ABSTRACT
Cohesin is crucial for genome stability, cell division, transcription and chromatin organization. Its functions critically depend on NIPBL, the cohesin-loader protein that is found to be mutated in >60% of the cases of Cornelia de Lange syndrome (CdLS). Other mutations are described in the cohesin subunits SMC1A, RAD21, SMC3 and the HDAC8 protein. In 25-30% of CdLS cases no mutation in the known CdLS genes is detected. Until now, functional elements in the noncoding genome were not characterized in the molecular etiology of CdLS and therefore are excluded from mutation screening, although the impact of such mutations has now been recognized for a wide range of diseases. We have identified different elements of the noncoding genome involved in regulation of the NIPBL gene. NIPBL-AS1 is a long non-coding RNA transcribed upstream and antisense to NIPBL. By knockdown and transcription blocking experiments, we could show that not the NIPBL-AS1 gene product, but its actual transcription is important to regulate NIPBL expression levels. This reveals a possibility to boost the transcriptional activity of the NIPBL gene by interfering with the NIPBL-AS1 lncRNA. Further, we have identified a novel distal enhancer regulating both NIPBL and NIPBL-AS1. Deletion of the enhancer using CRISPR genome editing in HEK293T cells reduces expression of NIPBL, NIPBL-AS1 as well as genes found to be dysregulated in CdLS.
Subject(s)
Enhancer Elements, Genetic , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/metabolism , Proteins/genetics , Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Segregation , De Lange Syndrome/genetics , Gene Expression Regulation , Genome, Human , HEK293 Cells , Humans , Mutation , Phenotype , Promoter Regions, Genetic , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Sequence Analysis, DNA , CohesinsABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adenosine Triphosphatases/metabolism , Arsenite Transporting ATPases/metabolism , Nuclear Proteins/metabolism , Vesicular Transport Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs , Amino Acid Sequence , Amyotrophic Lateral Sclerosis/genetics , Apoptosis Regulatory Proteins , Arsenite Transporting ATPases/chemistry , Hexosyltransferases , Humans , Immunoprecipitation , Mass Spectrometry , Models, Biological , Molecular Sequence Data , Mutation/genetics , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/pharmacology , Protein Binding/drug effects , Protein Structure, Tertiary , Proteolysis/drug effects , Ubiquitin/metabolism , Ubiquitination/drug effects , Vesicular Transport Proteins/chemistry , rab3 GTP-Binding Proteins/metabolismABSTRACT
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.
Subject(s)
DNA Damage/genetics , DNA Repair/genetics , DNA/genetics , Upstream Stimulatory Factors/genetics , Animals , Biopsy , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Survival/radiation effects , DNA/radiation effects , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Gene Expression Regulation/radiation effects , Genomic Instability , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Humans , Keratinocytes/cytology , Keratinocytes/metabolism , Keratinocytes/radiation effects , Mice , Mice, Knockout , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , RNA, Small Interfering , Ultraviolet RaysABSTRACT
In 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.
Subject(s)
DNA/genetics , DNA/metabolism , E-Box Elements/genetics , Gene Expression Regulation , Upstream Stimulatory Factors/metabolism , Base Sequence , Binding, Competitive , Cell Line , Cell Nucleus/metabolism , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Humans , Mutation , Promoter Regions, Genetic/genetics , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Substrate SpecificityABSTRACT
How transcription factors interpret the output from signal transduction pathways to drive distinct programs of gene expression is a key issue that underpins development and disease. The ubiquitously expressed basic-helix-loop-helix leucine zipper upstream stimulating factor-1 binds E-box regulatory elements (CANNTG) to regulate a wide number of gene networks. In particular, USF-1 is a key component of the tanning process. Following UV irradiation, USF-1 is phosphorylated by the p38 stress-activated kinase on threonine 153 and directly up-regulates expression of the POMC, MC1R, TYR, TYRP-1 and DCT genes. However, how phosphorylation on Thr-153 might affect the activity of USF-1 is unclear. Here we show that, in response to DNA damage, oxidative stress and cellular infection USF-1 is acetylated in a phospho-Thr-153-dependent fashion. Phospho-acetylated USF-1 is nuclear and interacts with DNA but displays altered gene regulatory properties. Phospho-acetylated USF-1 is thus proposed to be associated with loss of transcriptional activation properties toward several target genes implicated in pigmentation process and cell cycle regulation. The identification of this critical stress-dependent USF-1 modification gives new insights into understanding USF-1 gene expression modulation associated with cancer development.
