size of Mo5 neurons was similar between these mice (wild-type, mean +/- SD = 165 +/- 59 microm2, range = 65-326 microm2; knockout, mean +/- SD = 167 +/- 59 microm2, range = 71-327 microm2). Mo5 neurons were immunoreactive for calcitonin gene-related peptide and such immunoreactive neurons were abundant in both wild-type and mutant mice. In the mesencephalic tract nucleus (Mes5) of wild-type mice, many proprioceptors (mean number +/- SD per section = 56 +/- 19, range = 27-85) that contained parvalbumin immunoreactivity were also observed. In knockout mice, however, Mes5 neurons could not be detected. The area of brainstems which normally contained the Mes5 was devoid of parvalbumin-immunoreactive proprioceptors. The present study suggests that Brn-3a is required for the development of proprioceptors but not motoneurons in the trigeminal nervous system.

Axotomy-induced early down-regulation of POU-IV class transcription factors Brn-3a and Brn-3b in retinal ganglion cells.

It has been proposed that neurons being exposed to proapoptotic stimuli undergo dedifferentiation, a process that can either allow for regeneration and axon regrowth or, if remaining incomplete, can force the cell to activate apoptotic pathways. A pivotal step in the differentiation program from neuronal precursor cells to differentiated, postmitotic neurons is their exit from the cell cycle. The POU domain transcription factors Brn-3b and Brn-3a, which are expressed in retinal ganglion cells (RGCs) directly after the exit of RGC precursors from the cell cycle, can be employed as RGC-specific differentiation markers to study potential dedifferentiation of RGCs after axotomy. Here, we examined mRNAand protein expression of Brn-3a and -3b in rat RGCs following axonal lesion. We observed a rapid down-regulation of Brn-3a and -3b protein expression in axotomized RGCs, clearly preceding apoptosis of RGCs. Using real-time PCR, we show that regulation of Brn-3 expression occurred at the transcriptional level. The small subset of RGCs regenerating into a peripheral nerve graft did not (re-)express Brn-3a or -b. In conclusion, we found further evidence supporting the hypothesis of a dedifferentiation process in severed mature neurons. As Brn-3b expression has been shown to be a prerequisite for developmental survival of most RGCs and Brn-3a activates transcription of anti-apoptotic genes, down-regulation of Brn-3 transcription factors might be causally involved in the secondary death of adult RGCs following axotomy.

A GFP-based genetic screen reveals mutations that disrupt the architecture of the zebrafish retinotectal projection.

The retinotectal projection is a premier model system for the investigation of molecular mechanisms that underlie axon pathfinding and map formation. Other important features, such as the laminar targeting of retinal axons, the control of axon fasciculation and the intrinsic organization of the tectal neuropil, have been less accessible to investigation. In order to visualize these processes in vivo, we generated a transgenic zebrafish line expressing membrane-targeted GFP under control of the brn3c promoter/enhancer. The GFP reporter labels a distinct subset of retinal ganglion cells (RGCs), which project mainly into one of the four retinorecipient layers of the tectum and into a small subset of the extratectal arborization fields. In this transgenic line, we carried out an ENU-mutagenesis screen by scoring live zebrafish larvae for anatomical phenotypes. Thirteen recessive mutations in 12 genes were discovered. In one mutant, ddl, the majority of RGCs fail to differentiate. Three of the mutations, vrt, late and tard, delay the orderly ingrowth of retinal axons into the tectum. Two alleles of drg disrupt the layer-specific targeting of retinal axons. Three genes, fuzz, beyo and brek, are required for confinement of the tectal neuropil. Fasciculation within the optic tract and adhesion within the tectal neuropil are regulated by vrt, coma, bluk, clew and blin. The mutated genes are predicted to encode molecules essential for building the intricate neural architecture of the visual system.

Mesencephalic trigeminal nucleus development is dependent on Krox-20 expression.

Krox-20, a C2H2-type zinc-finger transcription factor, plays an important role in rhombomere development. This study reveals that the Krox-20 null mutation impacts the development of mesencephalic trigeminal (Me5) neurons, a cell group traditionally thought to emerge from the mesencephalon. Based on cell counting studies, we show that Krox-20 null mutants have twice as many Me5 neurons relative to wildtypes at E15, but by birth have half the number of Me5 cells as wildtypes. TUNEL studies reveal a period of increased apoptosis from E17-P0 in mutants. The mutation does not result in differences in Me5 cell size, morphology, gene expression or peripheral projection patterns between genotypes, as demonstrated by retrograde tracing and Brn3a immunohistochemistry. The data suggest that Krox-20 regulates the period and extent of Me5 apoptosis, impacting the final number of Me5 neurons. The loss of Me5 in Krox-20-/- mice may highlight species-specific differences in the origin of these cells.

Elevated expression of the Brn-3a and Brn-3b transcription factors in systemic lupus erythematosus correlates with antibodies to Brn-3 and overexpression of Hsp90.

OBJECTIVE: Important developmental and antiapoptotic roles have been described for the Brn-3 family of transcription factors in mammalian cells. Following a report of pathogenic autoantibody-inducing T cell reactivity to the Brn-3 transcription factors in murine lupus, we undertook this study to investigate serum levels of antibodies to Brn-3 and levels of expression of Brn-3 in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE). METHODS: Serum and PBMC samples were obtained from 87 SLE patients and 30 normal control subjects. Serum antibodies to the Brn-3a and Brn-3b transcription factors were measured by enzyme-linked immunosorbent assay. Levels of Brn-3a and Brn-3b messenger RNA (mRNA) in PBMCs were measured by reverse transcription and real-time quantitative polymerase chain reaction. RESULTS: Elevated serum levels of antibodies to Brn-3a and Brn-3b were found in 43% and 32%, respectively, of SLE patients. This elevation paralleled enhanced expression of Brn-3a and Brn-3b in PBMCs of 44% and 31%, respectively, of SLE patients. Furthermore, we observed a significant correlation (P = 0.002) between elevated levels of anti-Brn-3b antibodies and elevated levels of Brn-3b mRNA in individual patients. A preliminary analysis of possible target genes for Brn-3a and Brn-3b revealed a significant correlation (P = 0.01) between the level of Brn-3a mRNA and the level of Hsp90 protein (90-kd heat-shock protein, which is overexpressed in SLE) in PBMCs of SLE patients. In addition, we observed that overexpression of Brn-3a and Brn-3b in cultured cells enhanced expression of Hsp90 protein and transcription of Hsp90 promoter-reporter constructs. Finally, we observed an association between elevated levels of Brn-3a mRNA and active SLE (P = 0.002). CONCLUSION: Expression of both Brn-3a and Brn-3b was found to be enhanced in SLE, and this correlated with enhanced levels of autoantibodies to these proteins and with the previously reported overexpression of Hsp90, which was shown to be a novel gene regulated by Brn-3a and Brn-3b. The overexpression of Brn-3a correlated with active disease, suggesting that it may play a role in the disease process via its targeting by the immune system and its ability to induce the expression of specific genes.

Expression of the Brn-3b transcription factor correlates with expression of HSP-27 in breast cancer biopsies and is required for maximal activation of the HSP-27 promoter.

In breast cancer, overexpression of the small heat shock protein, HSP-27, is associated with increased anchorage-independent growth, increased invasiveness, and resistance to chemotherapeutic drugs and is associated with poor prognosis and reduced disease-free survival. Therefore, factors that increase the expression of HSP-27 in breast cancer are likely to affect the prognosis and outcome of treatment. In this study, we show a strong correlation between elevated levels of the Brn-3b POU transcription factor and high levels of HSP-27 protein in manipulated MCF-7 breast cancer cells as well as in human breast biopsies. Conversely, HSP-27 is decreased on loss of Brn-3b. In cotransfection assays, Brn-3b can strongly transactivate the HSP-27 promoter, supporting a role for direct regulation of HSP-27 expression. Brn-3b also cooperates with the estrogen receptor (ER) to facilitate maximal stimulation of the HSP-27 promoter, with significantly enhanced activity of this promoter observed on coexpression of Brn-3b and ER compared with either alone. RNA interference and site-directed mutagenesis support the requirement for the Brn-3b binding site on the HSP-27 promoter, which facilitates maximal transactivation either alone or on interaction with the ER. Chromatin immunoprecipitation provides evidence for association of Brn-3b with the HSP-27 promoter in the intact cell. Thus, Brn-3b can, directly and indirectly (via interaction with the ER), activate HSP-27 expression, and this may represent one mechanism by which Brn-3b mediates its effects in breast cancer cells.

Identification of an N-terminal transcriptional activation domain within Brn3b/POU4f2.

The POU-domain transcription factor Brn3b/ POU4f2 is an essential regulator of gene expression in mouse retinal ganglion cells. Although Brn3b's importance in the differentiation of these cells has been firmly established, the regions on Brn3b where transcriptional activation and/or repression domains reside are only vaguely defined, and conflicting publications report both activation and repression activities for Brn3b. To clarify its function, we monitored the transcriptional activity of Brn3b and Gal4 DNA-binding domain (DBD)-Brn3b fusion proteins in cotransfection experiments using either Brn3-consensus or Gal4 DNA-binding sites to drive reporter gene expression. At Gal4 DNA-binding sites, transrepression activity mapping to the POU domain within Brn3b's C-terminal region masked any transactivation activity. More detailed experiments revealed that expressing abnormally high levels of POU homeodomain- or other homeodomain-containing sequences caused fortuitous transrepression in the cotransfection assay. To avoid transrepression, Brn3b sequences lacking Brn3b's POU domain were fused to the Gal4 DBD to allow identification of regions that were responsible for transcriptional activation. Considerable transactivation activity was located between amino acid residues 100 and 239, although other regions also had activity. The transactivation domain synergized strongly with another transcription factor, LexA-VP16. At Brn3 DNA-binding sites, full-length Brn3b increased transcription more than 25-fold, and similar activation was observed with the closely related factor Brn3a/POU4f1. No transactivation activity was associated with the C-terminal POU domain-containing portion of Brn3b. The results demonstrate that Brn3b regulates gene expression through the action of a strong transcriptional activation domain within its N-terminal sequence.