SUMO-targeted ubiquitin ligases (STUbLs) reduce the toxicity and abnormal transcriptional activity associated with a mutant, aggregation-prone fragment of huntingtin
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE115990 | GEO | 2018/06/20
REPOSITORIES: GEO
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Project description:Cell viability and gene expression profiles are altered in cellular models of neurodegenerative disorders such as Huntington's Disease (HD). Using the yeast model system, we show that the SUMO-targeted ubiquitin ligase (STUbL) Slx5 reduces the toxicity and abnormal transcriptional activity associated with a mutant, aggregation-prone fragment of huntingtin (Htt), the causative agent of HD. We demonstrate that expression of an aggregation-prone Htt construct with 103 glutamine residues (103Q), but not the non-expanded form (25Q), results in severe growth defects in slx5Δ and slx8Δ cells. Since Slx5 is a nuclear protein and because Htt expression affects gene transcription, we assessed the effect of STUbLs on the transcriptional properties of aggregation-prone Htt. Expression of Htt 25Q and 55Q fused to the Gal4 activation domain (AD) resulted in reporter gene auto-activation. Remarkably, the auto-activation of Htt constructs was abolished by expression of Slx5 fused to the Gal4 DNA-binding domain (BD-Slx5). In support of these observations, RNF4, the human ortholog of Slx5, curbs the aberrant transcriptional activity of aggregation-prone Htt in yeast and a variety of cultured human cell lines. Functionally, we find that an extra copy of SLX5 specifically reduces Htt aggregates in the cytosol as well as chromatin-associated Htt aggregates in the nucleus. Finally, using RNA sequencing, we identified and confirmed specific targets of Htt's transcriptional activity that are modulated by Slx5. In summary, this study of STUbLs uncovers a conserved pathway that counteracts the accumulation of aggregating, transcriptionally active Htt (and possibly other poly-glutamine expanded proteins) on chromatin in both yeast and in mammalian cells.
| S-EPMC6154015 | biostudies-literature
Project description:Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post-translational modifications and the corresponding enzymatic machinery. Specifically, SUMO-targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance and transcription. However, how STUbLs select specific substrates amongst myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co-localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term ‘ubiquitin hotspots’. Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor-like Ymr111c/Euc1 to associate with these sites and be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO-interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1-ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z and Rpd3L pathways in specific stress responses. Thus, our data suggest that STUbL-dependent ubiquitin hotspots shape chromatin during stress adaptation.
2019-03-29 | GSE126526 | GEO
Project description:Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post-translational modifications and the corresponding enzymatic machinery. Specifically, SUMO-targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance and transcription. However, how STUbLs select specific substrates amongst myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co-localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term ‘ubiquitin hotspots’. Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor-like Ymr111c/Euc1 to associate with these sites and be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO-interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1-ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z and Rpd3L pathways in specific stress responses. Thus, our data suggest that STUbL-dependent ubiquitin hotspots shape chromatin during stress adaptation.
2019-03-29 | GSE118817 | GEO
Project description:Chromatin is a highly regulated environment, and protein association with chromatin is often controlled by post-translational modifications and the corresponding enzymatic machinery. Specifically, SUMO-targeted ubiquitin ligases (STUbLs) have emerged as key players in nuclear quality control, genome maintenance and transcription. However, how STUbLs select specific substrates amongst myriads of SUMOylated proteins on chromatin remains unclear. Here, we reveal a remarkable co-localization of the budding yeast STUbL Slx5/Slx8 and ubiquitin at seven genomic loci that we term ‘ubiquitin hotspots’. Ubiquitylation at these sites depends on Slx5/Slx8 and protein turnover on the Cdc48 segregase. We identify the transcription factor-like Ymr111c/Euc1 to associate with these sites and be a critical determinant of ubiquitylation. Euc1 specifically targets Slx5/Slx8 to ubiquitin hotspots via bipartite binding of Slx5 that involves the Slx5 SUMO-interacting motifs and an additional, novel substrate recognition domain. Interestingly, the Euc1-ubiquitin hotspot pathway acts redundantly with chromatin modifiers of the H2A.Z and Rpd3L pathways in specific stress responses. Thus, our data suggest that STUbL-dependent ubiquitin hotspots shape chromatin during stress adaptation.
2019-03-29 | GSE118771 | GEO
Project description:Huntington’s disease (HD) is a late onset neurological disorder for which no neuroprotective therapies efficiently delay onset or progression. A key pathological mechanism in disease involves the proteolysis of polyglutamine (polyQ)-expanded mutant huntingtin (mHTT) generating polyQ-containing N-terminal fragments that are crucial contributors to HD pathogenesis since inhibition of the cleavage at the putative caspase-6 site reduces pathology in a HD mouse model. Interestingly, a naturally occurring alternative spliced form of HTT lacking exon12 (HTTΔ12) leads to a protein with an internal deletion of the caspase-6 N-terminal cleavage site. Here, we have taken a multidisciplinary approach to characterize the therapeutic potential of targeting exon12 of HTT. We show that HTTΔ12 is fully resistant to caspase-6 cleavage in both cell-free and tissue lysate assays, while maintaining overall biochemical and structural properties similar to wild-type (wt)-HTT. We generated mouse deleted for exon12 and found that exon12 is dispensable for HTT main physiological functions including embryonic development and intracellular trafficking such as BDNF transport. Finally, we pharmacologically induced HTTΔ12 using an antisense oligonucleotide (ASO). QRX-704 ASO efficiently distributes in the whole brain, is stable several months, and reduces pathogenic proteolysis. Thus, ASO-induced exon12 deletion of HT We performed gene expression profiling analysis using data obtained from RNA-seq of brain tissue (cortex and striatum) samples originating from mouse littermates from HttΔ12 (Δ12;KI) and HttKO (KO) lines at 3 month of age.
2022-07-29 | GSE209893 | GEO
Project description:Many neurodegenerative diseases are characterized by the presence of intracellular protein aggregates resulting in alterations in autophagy. However, the consequences of impaired autophagy on neuronal function remain poorly understood. In this study, we used cell culture and mouse models of huntingtin protein aggregation, as well as post-mortem material from patients with Huntington's disease to demonstrate that Argonaute-2 (AGO2) accumulates in the presence of neuronal protein aggregates and that this is due to impaired autophagy. Accumulation of AGO2, a key factor of the RNAinduced silencing complex that executes microRNA functions, results in global alterations of microRNA levels and activity. Together these results demonstrate that impaired autophagy found in neurodegenerative diseases not only influences protein aggregation, but also directly contributes to global alterations of intracellular posttranscriptional networks.
2018-08-07 | GSE78928 | GEO
Project description:The polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington’s disease (HD). Htt is an essential gene as deletion of the mouse Htt gene homolog (Hdh) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD. To systematically search for a mouse Htt function, we took advantage of the Hdh +/- and Hdh-floxed mice and generated four mouse embryonic fibroblast (MEF) cells lines which contain a single copy of the Hdh gene (Hdh-HET) and four MEF lines in which the Hdh gene was deleted (Hdh-KO). The function of Htt in calcium (Ca2+) signaling was analyzed in Ca2+ imaging experiments with generated cell lines. We found that the cytoplasmic Ca2+ spikes resulting from the activation of inositol 1,4,5-trisphosphate receptor (InsP3R) and the ensuing mitochondrial Ca2+ signals were suppressed in the Hdh-KO cells when compared to Hdh-HET cells. Furthermore, in experiments with permeabilized cells we found that the InsP3-sensitivity of Ca2+ mobilization from endoplasmic reticulum was reduced in Hdh-KO cells. These results indicated that Htt plays an important role in modulating InsP3R-mediated Ca2+ signaling. To further evaluate function of Htt, we performed genome-wide transcription profiling of generated Hdh-HET and Hdh-KO cells by microarray. Our results revealed that 106 unique transcripts were downregulated by more than two-fold with p < 0.05 and 173 unique transcripts were upregulated at least two-fold with p < 0.05 in Hdh-KO cells when compared to Hdh-HET cells. The microarray results were confirmed by quantitative real-time PCR for a number of affected transcripts. Several signaling pathways affected by Hdh gene deletion were identified from annotation of the microarray results. The unbiased approach used in our study provides novel and unique information about the normal function of Htt in cells, which may contribute to our understanding and treatment of HD. Keywords: cell type comparison we generate four Hdh-HET MEF cell lines and four Hdh-KO MEF cell lines, and performed genome-wide transcription profiling of generated Hdh-HET and Hdh-KO cells by microarray.
2008-04-12 | E-GEOD-11139 | biostudies-arrayexpress
Project description:The Saccharomyces cerevisiae Slx5/8 complex is the founding member of a recently defined class of ubiquitin ligases (STUbLs) that target sumoylated substrates. Slx5/8 has been implicated in genome stability and transcription, but the precise contribution to these processes is not clear. To characterize Slx5/8 function, we determined genome-wide changes in gene expression upon loss of either subunit. The majority of mRNA changes are part of a general stress response, also exhibited by mutants of other genome integrity pathways and therefore indicative of an indirect effect on transcription. Genome-wide binding analysis reveals a uniquely centromeric location for Slx5. Detailed phenotype analyses of slx5D and slx8D mutants show severe mitotic defects that include aneuploidy, spindle mispositioning, fish hook spindles and aberrant spindle kinetics. This is associated with accumulation of the PP2A regulatory subunit Rts1 at centromeres prior to entry into anaphase. Knockdown of the human STUbL orthologue RNF4 also results in chromosome segregation defects due to formation of chromosome bridges. The study shows that STUbLs have a conserved role in maintenance of chromosomal stability and the results link SUMO-dependent ubiquitination to a centromere-specific function during mitosis.
2013-08-06 | GSE33929 | GEO
Project description:Huntington’s disease (HD), caused by a CAG repeat expansion in the huntingtin (HTT) gene, is characterized by abnormal protein aggregates and motor and cognitive dysfunction. Htt protein is ubiquitously expressed, but the striatal medium spiny neuron (MSN) is most susceptible to neuronal dysfunction and death. Abnormal gene expression represents a core pathogenic feature of HD, but the relative roles of cell-autonomous and non-cell-autonomous effects on transcription remain unclear. To determine the extent of cell-autonomous dysregulation in the striatum in vivo, we examined genome-wide RNA expression in symptomatic D9-N171-98Q (a.k.a. DE5) transgenic mice in which the forebrain expression of the first 171 amino acids of human Htt with a 98Q repeat expansion is limited to MSNs. Microarray data generated from these mice were compared to those generated on the identical array platform from a pan-neuronal HD mouse model, R6/2, carrying two different CAG repeat lengths, and a relatively high degree of overlap of changes in gene expression was revealed. We further focused on known canonical pathways associated with excitotoxicity, oxidative stress, mitochondrial dysfunction, dopamine signaling and trophic support, among others. While genes related to excitotoxicity, dopamine signaling and trophic support, were altered in both DE5 and R6/2 transgenic mice, which may be either cell-autonomous or non-cell-autonomous,, genes related to mitochondrial dysfunction, oxidative stress and the peroxisome proliferator-activated receptor are primarily affected in DE5 transgenic mice, indicating cell autonomous mechanisms Overall, our results demonstrate that HD-induced dysregulation of the striatal transcriptome can be largely attributed to intrinsic effects of mutant Htt,,such that mutant Htt-induced effects in cortical neurons is not necessary for striatal dysfunction/degeneration. Striatum from DE5 transgenic mice vs. wt littermate controls
2011-01-01 | E-GEOD-25232 | biostudies-arrayexpress
Project description:SUMO-targeted ubiquitin ligases (STUbLs) reduce the toxicity and abnormal transcriptional activity associated with a mutant, aggregation-prone fragment of huntingtin
| PRJNA476714 | ENA