Brn-3a Transcription Factor Blocks p53-mediated Activation of Proapoptotic Target Genes Noxa and Bax in Vitro and in Vivo to Determine Cell Fate

Abstract

The Brn-3a POU transcription factor is associated with survival and the differentiation of sensory neuronal cells during development. Brn-3a mediates its effects either by the direct regulation of target genes or indirectly upon interaction with proteins such as p53. Brn-3a differentially regulates p53-mediated gene expression and modifies its effect on cell fate. Here we show that, like Bax, Brn-3a antagonizes p53-mediated transcription of another proapoptotic target, Noxa, significantly reducing transactivation of the Noxa promoter by p53. This effect requires the p53 binding site, and electrophoretic mobility shift assay studies suggest that Brn-3a is associated with p53 when it is bound to its site in the Noxa promoter. The wild type but not the mutant promoter can be immunoprecipitated with Brn-3a in chromatin immunoprecipitation assays. Thus, Brn-3a may act by preventing the recruitment of cofactors required for p53 to transactivate this promoter. The co-expression of Brn-3a and p53 results in decreased endogenous Noxa protein in the neuronal cell line, ND7, suggesting a direct functional effect of this interaction. Moreover, there is a significant elevation of both proapoptotic Bax and Noxa proteins in sensory neuronal tissue taken from Brn-3a؊/؊ embryos during development, compared with wild type controls. Striking changes occurred at embryonic day 14.5, a time that precedes a significant loss of specific neurons in the mutant embryos, but not at embryonic day 16.5 when Brn-3a-expressing cells are already lost by apoptosis. Therefore, the lack of antagonism by Brn-3a on activation of proapoptotic p53 target genes may contribute to the increased apoptosis seen in the Brn-3a؊/؊ embryos. These results support a crucial role for Brn-3a in determining the pathway taken by p53 when co-expressed during development and thus in controlling the fate of these cells.During neuronal development, transcription factors, which are expressed in a temporal/spatial manner or in response to specific signals, play a critical role in determining the fate of specific progenitor cells in terms of proliferation, survival and differentiation, or apoptosis of excess cells. This role is necessary to achieve the correct balance of specific neurons required for the normal innervation and function. The pit-1, oct-1/2, and unc-86 (POU) transcription factor Brn-3a protein (also referred to as POU4f1 and Brn-3.0) is essential for the survival and differentiation of specific populations of sensory neuronal cells (1, 2), but the mechanism by which this is achieved has yet to be elucidated fully.Brn-3a is expressed in regions of the developing and adult central nervous system and peripheral nervous system (3, 4). During development, Brn-3a expression is initiated just before neurons exit the cell cycle in the peripheral nervous system and just after exiting the cell cycle in the central nervous system (5). This transcription factor is expressed in and acts as a marker of the earliest postmitotic sensory neurons to appear in primary cultures...

Brn-3a/POU4F1 interacts with and differentially affects p73-mediated transcription

“…20,21 However, since the expression pattern and neuronal phenotype of the Brn-3a KO are paralleled by neuronal loss seen in p73 KO, we believe that this interaction may be more physiologically relevant in specific neurons. At a functional level, similar to the effects seen with p53, Brn-3a alters p73 target gene expression in a complex manner: it antagonizes TAp73a and p73b activity on the bax promoter but co-operates with these isoforms to enhance p21 CIP1/Waf1 .…”

“…Brn-3aÀ/À mutants suffer significant loss of sensory neurons by apoptosis during development, and this correlated with elevated Bax expression. 20 These neurons are rescued during embryogenesis by the loss of bax as demonstrated in Brn-3a/Bax double KO. 3,4 Thus, the ability of Brn-3a to antagonize transactivation of Bax by p73 is likely to play an important role in neuronal survival during development.…”

“…Total cellular proteins were prepared from transfected cell lines as described 20 by harvesting into 2 Â Laemmli buffer and addition of 5% b-mercaptoethanol followed by heating to 95 1C for 5 min. Following centrifugation to remove cell debris, protein concentration was assessed using the Bradford assay or Coomassie Blue-stained PAGE.…”

“…For example, ASPP1/2 proteins interact with p53 and TAp73 proteins to enhance transcription of pro-apoptotic genes 19 whereas Brn-3a interacts with p53 but differentially regulates its ability to transactivate its target genes. [20][21][22] p73 KO mice display varying degrees of abnormal neurological development in different parts of the nervous system, which results in an increased death rate within weeks after birth. 23 This phenotype seems to be mainly related to the absence of DNp73, thus demonstrating specific functions for p73 isoforms in specific neuronal cells.…”

Stress via p53 pathway causes apoptosis by mitochondrial Noxa upregulation in doxorubicin-treated neuroblastoma cells

“…Noxa is a BH3-only member of Bcl-2 family proteins and its expression is induced by DNA damage such as that caused by etoposide or doxorubicin in a p53-dependent manner Shibue et al, 2003). Furthermore, several lines of evidence reported that Noxa is one of the most important cell death effectors in neuronal cell death, for example, nuclear factor-kappa B modulated cell death in mouse cortical neurons (Aleyasin et al, 2004), axotomized motor neurons of adult mouse (Kiryu-Seo et al, 2005), sensory neurons especially in trigeminal ganglia and cervical dorsal ganglia (Hudson et al, 2005) and arsenite-induced cortical neurons (Wong et al, 2005).…”