Short Isoform of POU Factor Brn-3b Can Form a Heterodimer with Brn-3a That Is Inactive for Octamer Motif Binding

Theil, Thomas; Rödel, Bernd; Spiegelhalter, Frank; Möröy, Tarik

doi:10.1074/jbc.270.52.30958

Cited by 18 publications

(15 citation statements)

References 26 publications

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“…Little is known about the protein-protein interactions involving POU4F family members. Previous studies have shown that POU4F1 and POU4F2 can heterodimerize with each other (33). Our studies have shown that mutant POU4F3 does not interfere with POU4F2 binding to DNA in vitro but, since it is reasonable to assume that POU4F3 does interact with other proteins, we cannot rule out the possibility that mutant POU4F3 interferes with these interactions.…”

Section: Discussionmentioning

confidence: 38%

The DFNA15 Deafness Mutation Affects POU4F3 Protein Stability, Localization, and Transcriptional Activity

Weiss

Gottfried

Mayrose

et al. 2003

Molecular and Cellular Biology

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A mutation in the POU4F3 gene (BRN-3.1, BRN3C) is responsible for DFNA15 (MIM 602459), autosomaldominant nonsyndromic hearing loss. POU4F3 is a member of the POU family of transcription factors and is essential for inner-ear hair cell maintenance. To test the potential effects of the human POU4F3 mutation, we performed a series of experiments in cell culture to mimic the human mutation. Mutant POU4F3 loses most of its transcriptional activity and most of its ability to bind to DNA and does not function in a dominantnegative manner. Moreover, whereas wild-type POU4F3 is found exclusively in the nucleus, our studies demonstrate that the mutant protein is localized both to the nucleus and the cytoplasm. Two nuclear localization signals were identified; both are essential for proper nuclear entry of POU4F3 protein. We found that the mutant protein half-life is longer than that of the wild type. We propose that the combination of defects caused by the mutation on the function of the POU4F3 transcription factor eventually leads to hair cell morbidity in affected family H members.The POU domain transcription factors play an important role in tissue-specific gene regulation. They were originally defined by sequence homology between four transcription factors: mammalian Pit-1, Oct-1, and Oct-2 and nematode Caenorhabditis elegans 7,15,16). The region of homology, referred to as the POU domain, is a bipartite DNAbinding domain that contains a POU-specific (POU S ) domain and the POU-homeodomain (POU HD ) joined by a variable linker. High-affinity DNA binding of POU transcription factors requires both domains (1, 32). Based on sequence homology in the POU domain, the POU transcription factors were divided into six subclasses from I to VI (37). The class IV POU subfamily includes POU4F1 (Brn3a/Brn-3.0), POU4F2 (Brn3b/ Brn-3.2), and POU4F3 (Brn3c/Brn-3.1) in mammals (11,14,21,25,35,44), C. elegans Unc-86, and Drosophila factors I-POU and tI-POU (37).The three mammalian members of the POU IV subfamily are expressed in distinct but overlapping populations of neuronal cells during development and in the adult organism (11,25,35). Gene-targeted mutagenesis in the mouse of each of these class IV transcription factors demonstrates that the cell type in which it is first expressed is the system that is most affected (6,10,23,39,40). In the inner ear, Pou4f3 is uniquely and strongly expressed in cochlear and vestibular hair cells. Targeted deletion of Pou4f3 results in profound deafness and impaired balance due to complete loss of auditory and vestibular hair cells. This is followed by a partial secondary loss of spiral and vestibular ganglion neurons (6,39,42). Pou4f3 is required for the final differentiation and survival of hair cells (41).A mutation in the POU4F3 gene is associated with hearing loss in a large Israeli Jewish family, family H (36). Affected members of family H suffer from progressive autosomal-dominant sensorineural hearing loss (9). In hearing impaired members of family H, an 8-bp deletion was identified in exon 2...

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Section: Discussionmentioning

confidence: 38%

The DFNA15 Deafness Mutation Affects POU4F3 Protein Stability, Localization, and Transcriptional Activity

Weiss

Gottfried

Mayrose

et al. 2003

Molecular and Cellular Biology

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“…The ERE luciferase vector contains the ERE sequence (consensus sequence underlined) 5Ј-CTAG AAAGTCAGGTCACAGTGACCTGATCAAT-3Ј cloned into the pGL2 vector (Promega). The coding sequences of Brn-3a and Brn-3b were cloned into Bluescript and used for in vitro translation (67). The coding sequences of Brn-3a or Brn-3b and the POU domains were cloned under the control of the Moloney murine leukemia virus promoter into the pLTR expression vector, which was modified by a cryptic splice site in the simian virus 40 3Ј untranslated region (65).…”

Section: Methodsmentioning

confidence: 99%

“…The EMSA was carried out as described by Theil et al (67). Briefly, 3 l of the in vitro-translated ER, Brn-3 proteins, or luciferase (control) protein was added as indicated to 2 l of 10ϫ EMSA buffer (10 mM HEPES [pH 7.9], 60 mM KCl, 4% Ficoll, 1 mM dithiothreitol, 1 mM EDTA) containing 2 g of poly(dI-dC) to prevent nonspecific interactions, specific or nonspecific competitor oligonucleotides, or antibodies (where specified) and kept at room temperature for 5 min.…”

Section: Methodsmentioning

confidence: 99%

POU Transcription Factors Brn-3a and Brn-3b Interact with the Estrogen Receptor and Differentially Regulate Transcriptional Activity via an Estrogen Response Element

Budhram-Mahadeo

Parker

Latchman

1998

Molecular and Cellular Biology

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The estrogen receptor (ER) modulates transcription by forming complexes with other proteins and then binding to the estrogen response element (ERE). We have identified a novel interaction of this receptor with the POU transcription factors Brn-3a and Brn-3b which was independent of ligand binding. By pull-down assays and the yeast two-hybrid system, the POU domain of Brn-3a and Brn-3b was shown to interact with the DNA-binding domain of the ER. Brn-3-ER interactions also affect transcriptional activity of an ERE-containing promoter, such that in estradiol-stimulated cells, Brn-3b strongly activated the promoter via the ERE, while Brn-3a had a mild inhibitory effect. The POU domain of Brn-3b which interacts with the ER was sufficient to confer this activation potential, and the change of a single amino acid in the first helix of the POU homeodomain of Brn-3a to its equivalent in Brn-3b can change the mild repressive effect of Brn-3a to a stimulatory Brn-3b-like effect. These observations and their implications for transcriptional regulation by the ER are discussed.Transcriptional regulation by the complex interaction of different classes of transcription factors allows a limited number of proteins to elicit diverse effects on gene expression, depending on the expression of other proteins, such as tissue-specific factors and signals which may influence their interactions (reviewed in references 39, 40, 59, and 68 and references therein). We were interested in looking at proteins which interact with the transcription factors Brn-3a and Brn-3b and modulate the regulation of gene expression by these proteins. These two proteins belong to the POU (Pit-Oct-Unc) family of transcription factors (21,25,26,42,66,70,73,76). Members of this class of transcription factors are defined on the basis of the common POU domain, which consists of two highly conserved regions, the POU-specific domain and the POU homeodomain, which are separated by a poorly conserved linker region. The POU domain acts as the DNA-binding domain which recognizes and binds specific DNA sequences present in target gene promoters but is also involved in protein-protein interactions (3,72,73). There are three known members of the Brn-3 family of transcription factors, namely, Brn-3a (also known as Brn-3.0) (21, 42, 65), Brn-3b (also called Brn-3.2) (42, 65, 70), and Brn-3c (also known as Brn-3.1) (21, 52), which are encoded by different genes (65, 77). Furthermore, different isoforms of Brn-3a and Brn-3b which result from alternative splicing of the genes encoding these two proteins have been identified (21,43,65,70).The Brn-3 proteins show restricted homology outside the conserved carboxyl-terminal POU domain and the amino-terminal POU IV box (21,65,70). Since the studies reported here were carried out with Brn-3a and Brn-3b, references to Brn-3 proteins will pertain to observations with Brn-3a or Brn-3b and not Brn-3c. Sequence differences between Brn-3a and Brn-3b proteins are paralleled by different effects on promoters which contain binding sites recog...

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“…Two isoforms of Brn-3b have been identified (Theil et al 1995), with the longer 43 kDa Brn-3b(l) protein containing an additional amino terminal domain that is absent in the shorter 30-35 kDa Brn-3b(s) isoform. Both Brn-3b isoforms have been detected in breast cancer cells (Budhram-Mahadeo et al 1999).…”

Section: Introductionmentioning

confidence: 99%

Hsp-27 induction requires POU4F2/Brn-3b TF in doxorubicin-treated breast cancer cells, whereas phosphorylation alters its cellular localisation following drug treatment

Fujita

Ounzain

Wang

et al. 2011

Cell Stress and Chaperones

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POU4F2/Brn-3b transcription factor (referred to as Brn-3b) is elevated in >60% of breast cancers and profoundly alters growth and behaviour of cancer cells by regulating distinct subsets of target genes. Previous studies showed that Brn-3b was required to maximally transactivate small heat shock protein, HSPB1/Hsp-27 (referred to as , and consequently, Brn-3b expression correlated well with Hsp27 levels in human breast biopsies. In these studies, we showed that Brn-3b is increased in MCF7 breast cancer cells that survive following treatment with chemotherapeutic drug doxorubicin (Dox) with concomitant increases in Hsp-27 expression. Targeting of Brn3b using short interfering RNA reduced Hsp-27 in Doxtreated cells, suggesting that Brn-3b regulates Hsp-27 expression under these conditions. Wound healing assays showed increased Brn-3b in Dox-treated migratory cells that also express Hsp-27. Interestingly, Hsp-27 phosphorylation and cellular localisation are also significantly altered at different times following Dox treatment. Thus, phosphoHsp-27 (p-Hsp27) protein displayed widespread distribution after 24 hrs of Dox treatment but was restricted to the nucleus after 5 days. However, in drug-resistant cells (grown in Dox for >1 month), p-Hsp-27 was excluded from nuclei and most of the cytoplasm and appeared to be associated with the cell membrane. Studies to determine how this protein promotes survival and migration in breast cancer cells showed that the protective effects were conferred by unphosphorylated Hsp-27 protein.

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Short Isoform of POU Factor Brn-3b Can Form a Heterodimer with Brn-3a That Is Inactive for Octamer Motif Binding

Cited by 18 publications

References 26 publications

The DFNA15 Deafness Mutation Affects POU4F3 Protein Stability, Localization, and Transcriptional Activity

The DFNA15 Deafness Mutation Affects POU4F3 Protein Stability, Localization, and Transcriptional Activity

POU Transcription Factors Brn-3a and Brn-3b Interact with the Estrogen Receptor and Differentially Regulate Transcriptional Activity via an Estrogen Response Element

Hsp-27 induction requires POU4F2/Brn-3b TF in doxorubicin-treated breast cancer cells, whereas phosphorylation alters its cellular localisation following drug treatment

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