Transcriptional repression of the silent mating-type loci in Saccharomyces cerevisiae requires a cell cycle-dependent establishment step that is commonly assumed to involve DNA replication. Using site-specific recombination, we created a nonreplicating DNA ring in vivo to test directly the role of replication in establishment of silencing. Sir1 was tethered to the ring following excision from the chromosome to activate a dormant silencer. We show here that silencing can be established in DNA that does not replicate. The silenced ring adopted structural features characteristic of bona fide silent chromatin, including an altered level of DNA supercoiling and reduced histone acetylation. In addition, the process required silencing factors Sir2, Sir3, and Sir4 and progression between early S and M phases of the cell cycle. The results indicate that passage of a replication fork is not the cell-cycle event required for establishment of silencing in yeast.

Lengthy trinucleotide repeats encoding polyglutamine (polyQ) stretches characterize the variant proteins of Huntington's disease and certain other inherited neurological disorders. Using a phenotypic screen to identify events that restore functionality to polyQ proteins in S. cerevisiae, we discovered that transcription elongation factor Spt4 is required to transcribe long trinucleotide repeats located either in ORFs or nonprotein-coding regions of DNA templates. Mutation of SPT4 selectively decreased synthesis of and restored enzymatic activity to expanded polyQ protein without affecting protein lacking long-polyQ stretches. RNA-seq analysis revealed limited effects of Spt4 on overall gene expression. Inhibition of Supt4h, the mammalian ortholog of Spt4, reduced mutant huntingtin protein in neuronal cells and decreased its aggregation and toxicity while not altering overall cellular mRNA synthesis. Our findings identify a cellular mechanism for transcription through repeated trinucleotides and a potential target for countermeasures against neurological disorders attributable to expanded trinucleotide regions.

Production of protein containing lengthy stretches of polyglutamine encoded by multiple repeats of the trinucleotide CAG is a hallmark of Huntington’s disease (HD) and of a variety of other inherited degenerative neurological and neuromuscular disorders. Earlier work has shown that interference with production of the transcription elongation protein SUPT4H results in decreased cellular capacity to transcribe mutant huntingtin gene (Htt) alleles containing long CAG expansions, but has little effect on expression of genes containing short CAG stretches. zQ175 and R6/2 are genetically engineered mouse strains whose genomes contain human HTT alleles that include greatly expanded CAG repeats and which are used as animal models for HD. Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2’-O-methoxyethyl oligonucleotides (ASOs) directed against Supt4h, or in R6/2 mice by deletion of one copy of the Supt4h gene, results in a decrease in mRNA and protein encoded specifically by mutant Htt alleles. We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals. Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.

Expression of p53 protein correlates with increased MDM2 and p14(ARF) protein levels in ESCC. In addition, status of p53 (wild-type versus mutant) rather than expression level of p53, MDM2, or p14(ARF) is likely to be the more critical determinant of clinical outcome.

Background Polyglutamine (polyQ) diseases are dominant neurodegenerative diseases caused by an expansion of the polyQ‐encoding CAG repeats in the disease‐causing gene. The length of the CAG repeats is the major determiner of the age at onset (AO) of polyQ diseases, including Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3). Objective We set out to identify common genetic variant(s) that may affect the AO of polyQ diseases. Methods Three hundred thirty‐seven patients with HD or SCA3 were enrolled for targeted sequencing of 583 genes implicated in proteinopathies. In total, 16 genes were identified as containing variants that are associated with late AO of polyQ diseases. For validation, we further investigate the variants of PIAS1 because PIAS1 is an E3 SUMO (small ubiquitin‐like modifier) ligase for huntingtin (HTT), the protein linked to HD. Results Biochemical analyses revealed that the ability of PIAS1S510G to interact with mutant huntingtin (mHTT) was less than that of PIAS1WT, resulting in lower SUMOylation of mHTT and lower accumulation of insoluble mHTT. Genetic knock‐in of PIAS1S510G in a HD mouse model (R6/2) ameliorated several HD‐like deficits (including shortened life spans, poor grip strength and motor coordination) and reduced neuronal accumulation of mHTT. Conclusions Our findings suggest that PIAS1 is a genetic modifier of polyQ diseases. The naturally occurring variant, PIAS1S510G, is associated with late AO in polyQ disease patients and milder disease severity in HD mice. Our study highlights the possibility of targeting PIAS1 or pathways governing protein homeostasis as a disease‐modifying approach for treating patients with HD. © 2021 International Parkinson and Movement Disorder Society