The class III POU factor Brn-4 interacts with other class III POU factors and the heterogeneous nuclear ribonucleoprotein U

doi:10.1016/S0169-328X(96)00238-0

Molecular Brain Research

Volume 45, Issue 1, April 1997, Pages 99-107

https://doi.org/10.1016/S0169-328X(96)00238-0 Get rights and content

Abstract

The class III POU proteins are expressed throughout the central nervous system, including the hypothalamus, where they are often co-localized. Presumably, these POU proteins (Brain-1, Brain-2, Brain-4 and SCIP) serve as transcriptional transactivators. That they are co-expressed in some neurons suggests that, if they were to form homomeric and heteromeric complexes with each other, depending on the particular combination, they might have different DNA-binding specificities and, thus, activate different genes. We used purified fusion proteins of the four class III POU proteins in far-western assays to show that the proteins can interact. We confirmed their interactions using a two-hybrid system. Both techniques indicate that the interaction occurs through the POU domain. The far-western technique also allowed us to identify a 120-kDa nuclear protein that interacts with Brain-4. Subsequent affinity purification and microsequencing identified the protein as the heterogeneous nuclear ribonucleoprotein U (hnRNP U). This result suggests another mechanism by which a POU protein can influence gene expression by facilitating the processing of pre-mRNA whose transcription it has stimulated.

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Insulin expression is highly controlled on the posttranscriptional level. The RNA binding proteins (RBPs) responsible for this result are still largely unknown.
To identify RBPs that bind to insulin mRNA we performed mass spectrometry analysis on proteins that bound synthetic oligonucloetides mimicing the 5′- and the 3′-untranslated regions (UTRs) of rat and human insulin mRNA in vitro. We observed that the RBPs heterogeneous nuclear ribonucleoprotein (hnRNP) U, polypyrimidine tract binding protein (PTB), hnRNP L and T-cell restricted intracellular antigen 1-related protein (TIA-1-related protein; TIAR) bind to insulin mRNA sequences, and that the in vitro binding affinity of these RBPs changed when INS-1 cells were exposed to glucose, 3-isobutyl-1-methylxanthine (IBMX) or nitric oxide. High glucose exposure resulted in a modest increase in PTB and TIAR binding to an insulin mRNA sequence. The inducer of nitrosative stress DETAnonoate increased markedly hnRNP U and TIAR mRNA binding. An increased PTB to TIAR binding ratio in vitro correlated with higher insulin mRNA levels and insulin biosynthesis rates in INS-1 cells. To further investigate the importance of RNA-binding proteins for insulin mRNA stability, we decreased INS-1 and EndoC-βH1 cell levels of PTB and TIAR by RNAi. In both cell lines, decreased levels of PTB resulted in lowered insulin mRNA levels while decreased levels of TIAR resulted in increased insulin mRNA levels. Thapsigargin-induced stress granule formation was associated with a redistribution of TIAR from the cytosol to stress granules.
These experiments indicate that alterations in insulin mRNA stability and translation correlate with differential RBP binding. We propose that the balance between PTB on one hand and TIAR on the other participates in the control of insulin mRNA stability and utilization for insulin biosynthesis.
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hnRNP U functions in pre-mRNA processing together with other hnRNP proteins and/or acts in the higher-order organization of chromatin [15]. Recent studies have shown that hnRNP U may also interact with both specific and general transcription factors, such as the glucocorticoid hormone receptor, BRN4, SOX2 and OCT4 [16–18]. In addition, a subfraction of hnRNP U co-purifies with RNA Pol II and enters into the preinitiation complex, consequently regulating transcriptional elongation [19].
H19 was one of the earliest identified, and is the most studied, long noncoding RNAs. It is presumed that H19 is essential for regulating development and disease conditions, and it is associated with carcinogenesis for many types. However the biological function and regulatory mechanism of this conserved RNA, particularly with respect to its effect on transcription, remain largely unknown.
We performed RNA pulldown, RNA immunoprecipitation and deletion mapping to identify the proteins that are associated with H19. In addition, we employed EU (5-ethynyl uridine) incorporation, immunoprecipitation and Western blotting to investigate the functional aspects of H19.
Our research further verifies that H19 is bound to hnRNP U, and this interaction is located within the 5′ 882 nt region of H19. Moreover, H19 disrupts the interaction between hnRNP U and actin, which inhibits phosphorylation at Ser5 of the RNA polymerase II (Pol II) C-terminal domain (CTD), consequently preventing RNA Pol II-mediated transcription. We also showed that hnRNP U is essential for H19-mediated transcription repression.
In this study, we demonstrate that H19 inhibits RNA Pol II-mediated transcription by disrupting the hnRNP U–actin complex.
These data suggest that H19 regulates general transcription and exerts wide-ranging effects in organisms.
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This study pertains to the regulatory network of neurogenin3 (NGN3, approved symbol: NEUROG3), the main regulator of insulin producing cells’ formation.
In silico regulatory region analyses of known and novel targets of NGN3 revealed the presence of two variants of a regulatory module that appeared conserved at the most phylogenetically distant species with pancreas. Both variants of this module contained binding sites of six transcription factors implicated in pancreas development. Nevertheless, an additional factor was found only into the module of the down-regulated by NGN3 genes. Whole genome analyses confirmed the statistical significance of these regulatory modules. Investigation of protein–protein interactions among the factors bound into these sequences indicated the formation of alternative protein complexes resulting into the up- or down-regulation of the respective genes.
Subsequently, an NGN3-guided regulatory network, was modeled, describing the interactions among the analyzed genes with their transcriptional regulators, leading into the differentiation of cells capable of producing insulin.
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Sequence analysis brought to the identification of five nuclear proteins: heterogenous nuclear ribonucleoprotein M (P52272, 77.4 kDa), which has been proposed to play a role in mRNA splicing [14], Matrin-3 (P43243, 94.6 kDa), a component of the nuclear matrix which binds DNA as well as RNA [15], DNA topoisomerases IIα (P11388, 174.3 kDa) and IIβ (Q02880, 183.3 kDa), which are required to relax supercoiling generated by transcription [16], and SAF-A (Q00839, 88.8 kDa), also known as heterogenous nuclear ribonucleoprotein U [17]. SAF-A binds AT-rich regions within the genome called scaffold attachment regions, and has been shown to interact with Tfs such as the glucocorticoid receptor, a member of the superfamily of hormone nuclear receptors [18], and the class III POU Brain-4 factor [19], and also with the Yes-associated protein, a modulator of multiple transcription factors in a variety of cell types [20]. The liaison of SAF-A with the glucocorticoid receptor takes place through its DNA-binding and τ2 transactivation domains [21], repressing glucocorticoid induced activation by sequestrating the glucocorticoid receptor on the nuclear matrix [22].
Development proceeds through successive activation of different sets of genes by specific transcription factors as a consequence of cell interactions and signaling. It is thus of primary interest to identify new putative transcriptional regulators. We report here the isolation of chicken clones bearing sequences coding for a chicken zinc finger protein (chZFp) which contains four pairs of zinc fingers of mixed type C₂–H–C/C₂–H₂. At least five chZFp isoforms are produced through differential splicing of four small exons. The amino acid domains encoded by these four exons are highly conserved across species. Northern blot analysis and RNase-protection assays showed that chZFp transcripts are present in brain, heart, skin and liver during chick development. Reverse transcription mediated polymerase chain reaction (RT-PCR) experiments suggested that the relative amount of some chZFp isoforms increases at critical stages of development and skin morphogenesis. Finally, the main chZFp isoforms are able to directly interact in vitro with the scaffold attachment factor-A (SAF-A, also known as heterogenous nuclear ribonucleoprotein U) through both their aminoterminal and carboxyterminal domains.
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Members of the POU family of transcription factors are important regulators of gene expression during development and several, particularly the Brn proteins, play a critical role in brain development (Latchman 1999; Phillips and Luisi 2000). All POU proteins share a common bipartate DNA binding region consisting of a POU domain and a homeobox domain separated by a short amino acid linker or hinge (Herr and Cleary 1995; Malik et al 1997). A number of genes expressed in the brain that are critical for developmental and postdevelopmental neuronal function are influenced by POU proteins including SNAP-25, neurofilaments, nicotinic receptor, KCNN3, and Bcl-2 (Latchman 1999).
Genes involved in phosphoinositide (PI) lipid metabolism are excellent candidates to consider in the pathogenesis of bipolar disorder (BD) and schizophrenia (SZ). One is PIK3C3, a member of the phosphatidylinositide 3-kinase family that maps closely to markers on 18q linked to both BD and SZ in a few studies.
The promoter region of PIK3C3 was analyzed for mutations by single-strand conformation polymorphism analysis and sequencing. A case–control association study was conducted to determine the distribution of variant alleles in unrelated patients from three cohorts. Electromobility gel shift assays (EMSA) were performed to assess the functional significance of variants.
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Cloning of rat ARHGAP4/C1, a RhoGAP family member expressed in the nervous system that colocalizes with the Golgi complex and microtubules
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The Rho GTPase family of intracellular molecular switches control multiple cellular functions via the regulation of the actin cytoskeleton. Increasing evidence implicates a critical involvement of these molecules in the nervous system, particularly during neuronal migration and polarity, axon and growth cone guidance, dendritic arborization and synaptic formation. However, the molecules regulating Rho GTPase activities in the nervous system are less known. Here, we present the cloning of rat ARHGAP4, a member of the Rho GTPase activating protein family, and also demonstrate its close linkage to the vasopressin 2 receptor gene. In vitro, recombinant ARHGAP4 stimulated the GTPase activity of three members of Rho GTPases, Rac1, Cdc42 and RhoA. ARHGAP4 mRNA expression was observed in multiple tissues with marked expression throughout the developing and adult nervous systems. On closer analysis of protein levels, ARHGAP4 was significantly restricted to specific regions in the nervous system. These included the stratum lucidem in the CA3 area of the hippocampus, neuronal fibers in the ventral region of the brainstem and striatum, and in the cerebellar granule cells. Subcellularly, endogenous ARHGAP4 expression localized to the Golgi complex and could redistribute to the microtubules, for example during mitosis. In addition, distinct protein expression was observed in the tips of differentiating neurites of PC12 cells. Collectively, these results demonstrate that ARHGAP4 is more widely expressed than previously thought but potentially possesses specialized activity in regulating members of the Rho GTPase family in specific cellular compartments of the nervous system.

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Research reportThe class III POU factor Brn-4 interacts with other class III POU factors and the heterogeneous nuclear ribonucleoprotein U

Abstract

Anal. Biochem.

Cell

Cell

Mol. Brain Res.

J. Biol. Chem.

Cell

Biochim. Biophys. Acta

Curr. Opin. Cell Biol.

Model of forebrain regionalization based on spatiotemporal patterns of POU-III homeobox gene expression, birthdates, and morphological features

J. Comp. Neurol.

Interaction cloning: identification of a helix-loop-helix zipper protein that interacts with c-Fos

Science

Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei

Nucleic Acids Res.

hnRNP proteins and the biogenesis of mRNA

Annu. Rev. Biochem.

A sequence-specific, single-strand binding protein activates the far upstream element of c-myc and defines a new DNA-binding motif

Genes Dev.

Research report
The class III POU factor Brn-4 interacts with other class III POU factors and the heterogeneous nuclear ribonucleoprotein U