Distinct promoter elements mediate the co-operative effect of Brn-3a and p53 on the p21 promoter and their antagonism on the Bax promoter

Although the promoters of both the Bax and p21 genes are activated by p53, they differ in the effect on this activation of the POU family transcription factor Brn-3a. Thus, Brn-3a inhibits activation of the Bax promoter by p53 but enhances the ability of p53 to activate the p21 promoter. We demonstrate that repression of p53-mediated activation of the Bax promoter involves a complex upstream sequence in which two Brn-3a response elements flank the p53 response element. In contrast, a minimal p21 promoter is activated by Brn-3a and such activation cannot be abolished without abolishing basal promoter activity. Moreover, synergistic activation by Brn-3a and p53 continues to be observed when the p53-binding sites in the p21 promoter are substituted by the Bax p53 site or by the region of the Bax promoter essential for Brn-3a-mediated repression, indicating that the p21 core promoter plays a central role in this response. The significance of these effects is discussed in terms of the different responses of the Bax and p21 promoters and the overlapping but distinct roles of Brn-3a and p53 in neuronal growth arrest and apoptosis

Linking metabolic dysfunction with cardiovascular diseases: Brn-3b/POU4F2 transcription factor in cardiometabolic tissues in health and disease.

Metabolic and cardiovascular diseases are highly prevalent and chronic conditions that are closely linked by complex molecular and pathological changes. Such adverse effects often arise from changes in the expression of genes that control essential cellular functions, but the factors that drive such effects are not fully understood. Since tissue-specific transcription factors control the expression of multiple genes, which affect cell fate under different conditions, then identifying such regulators can provide valuable insight into the molecular basis of such diseases. This review explores emerging evidence that supports novel and important roles for the POU4F2/Brn-3b transcription factor (TF) in controlling cellular genes that regulate cardiometabolic function. Brn-3b is expressed in insulin-responsive metabolic tissues (e.g. skeletal muscle and adipose tissue) and is important for normal function because constitutive Brn-3b-knockout (KO) mice develop profound metabolic dysfunction (hyperglycaemia; insulin resistance). Brn-3b is highly expressed in the developing hearts, with lower levels in adult hearts. However, Brn-3b is re-expressed in adult cardiomyocytes following haemodynamic stress or injury and is necessary for adaptive cardiac responses, particularly in male hearts, because male Brn-3b KO mice develop adverse remodelling and reduced cardiac function. As a TF, Brn-3b regulates the expression of multiple target genes, including GLUT4, GSK3β, sonic hedgehog (SHH), cyclin D1 and CDK4, which have known functions in controlling metabolic processes but also participate in cardiac responses to stress or injury. Therefore, loss of Brn-3b and the resultant alterations in the expression of such genes could potentially provide the link between metabolic dysfunctions with adverse cardiovascular responses, which is seen in Brn-3b KO mutants. Since the loss of Brn-3b is associated with obesity, type II diabetes (T2DM) and altered cardiac responses to stress, this regulator may provide a new and important link for understanding how pathological changes arise in such endemic diseases

POU4F2/Brn-3b transcription factor is associated with survival and drug resistance in human ovarian cancer cells

The development of drug resistance following treatment with chemotherapeutic agents such as cisplatin (cis) and paclitaxel (pax) contributes to high morbidity and mortality in ovarian cancers. However, the molecular mechanisms underlying such changes are not well understood. In this study, we demonstrate that the Brn-3b transcription factor was increased in different ovarian cancer cells including SKOV3 and A2780 following treatment with cis and pax. Furthermore, sustained increases in Brn-3b were associated with survival in drug resistant cells and correlated with elevated HSP27 expression. In contrast, targeting Brn-3b for reduction using short interfering RNA (siRNA) also resulted in attenuated HSP27 expression. Importantly, blocking Brn-3b expression with siRNA in SKOV3 cells was associated with reduced cell numbers at baseline but also increased cell death after further treatment, indicating sensitization of cells. Similar results were obtained in the metastatic IP1 cell line derived from ascites of mice bearing SKOV3 tumours. These findings suggest that increased Brn-3b may confer resistance to chemotherapeutic drugs in ovarian cancer cells by regulating key target genes such as HSP27 and that targeting Brn-3b may provide a novel mechanism for treatment of drug resistant ovarian cancers

Essential but partially redundant roles for POU4F1/Brn-3a and POU4F2/Brn-3b transcription factors in the developing heart

Congenital heart defects contribute to embryonic or neonatal lethality but due to the complexity of cardiac development, the molecular changes associated with such defects are not fully understood. Here, we report that transcription factors (TFs) Brn-3a (POU4F1) and Brn-3b (POU4F2) are important for normal cardiac development. Brn-3a directly represses Brn-3b promoter in cardiomyocytes and consequently Brn-3a knockout (KO) mutant hearts express increased Brn-3b mRNA during mid-gestation, which is linked to hyperplastic growth associated with elevated cyclin D1, a known Brn-3b target gene. However, during late gestation, Brn-3b can cooperate with p53 to enhance transcription of pro-apoptotic genes e.g. Bax, thereby increasing apoptosis and contribute to morphological defects such as non-compaction, ventricular wall/septal thinning and increased crypts/fissures, which may cause lethality of Brn-3a KO mutants soon after birth. Despite this, early embryonic lethality in e9.5 double KO (Brn-3a(-/-) : Brn-3b(-/-)) mutants indicate essential functions with partial redundancy during early embryogenesis. High conservation between mammals and zebrafish (ZF) Brn-3b (87%) or Brn-3a (76%) facilitated use of ZF embryos to study potential roles in developing heart. Double morphant embryos targeted with morpholino oligonucleotides to both TFs develop significant cardiac defects (looping abnormalities and valve defects) suggesting essential roles for Brn-3a and Brn-3b in developing hearts

Verification of genes differentially expressed in neuroblastoma tumours: a study of potential tumour suppressor genes

<p>Abstract</p> <p>Background</p> <p>One of the most striking features of the childhood malignancy neuroblastoma (NB) is its clinical heterogeneity. Although there is a great need for better clinical and biological markers to distinguish between tumours with different severity and to improve treatment, no clear-cut prognostic factors have been found. Also, no major NB tumour suppressor genes have been identified.</p> <p>Methods</p> <p>In this study we performed expression analysis by quantitative real-time PCR (QPCR) on primary NB tumours divided into two groups, of favourable and unfavourable outcome respectively. Candidate genes were selected on basis of lower expression in unfavourable tumour types compared to favourables in our microarray expression analysis. Selected genes were studied in two steps: (1) using TaqMan Low Density Arrays (TLDA) targeting 89 genes on a set of 12 NB tumour samples, and (2) 12 genes were selected from the TLDA analysis for verification using individual TaqMan assays in a new set of 13 NB tumour samples.</p> <p>Results</p> <p>By TLDA analysis, 81 out of 87 genes were found to be significantly differentially expressed between groups, of which 14 have previously been reported as having an altered gene expression in NB. In the second verification round, seven out of 12 transcripts showed significantly lower expression in unfavourable NB tumours, <it>ATBF1</it>, <it>CACNA2D3</it>, <it>CNTNAP2</it>, <it>FUSIP1</it>, <it>GNB1</it>, <it>SLC35E2</it>, and <it>TFAP2B</it>. The gene that showed the highest fold change in the TLDA analysis, <it>POU4F2</it>, was investigated for epigenetic changes (CpG methylation) and mutations in order to explore the cause of the differential expression. Moreover, the fragile site gene <it>CNTNAP2 </it>that showed the largest fold change in verification group 2 was investigated for structural aberrations by copy number analysis. However, the analyses of <it>POU4F2 </it>and <it>CNTNAP2 </it>showed no genetic alterations that could explain a lower expression in unfavourable NB tumours.</p> <p>Conclusion</p> <p>Through two steps of verification, seven transcripts were found to significantly discriminate between favourable and unfavourable NB tumours. Four of the transcripts, <it>CACNA2D3</it>, <it>GNB1</it>, <it>SLC35E2</it>, and <it>TFAP2B</it>, have been observed in previous microarray studies, and are in this study independently verified. Our results suggest these transcripts to be markers of malignancy, which could have a potential usefulness in the clinic.</p

The POU4F2/Brn-3b transcription factor is required for the hypertrophic response to angiotensin II in the heart

Adult hearts respond to increased workload such as prolonged stress or injury, by undergoing hypertrophic growth. During this process, the early adaptive responses are important for maintaining cardiac output whereas at later stages, pathological responses such as cardiomyocyte apoptosis and fibrosis cause adverse remodelling, that can progress to heart failure. Yet the factors that control transition from adaptive responses to pathological remodelling in the heart are not well understood. Here we describe the POU4F2/Brn-3b transcription factor (TF) as a novel regulator of adaptive hypertrophic responses in adult hearts since Brn-3b mRNA and protein are increased in angiotensin-II (AngII) treated mouse hearts with concomitant hypertrophic changes [increased heart weight:body weight (HW:BW) ratio]. These effects occur specifically in cardiomyocytes because Brn-3b expression is increased in AngII-treated primary cultures of neonatal rat ventricular myocytes (NRVM) or foetal heart-derived H9c2 cells, which undergo characteristic sarcomeric re-organisation seen in hypertrophic myocytes and express hypertrophic markers, ANP/βMHC. The Brn-3b promoter is activated by known hypertrophic signalling pathways e.g. p42/p44 mitogen-activated protein kinase (MAPK/ERK1/2) or calcineurin (via NFAT). Brn-3b target genes, e.g. cyclin D1, GLUT4 and Bax, are increased at different stages following AngII treatment, supporting distinct roles in cardiac responses to stress. Furthermore, hearts from male Brn-3b KO mutant mice display contractile dysfunction at baseline but also attenuated hypertrophic responses to AngII treatment. Hearts from AngII-treated male Brn-3b KO mice develop further contractile dysfunction linked to extensive fibrosis/remodelling. Moreover, known Brn-3b target genes, e.g. GLUT4, are reduced in AngII-treated Brn-3b KO hearts, suggesting that Brn-3b and its target genes are important in driving adaptive hypertrophic responses in stressed hear

Cardiac expression of Brn-3a and Brn-3b POU transcription factors and regulation of Hsp27 gene expression

The Brn-3 family of transcription factors play a critical role in regulating expression of genes that control cell fate, including the small heat shock protein Hsp27. The aim of this study was to investigate the relationship between Brn-3a and Brn-3b and Hsp27 expression in the developing rodent heart. Brn-3a and Brn-3b were detected from embryonic days 9.5–10.5 (E9.5–E10.5) in the mouse heart, with significant increases seen later during development. Two isoforms (long and short) of each protein were detected during embryogenesis and postnatally. Brn-3a messenger RNA (mRNA) and protein were localized by E13.0 to the atrio-ventricular (AV) valve cushions and leaflets, outflow tract (OFT), epicardium and cardiac ganglia. By E14.5, Brn-3a was also localised to the septa and compact ventricular myocardium. An increase in expression of the long Brn-3a(l) isoform between E17 and adult coincided with a decrease in expression of Brn-3b(l) and a marked increase in expression of Hsp27. Hearts from Brn-3a−/− mice displayed a partially penetrant phenotype marked by thickening of the endocardial cushions and AV valve leaflets and hypoplastic ventricular myocardium. Loss of Brn-3a was correlated with a compensatory increase in Brn-3b and GATA3 mRNA but no change in Hsp27 mRNA. Reporter assays in isolated cardiomyocytes demonstrated that both Brn-3a and Brn-3b activate the hsp27 promoter via a consensus Brn-3-binding site. Therefore, Brn-3 POU factors may play an important role in the development and maintenance of critical cell types and structures within the heart, in part via developmental regulation of myocardial Hsp27 expression. Furthermore, Brn-3a may be necessary for correct valve and myocardial remodelling and maturation