scholarly journals Mutations in SKI in Shprintzen-Goldberg syndrome lead to attenuated TGF-β responses through SKI stabilization

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Ilaria Gori ◽  
Roger George ◽  
Andrew G Purkiss ◽  
Stephanie Strohbuecker ◽  
Rebecca A Randall ◽  
...  
Keyword(s):  
Connective Tissue ◽  
Genome Editing ◽  
Structural Biology ◽  
Point Mutations ◽  
Connective Tissue Disorder ◽  
Negative Regulator ◽  
Transcriptional Corepressor ◽  
Transcriptional Responses ◽  
Ligand Stimulation

Shprintzen-Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys-Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-β signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional corepressor SKI, which is a negative regulator of TGF-b signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-β responses, in both knockin cells expressing an SGS mutation, and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-b-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.

scholarly journals Mutations in SKI in Shprintzen-Goldberg syndrome lead to attenuated TGF-β responses through SKI stabilization

2020 ◽  
Author(s):  
Ilaria Gori ◽  
Roger George ◽  
Andrew G. Purkiss ◽  
Stephanie Strohbuecker ◽  
Rebecca A. Randall ◽  
...  
Keyword(s):  
Connective Tissue ◽  
Genome Editing ◽  
Structural Biology ◽  
Point Mutations ◽  
Connective Tissue Disorder ◽  
Negative Regulator ◽  
Transcriptional Corepressor ◽  
Transcriptional Responses ◽  
Ligand Stimulation

ABSTRACTShprintzen-Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys-Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-β signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional corepressor SKI, which is a negative regulator of TGF-β signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-β responses, in both knockin cells expressing an SGS mutation, and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-β-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.

scholarly journals Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuu Asano ◽  
Kensuke Yamashita ◽  
Aoi Hasegawa ◽  
Takanori Ogasawara ◽  
Hoshie Iriki ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dictyostelium Discoideum ◽  
Streptococcus Pyogenes ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Knock Out ◽  
Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

scholarly journals Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis

2021 ◽  
Vol 7 (2) ◽  
pp. 149
Author(s):  
Sarah-Maria Wege ◽  
Katharina Gejer ◽  
Fabienne Becker ◽  
Michael Bölker ◽  
Johannes Freitag ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Biology ◽  
Gene Deletion ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Model Organism ◽  
Ustilago Maydis ◽  
Genomic Locus ◽  
Targeted Gene Deletion ◽  
Molecular Cell Biology

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

scholarly journals Atypical presentation of rheumatoid arthritis: a case of massive bilateral pleural effusions in a patient with Caplan syndrome

2019 ◽  
Vol 12 (10) ◽  
pp. e231899
Author(s):  
Yinglun Wu ◽  
Philip C Dittmar
Keyword(s):  
Rheumatoid Arthritis ◽  
Atrial Fibrillation ◽  
Pleural Effusion ◽  
Connective Tissue ◽  
Minimal Change Disease ◽  
Connective Tissue Disorder ◽  
Minimal Change ◽  
Atypical Presentation ◽  
Pleural Effusions ◽  
Recent Treatment

Rheumatoid arthritis (RA) is a common connective tissue disorder affecting the synovial joints. In patients with RA, involvement of the lungs occurs in 30%–40% of cases while pleural effusions occur in only 3%–5%. However, the majority of RA-associated pleural effusions are small, unilateral and asymptomatic. We present a case of massive bilateral pleural effusions in a patient with established rheumatoid pneumoconiosis (Caplan syndrome). Interestingly, the pleural effusion occurred following recent treatment for minimal change disease and atrial fibrillation.

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