Cross-linking and immunoprecipitation coupled with high-throughput sequencing was used to identify binding sites within 6,304 genes as the brain RNA targets for TDP-43, an RNA binding protein which when mutated causes Amyotrophic Lateral Sclerosis (ALS). Use of massively parallel sequencing and splicing-sensitive junction arrays revealed that levels of 601 mRNAs are changed (including Fus/Tls, progranulin, and other transcripts encoding neurodegenerative disease-associated proteins) and 965 altered splicing events are detected (including in sortilin, the receptor for progranulin), following depletion of TDP-43 from mouse adult brain with antisense oligonucleotides. RNAs whose levels are most depleted by reduction in TDP-43 are derived from genes with very long introns and which encode proteins involved in synaptic activity. Lastly, TDP-43 was found to auto-regulate its synthesis, in part by directly binding and enhancing splicing of an intron within the 3′ untranslated region of its own transcript, thereby triggering nonsense mediated RNA degradation. (147 words)

FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from >5,500 genes in mouse and human brain, primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern, consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of >950 mRNAs, most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain, but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in TDP-43 aggregate–containing motor neurons in sporadic ALS, supporting a common loss-of-function pathway as one component underlying motor neuron death from misregulation of TDP-43 or FUS/TLS.

Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.E xpanded hexanucleotide repeats in the first intron of the chromosome 9 open reading frame 72 (C9orf72) gene were recently identified (1, 2) as the most common genetic cause of ALS, frontotemporal degeneration (FTD), or concomitant ALS/ FTD (3, 4). The mechanisms by which the expanded repeats cause neurodegeneration are unknown, but leading candidate mechanisms are RNA-mediated toxicity, loss of the C9orf72 gene function (from reduced C9orf72 produced by the allele with the expansion), or a combination of the two.RNA-mediated toxicity from nucleotide repeat expansion was initially described for CUG expansion in the RNA encoded by the DMPK gene in myotonic muscular dystrophy (5). A consensus view is that RNA toxicity plays a crucial role in a variety of repeat expansion disorders (6). A hallmark of these disorders is the accumulation of expanded transcripts into nuclear RNA foci (7). RNAs harboring a long stretch of repeats are thought to fold into stable structures and sequester RNA binding proteins, which, in turn, sets off a molecular cascade leading to neurodegeneration (7). In myotonic dystrophy, sequestration and functional disruption of the muscleblind-like family of RNA binding proteins are associated with specific splicing and expression changes in affected tissues (5,(8)(9)(10)(11)(12).Sequestration of one or more RNA binding proteins into pathological RNA foci has ...

Summary Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded-AR first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic machinery induced by genotoxic stress and liganded-AR, including Activation-Induced Cytidine Deaminase (AID) and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymatic machineries synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by Non-Homologous Ending Joining (NHEJ) pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded-nuclear receptor and genotoxic stress underlie non-random tumor translocations, which may function in many types of tumors and pathological processes.

Nuclear receptors undergo ligand-dependent conformational changes that are required for corepressor-coactivator exchange, but whether there is an actual requirement for specific epigenetic landmarks to impose ligand dependency for gene activation remains unknown. Here we report an unexpected and general strategy that is based on the requirement for specific cohorts of inhibitory histone methyltransferases (HMTs) to impose gene-specific gatekeeper functions that prevent unliganded nuclear receptors and other classes of regulated transcription factors from binding to their target gene promoters and causing constitutive gene activation in the absence of stimulating signals. This strategy, based at least in part on an HMT-dependent inhibitory histone code, imposes a requirement for specific histone demethylases, including LSD1, to permit ligand- and signal-dependent activation of regulated gene expression. These events link an inhibitory methylation component of the histone code to a broadly used strategy that circumvents pathological constitutive gene induction by physiologically regulated transcription factors.