The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function

High Mobility Group A (HMGA) is a family of architectural nuclear factors which play an important role in neoplastic transformation. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes including transcription. HMGA localization is exclusively nuclear but, to date, the mechanism of nuclear import for these proteins remains unknown. Here, we report the identification and characterization of a nuclear localization signal (NLS) for HMGA2, a member of the HMGA family. The NLS overlaps with the second of the three AT-hooks, the DNA-binding domains characteristic for this group of proteins. The functionality of this NLS was demonstrated by its ability to target a heterologous β-galactosidase/green fluorescent protein fusion protein to the nucleus. Mutations to alanine of basic residues within the second AT-hook resulted in inhibition of HMGA2 nuclear localization and impairment of its function in activating the cyclin A promoter. In addition, HMGA2 was shown to directly interact with the nuclear import receptor importin-α2 via the second AT-hook. HMGA proteins are overexpressed and rearranged in a variety of tumors; our findings can thus help elucidating their role in neoplastic transformation

The AT-hook of the Chromatin Architectural Transcription Factor High Mobility Group A1a Is Arginine-methylated by Protein Arginine Methyltransferase 6

The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg(57) and Arg(59), two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization

Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells

© 2018 Sutanto et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAEC CF ) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508-del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAEC CF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAEC CF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAEC CF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations

Correlation between serum ghrelin levels and cocaine-seeking behaviour triggered by cocaine-associated conditioned stimuli in rats

Ghrelin is a brain\u2013gut peptide with growth hormone-releasing and appetite-inducing activities. A growing body of evidence suggests that ghrelin may affect the central reward system and modulate the activity of the mesolimbic system. Recent clinical studies also showed a significant positive correlation between plasma ghrelin levels and craving in alcoholics. Accordingly, the present study investigated the potential role of serum ghrelin levels in the reinstatement of cocaine-seeking behaviour triggered by cocaine-associated cues. In addition, serum corticosterone levels were determined in the light of evidence suggesting that corticosterone plays a modulatory role in cocaine-seeking behaviour. Male Lister Hooded rats under a restricted diet regime were first trained to intravenously self-administer cocaine under a fixed ratio-1 schedule of reinforcement. Conditioned stimuli (CS: tone and cue-light on for 5 seconds) were presented contingently with cocaine delivery. Once a stable baseline of cocaine self-administration was observed, lever presses were extinguished to less than 30% of baseline rates by removing both cocaine and CS. Reinstatement of responding was then induced by re-exposure to cocaine-associated CS. Blood samples for the enzyme immunoassay determination of serum ghrelin and the radioimmunoassay determination of serum corticosterone levels were collected 30 minutes before the beginning of reinstatement sessions. Rats significantly reinstated their responding when exposed to CS. A positive and significant correlation was observed between ghrelin levels (r = 0.64; P < 0.05), but not corticosterone (r = 0.37; NS), and the increased active lever presses only in animals exposed to CS. These findings suggest a potential role of ghrelin in the modulation of cue-triggered reinstatement of cocaine-seeking behaviour

Identification and validation of novel drug targets to treat Cystic Fibrosis: modifiers of the trafficking defect of DF508-CFTR

Abnormal retention of the CFTR ΔF508 mutated protein in lung epithelial cells underlies the pathology in a large proportion of individuals with cystic fibrosis (CF). A drug discovery alliance between CFFT and Galapagos was initiated with the aim to identify novel genes which upon shRNAmediated knockdown were able to efficiently restore ΔF508 CFTR activity. The final goal of this program is to prioritize these targets for entry into drug discovery. BioFocus DPI’s proprietary adenoviral shRNA libraries totaling 11,334 viruses against the human druggable genome were screened in a highthroughput functional assay in a human cystic fibrosis bronchial epithelial 2009 Cystic Fibrosis Conference 216 cell line (CFBE41o-). A total of 354 hits were identified and confirmed in the primary assay. Validation of the hits included a lack of cytotoxicity of the shRNA, expression of target in airway epithelial cells, identification of a second shRNA against the same target, and cell surface expression by biotinylation of CFTR ΔF508 upon shRNA-mediated knock-down of the target. Most importantly, these shRNAs restored functional activity of the mutant channel in primary bronchial epithelial cells from ΔF508/ΔF508 CF patients in transepithelial CFTR mediated current assays (Table 1). There was a very strong correlation between fully glycosylated band C expression in the CFBE41o- cells and functional activity in the primary cells of CF patients. We will present an overview of the target discovery and validation program, resulting in a portfolio of 19 novel drug targets to treat CF. The identity of these novel targets also sheds light on the biology of trafficking of CFTR. For example, these data point to a role of TGF-beta signaling and inflammatory mediators in airways in the regulation of CFTR trafficking. Acknowledgments: We thank Drs. Bill Guggino, Ineke Braakman, Kevin Foskett, John Hanrahan and Hugo De Jonge for helpful discussions. We acknowledge the support of Cystic Fibrosis Foundation Therapeutics

HMGA2-mediated transcriptional activation is dependent upon basic residues within the second AT-hook

<p><b>Copyright information:</b></p><p>Taken from "The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function"</p><p></p><p>Nucleic Acids Research 2007;35(6):1751-1760.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874589.</p><p>© 2007 The Author(s)</p> () Diagram of luciferase reporter gene under the transcriptional regulation of the cyclin A promoter and the HMGA2 wild type and mutants expressed by the vectors used. () CHO cells were transiently cotransfected with 1 µg of the CycA luciferase reporter plasmid (bars 1–4) and with 3 µg of HMGA2 wt-, mS5- and mT6- EGFP expression vectors (bars 2–4 respectively). 0.1 µg of pRL-CMV Renilla luciferase expression vector was included to normalize for transfection efficiencies. Values are reported as relative luciferase activity. Standard deviations are indicated for experiments repeated three times. The amount of HMGA2 wt-, mS5- and mT6-EGFP expression was assayed by Western blot analysis using a polyclonal α-HMGA2 antibody. The subcellular localization of the expressed proteins in CHO cells was confirmed by confocal microscopy (data not shown)

Identification of HMGA2 nuclear localization signal by deletion mutagenesis

<p><b>Copyright information:</b></p><p>Taken from "The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function"</p><p></p><p>Nucleic Acids Research 2007;35(6):1751-1760.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874589.</p><p>© 2007 The Author(s)</p> () Schematic representation of HMGA2 deletion mutants; each deletion mutant is fused to β-gal at the N-terminus, and to GFP at the C-terminus. Summary of intracellular localization is indicated at the right. () NIH-3T3 cells were transfected with HMGA2 deletion mutants constructs, and fusion proteins were visualized by confocal laser microscopy. Propidium iodide (PI) staining of the same nuclei is shown. At least one hundred cells per transfection were analyzed in three different experiments. Bars, 10 μm. () Western blot analysis performed with an anti-GFP antibody