SUMMARY Acetylation of histones at DNA regulatory elements plays a critical role in transcriptional activation. Histones are also modified by other acyl moieties, including crotonyl, yet the mechanisms that govern acetylation versus crotonylation and the functional consequences of this “choice” remain unclear. We show that the coactivator p300 has both crotonyltransferase and acetyltransferase activities and that p300-catalyzed histone crotonylation directly stimulates transcription to a greater degree than histone acetylation. Levels of histone crotonylation are regulated by the cellular concentration of crotonyl-CoA, which can be altered through genetic and environmental perturbations. In a cell-based model of transcriptional activation, increasing or decreasing the cellular concentration of crotonyl-CoA leads to enhanced or diminished gene expression, respectively, which correlates with the levels of histone crotonylation flanking the regulatory elements of activated genes. Our findings support a general principle wherein differential histone acylation (i.e. acetylation versus crotonylation) couples cellular metabolism to the regulation of gene expression.

Neuronal intranuclear inclusion disease (NIID) is a slowly progressing neurodegenerative disease characterized by eosinophilic intranuclear inclusions in the nervous system and multiple visceral organs. The clinical manifestation of NIID varies widely, and both familial and sporadic cases have been reported. Here we have performed genetic linkage analysis and mapped the disease locus to 1p13.3-q23.1; however, whole-exome sequencing revealed no potential disease-causing mutations. We then performed long-read genome sequencing and identified a large GGC repeat expansion within human-specific NOTCH2NLC. Expanded GGC repeats as the cause of NIID was further confirmed in an additional three NIID-affected families as well as five sporadic NIID-affected case subjects. Moreover, given the clinical heterogeneity of NIID, we examined the size of the GGC repeat among 456 families with a variety of neurological conditions with the known pathogenic genes excluded. Surprisingly, GGC repeat expansion was observed in two Alzheimer disease (AD)-affected families and three parkinsonism-affected families, implicating that the GGC repeat expansions in NOTCH2NLC could also contribute to the pathogenesis of both AD and PD. Therefore, we suggest defining a term NIID-related disorders (NIIDRD), which will include NIID and other related neurodegenerative diseases caused by the expanded GGC repeat within human-specific NOTCH2NLC.

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Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a premutation CGG repeat expansion (55–200 repeats) within the 5′ UTR of the fragile X gene (FMR1). FXTAS is characterized by intension tremor, cerebellar ataxia, progressive neurodegeneration, parkinsonism and cognitive decline. The development of transgenic mouse and Drosophila melanogaster models carrying an expanded CGG repeat has yielded valuable insight into the pathophysiology of FXTAS. To date, we know of two main molecular mechanisms of this disorder: (1) a toxic gain of function of the expanded CGG-repeat FMR1 mRNA, which results in the binding/sequestration of the CGG-binding proteins; and (2) CGG repeat-associated non-AUG-initiated (RAN) translation, which generates a polyglycine peptide toxic to cells. Besides these CGG-mediated mechanisms, recent studies have shed light on additional mechanisms of pathogenesis, such as the antisense transcript ASFMR1, mitochondrial dysfunction, DNA damage from R-loop formation and 5-hydroxymethylcytosine (5hmC)-mediated epigenetic modulation. Here we summarize the recent progress towards understanding the etiology of FXTAS and provide an overview of potential treatment strategies.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects premutation carriers (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Much remains unknown regarding the metabolic alterations associated with FXTAS, especially in the brain, and the most affected region, the cerebellum. Investigating the metabolic changes in FXTAS will aid in the identification of biomarkers as well as in understanding the pathogenesis of disease. To identify the metabolic alterations associated with FXTAS, we took advantage of our FXTAS mouse model that expresses 90 CGG repeats in cerebellar Purkinje neurons and exhibits the key phenotypic features of FXTAS. We performed untargeted global metabolic profiling of age-matched control and FXTAS mice cerebella at 16-20 weeks and 55 weeks. Out of 506 metabolites measured in cerebellum, we identified 186 metabolites that demonstrate significant perturbations due to the (CGG) 90 repeat (P<0.05) and found that these differences increase dramatically with age. To identify key metabolic changes in FXTAS pathogenesis, we performed a genetic screen using a Drosophila model of FXTAS. Out of 28 genes that we tested in the f ly, 8 genes showed significant enhanced neuronal toxicity associated with CGG repeats, such as Schlank (ceramide synthase), Sk2 (sphingosine kinase) and Ras (IMP dehydrogenase). By combining metabolic profiling with a Drosophila genetic screen to identify genetic modifiers of FXTAS, we demonstrate an effective method for functional validation of high-throughput metabolic data and show that sphingolipid and purine metabolism are significantly perturbed in FXTAS pathogenesis.

Significance Expansion of 55-200 CGG repeats in the 5′ untranslated region of FMR1 predisposes carriers to fragile X–associated tremor/ataxia syndrome (FXTAS), a late-onset neurodegenerative disorder. FXTAS demonstrates incomplete penetrance, which strongly suggests the presence of genetic modifiers. We performed whole-genome sequencing (WGS) on male premutation carriers (CGG 55–200 ) followed by a functional screen in Drosophila and identified PSMB5 as a strong suppressor of CGG-associated neurodegeneration, thereby presenting a therapeutic strategy for FXTAS.

Eukaryotic genomes are dynamically regulated through a host of epigenetic stimuli. The substrate for these epigenetic transactions, chromatin, is a polymer of nucleosome building blocks. In native (i.e. cellular) chromatin, each nucleosome can differ from its neighbors through the localized installation of covalent modifications to both the genomic DNA and the histone packaging proteins. The heterotypic nature of chromatin presents a formidable obstacle to biochemical studies seeking to understand the role of context on epigenetic regulation and that, as a consequence, must employ compositionally defined chromatin substrates. Here, we introduce a chemical approach to the production of heterotypic ‘designer’ chromatin that can be used in such studies. Our method involves attachment of a user-defined modified histone peptide to a designated nucleosome within the polymer by using a Peptide Nucleic Acid (PNA) targeting compound. We apply this strategy to dissect the role of chromatin context on both the activation and inhibition of the histone methyltransferase, PRC2, which methylates Lys 27 of histone H3 (H3K27). Our studies show that PRC2 can be stimulated to produce de novo H3K27 methylation from a defined nucleation site. More generally, this technology promises to facilitate biochemical studies that require the use of heterotypic chromatin substrates.