VAPB/ALS8 interacts with FFAT-like proteins including the p97 cofactor FAF1 and the ASNA1 ATPase

BACKGROUND: FAF1 is a ubiquitin-binding adaptor for the p97 ATPase and belongs to the UBA-UBX family of p97 cofactors. p97 converts the energy derived from ATP hydrolysis into conformational changes of the p97 hexamer, which allows the dissociation of its targets from cellular structures or from larger protein complexes to facilitate their ubiquitin-dependent degradation. VAPB and the related protein VAPA form homo- and heterodimers that are anchored in the endoplasmic reticulum membrane and can interact with protein partners carrying a FFAT motif. Mutations in either VAPB or p97 can cause amyotrophic lateral sclerosis, a neurodegenerative disorder that affects upper and lower motor neurons. RESULTS: We show that FAF1 contains a non-canonical FFAT motif that allows it to interact directly with the MSP domain of VAPB and, thereby, to mediate VAPB interaction with p97. This finding establishes a link between two proteins that can cause amyotrophic lateral sclerosis when mutated, VAPB/ALS8 and p97/ALS14. Subsequently, we identified a similar FFAT-like motif in the ASNA1 subunit of the transmembrane-domain recognition complex (TRC), which in turn mediates ASNA1 interaction with the MSP domain of VAPB. Proteasome inhibition leads to the accumulation of ubiquitinated species in VAPB immunoprecipitates and this correlates with an increase in FAF1 and p97 binding. We found that VAPB interaction with ubiquitinated proteins is strongly reduced in cells treated with FAF1 siRNA. Our efforts to determine the identity of the ubiquitinated targets common to VAPB and FAF1 led to the identification of RPN2, a subunit of an oligosaccharyl-transferase located at the endoplasmic reticulum, which may be regulated by ubiquitin-mediated degradation. CONCLUSIONS: The FFAT-like motifs we identified in FAF1 and ASNA1 demonstrate that sequences containing a single phenylalanine residue with the consensus (D/E)(D/E)FEDAx(D/E) are also proficient to mediate interaction with VAPB. Our findings indicate that the repertoire of VAPB interactors is more diverse than previously anticipated and link VAPB to the function of ATPase complexes such as p97/FAF1 and ASNA1/TRC

USF-1 Is Critical for Maintaining Genome Integrity in Response to UV-Induced DNA Photolesions

An important function of all organisms is to ensure that their genetic material remains intact and unaltered through generations. This is an extremely challenging task since the cell's DNA is constantly under assault by endogenous and environmental agents. To protect against this, cells have evolved effective mechanisms to recognize DNA damage, signal its presence, and mediate its repair. While these responses are expected to be highly regulated because they are critical to avoid human diseases, very little is known about the regulation of the expression of genes involved in mediating their effects. The Nucleotide Excision Repair (NER) is the major DNA–repair process involved in the recognition and removal of UV-mediated DNA damage. Here we use a combination of in vitro and in vivo assays with an intermittent UV-irradiation protocol to investigate the regulation of key players in the DNA–damage recognition step of NER sub-pathways (TCR and GGR). We show an up-regulation in gene expression of CSA and HR23A, which are involved in TCR and GGR, respectively. Importantly, we show that this occurs through a p53 independent mechanism and that it is coordinated by the stress-responsive transcription factor USF-1. Furthermore, using a mouse model we show that the loss of USF-1 compromises DNA repair, which suggests that USF-1 plays an important role in maintaining genomic stability

Régulation transcriptionnelle UV-induite de gènes de la réparation de l'ADN par le facteur de transcription USF-1 (Upstream Stimulating Factor 1)

Skin is the first body barrier exposed to various environmental hazardous including solar UV irradiations wich can alter DNA structure. To protect and maintain the integrity of the genome, cells are equipped with the UV-inducible skin pigmentation pathway and specific defense machinery including Nucleotide Excision Repair (NER). NER is one of the most complexe and versatile DNA repair system, highly conserved during evolution, wich requires several actors whose mutations lead to extrem cutaneous photosensibility. In parallel of the UV-inducible and USF-1 (Upstream Stimulating Factor 1) dependante transcriptional regulation of actors of the pigmentation pathway, we have investigated transcriptional induction of genes encoding NER actors. Using an approach of combined in-vivo (RT-QPCR and ChIP) and in-vitro (EMSA, luciferase assay) experiments, we show that USF-1 is directly implicated in transcriptional induction of UV induced DNA damages recognition step of NER. This model is confirmed by using USF-1 KO mice. Data obtained during my PhD complete the listing of USF-1 target genes and expand implicated pathways. These results suggest a potential role of this transcription factor in skin cancer processes.La peau est la première barrière de protection de l'organisme face aux agressions de l'environnement incluant notamment les radiations UV solaires, responsables de nombreux dommages sur l'ADN. Afin de prévenir l'apparition de lésions et de maintenir l'intégrité du génome, la cellule a mis en place des systèmes de protection avec la réponse pigmentaire, et de réparation avec le mécanisme du NER (nucleotide excision repair). Le NER est un mécanisme complexe et versatile, conservé au cours de l'évolution, qui repose sur l'action coordonnée d'acteurs protéiques dont les mutations conduisent à l'apparition de syndromes d'hyper-sensibilité aux UV. Ainsi, parallèlement à la régulation transcriptionnelle UV induite dépendante du facteur USF-1 (Upstream Stimulating Factor 1) des gènes clés de la pigmentation, nous avons étudié l'induction transcriptionnelle potentielle de gènes codant pour les acteurs du NER. Par une approche combinant expériences in-vivo (RT-QPCR et ChIP) et in-vitro (EMSA, luciférase essais), nous impliquons le facteur USF-1 dans l'induction transcriptionnelle de gènes codant deux acteurs des étapes de reconnaissance des lésions UV-induites par le NER. L'utilisation de souris invalidées pour le gène USF-1 confirme le modèle observé. L'ensemble de ces données obtenues au cours de ma thèse vient compléter la liste des gènes cibles du facteur de transcription USF-1 et élargit son réseau d'implication en réponse aux UV. Ces résultats suggèrent un rôle potentiel du facteur USF-1 dans le processus tumoral des cancers cutanés UV-induits

Identification of specific protein/E-box-containing DNA complexes: lessons from the ubiquitously expressed USF transcription factors of the b-HLH-LZ super family.

In : Transcription Factors: Methods and Protocols Higgins, Paul J. (Ed.) 2010, 377 p. 172 illus., 86 in color., ISBN 978-1-60761-737-2 (print), 978-1-60761-738-9 (electronic), HardcoverInternational audienceIn order to determine how gene expression is regulated in response to environmental cues, it is necessary to identify the specific interaction between transcription factors and their cognate cis-regulatory DNA elements. Here we have out-lined electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) protocols to define in vitro and in vivo USFs specific interacting sequences. The proposed procedures have been optimized for the USFs transcription factor family, allowing the identification of USF-specific targets

Régulation transcriptionnelle UV-induite de gènes de la réparation de l'ADN par le facteur de transcription USF-1 (Upstream Stimulating Factor 1)

La peau est la première barrière de protection de l'organisme face aux agressions de l'environnement incluant les radiations UV solaires responsables de dommages sur l'ADN. Afin de prévenir l'apparition de lésions et de maintenir l'intégrité du génome, la cellule a mis en place des systèmes de protection, la réponse pigmentaire, et de réparation, le NER. Parallèlement à la régulation transcriptionnelle UV induite dépendante du facteur USF-1 des gènes clés de la pigmentation, nous avons étudié l'induction transcriptionnelle potentielle de gènes codant pour les acteurs du NER. Par une approche combinant expériences in-vivo (RT-QPCR, ChIP, souris KO USF-1) et in-vitro (EMSA, luciférase essais), nous impliquons le facteur USF-1 dans l'induction transcriptionnelle en réponse aux UV de deux acteurs des étapes de reconnaissances des lésions par le NER. L'ensemble de ces données obtenues au cours de ma thèse vient compléter la liste des gènes cibles du facteur de transcription USF-1 et élargit son réseau d'implication en réponse aux UV. Ces résultats suggèrent un rôle potentiel du facteur USF-1 dans le processus tumoral des cancers cutanés UV-induits.Skin is the first body barrier exposed to various environmental hazardous including solar UV irradiations wich can alter DNA structure. To protect and maintain the integrity of the genome, cells are equipped with the UV-inducible skin pigmentation pathway and specific defense machinery including Nucleotide Excision Repair. In parallel of the UV-inducible and USF-1 dependante transcriptional regulation of actors of the pigmentation pathway, we have investigated transcriptional induction of genes encoding NER actors. Using an approach of combined in-vivo (RT-QPCR and ChIP) and in-vitro (EMSA, luciferase assay) experiments, we show that USF-1 is directly implicated in transcriptional induction of UV induced DNA damages recognition step of NER. This model is confirmed by using USF-1 KO mice. Data obtained during my PhD complete the listing of USF-1 target genes and expand implicated pathways. These results suggest a potential role of this transcription factor in skin cancer processes.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

<i>CSA</i> and <i>HR23A</i> expression is up-regulated in XB2 mouse keratinocytes after repetitive UV irradiation.

<p>(A) Quantification of <i>CSA</i> and <i>CSB</i> expression following UV-irradiation (8×10 J/m²) was determined by RT-qPCR (ΔΔCT method). Results are expressed relative to control (no UV treatment) and normalized to an <i>HPRT</i> transcript standard (comparable results were obtained with other reference genes: <i>GAPDH</i>) n = 3. (B) Western blotting analysis and quantification of CSA protein level in XB2 cells irradiated as previously described and in UV-irradiated cells following a pretreatment or not with α-amanitin. Tubulin (α-Tub) is included as a loading control. Signals are detected using LAS-3000 Imaging System (Fujifilm) and quantified with ImageJ. CSA quantified data are reported in the subpanel, where (▪) corresponds to UV-irradiated samples and () to UV-irradiated samples pre-treated with α-amanitin. The bar graphs compare the intensity of CSA protein normalized to the loading control. (C) Quantification of <i>HR23A</i> and <i>HR23B</i> mRNA expression following UV-irradiation (8×10 J/m²) determined as previously by RT-qPCR (ΔΔCT method) n = 4. (D) Western blotting analysis of HR23A and HR23B protein levels as described previously in irradiated XB2 cells, pre-treated (▪) or not with α-amanitin (). HSC70 is included as a loading control. For all results errors bars indicate s.e.m.; one asterisk, <i>P</i><0,05 <i>n</i> = 3; two asterisks, <i>P</i><0.01; three asterisks, <i>P</i><0.001.</p

USF family members interact with <i>CSA</i> and <i>HR23A</i> proximal promoters.

<p>(A) Graphic representation of human, mouse, dog and zebrafish <i>CSA</i> proximal promoter. Conserved E-boxes are represented in dark grey. (B) <i>In vivo</i> chromatin immunoprecipitation assays (ChIP) with HaCaT cells using USF-1, USF-2 antibodies or non-specific IgG. Recovered DNA under basal or UV-irradiation conditions was subjected to PCR or quantitative PCR using specific primers of both proximal and distal region (negative control) of the <i>CSA</i> promoter. (C) <i>In vitro</i> Electrophoretic Mobility Shift Assay (EMSA) experiments were performed using HaCaT nuclear extract and radiolabeled probes centered on the E-box motif present in the <i>CSA</i> proximal promoter (−246) (shifted complex (→)). Competition assays were performed in the presence or not of cold competitors (WT or mutated cold probe). Supershift assays were obtained in the presence of anti-USF-1, anti-USF-2, and anti-TBX2 antibodies or IgG as non-specific controls ( = >). (D) Graphic representation of human, mouse and dog <i>HR23A</i> and human <i>HR23B</i> proximal promoters. Conserved E-box motifs are represented in dark grey and GC-rich regions in light grey. (E) ChIP assays were performed as in (B) targeting proximal <i>HR23A</i> or <i>HR23B</i> promoters and the distal region of <i>HR23A</i> promoter (−3 kb). (F) EMSA experiments were performed as described in (C) using radiolabeled probes centered on each E-box motif (−154 and −36) present in the <i>HR23A</i> proximal promoter. (G) ChIP assay using SP3 antibody or non-specific IgG were performed as previously described for <i>HR23A</i> and <i>HR23B</i> promoter occupancy. (H) EMSA experiments were performed as previously with HaCaT nuclear extract and radiolabelled probes centered on the GC box present in the <i>HR23A</i> proximal promoter (−131) (shifted complex (→)).</p

<i>In vitro</i> transcriptional regulation of human <i>CSA</i> and <i>HR23A</i> by USF.

<p>(A) Schematic representation of the <i>CSA</i> promoter-luciferase constructs. The construct contains the sequence from −847 to +1 of the <i>CSA</i> promoter linked to the luciferase reporter. E-box is represented in dark gray and its position is indicated on top. Cross shows mutated E-box. (B) <i>CSA</i> promoter-luciferase activity measured after co-transfection of XB2 keratinocytes with WT or mutated CSA promoter-luciferase constructs with pCMV-USF-1, pCMV-USF-2 expression vectors or pCMV empty vector (control). (C) <i>CSA</i> promoter-luciferase activity measured 30 min or 5 h after UV induction (6×10 J/m²) of XB2 cells transfected with WT or mutated CSA promoter-luciferase constructs. (D) Schematic representation of the <i>HR23A</i> promoter-luciferase constructs. The construct contains the sequences from −186 to +73 of the <i>HR23A</i> promoter linked to the luciferase reporter. E-boxes are represented in dark gray, GC-box in light gray, and positions are indicated on top. Crosses show mutated boxes. (E) <i>HR23A</i> promoter-luciferase activity measured after co-transfection of XB2 keratinocytes with pCMV-USF-1 or pCMV-USF-2 expression vectors or empty vector. (F) WT and mutated <i>HR23A</i> promoter-luciferase activities in XB2 cells following UV-irradiation (6×10 J/m²). (G) WT and mutated <i>HR23A</i> promoter-luciferase activities transfected in XB2 cells with pCMV-USF-1 expression vector and UV irradiated (6×10 J/m²). Error bars indicate s.e.m.; <i>n</i> = 3; one asterisk, <i>P</i><0.05, two asterisks, <i>P</i><0.01, three asterisks, <i>P</i><0.001.</p

UV-induced <i>CSA</i> and <i>HR23A</i> expression is impaired in <i>USF-1</i> knock-out (KO) mice.

<p>(A) Expression analysis of <i>CSA</i> and <i>CSB</i> were performed by RT-qPCR after UV-irradiation (4×50 J/m²) of cultured punch biopsy samples from WT (dark color) or <i>USF</i>-1 KO mice (light color). Results for UV-treated samples are expressed relative to controls (no irradiation) with the <i>HPRT</i> transcript used as a standard. (B) Expression analysis of <i>HR23A</i> and <i>HR23B</i> were performed by RT-qPCR as previously described. (C) Expression analysis of the UV response positive control gene, <i>Gadd45α</i>, was performed by RT-qPCR as previously described. (D) 36 hours kinetics of CPD DNA-damage removal (ELISA quantification) in cultured skin punch biopsies from WT (black) or <i>USF-1</i> KO mice (grey). The yellow band corresponds to the irradiation protocol (4×50 J/m²). Skin punch biopsies were analyzed immediately after 3 UV-pulses (3×50 J/m²) (time 1 h), and after 4 UV-pulses (4×50 J/m²) (at the following times: 3–7–24–36 h). Error bars indicate s.e.m.; <i>n</i> = 3.</p