Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3′ untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.National Institutes of Health (U.S.) (Grant HG007005

Decoding a signature-based model of transcription cofactor recruitment dictated by cardinal cis-regulatory elements in proximal promoter regions.

Genome-wide maps of DNase I hypersensitive sites (DHSs) reveal that most human promoters contain perpetually active cis-regulatory elements between -150 bp and +50 bp (-150/+50 bp) relative to the transcription start site (TSS). Transcription factors (TFs) recruit cofactors (chromatin remodelers, histone/protein-modifying enzymes, and scaffold proteins) to these elements in order to organize the local chromatin structure and coordinate the balance of post-translational modifications nearby, contributing to the overall regulation of transcription. However, the rules of TF-mediated cofactor recruitment to the -150/+50 bp promoter regions remain poorly understood. Here, we provide evidence for a general model in which a series of cis-regulatory elements (here termed 'cardinal' motifs) prefer acting individually, rather than in fixed combinations, within the -150/+50 bp regions to recruit TFs that dictate cofactor signatures distinctive of specific promoter subsets. Subsequently, human promoters can be subclassified based on the presence of cardinal elements and their associated cofactor signatures. In this study, furthermore, we have focused on promoters containing the nuclear respiratory factor 1 (NRF1) motif as the cardinal cis-regulatory element and have identified the pervasive association of NRF1 with the cofactor lysine-specific demethylase 1 (LSD1/KDM1A). This signature might be distinctive of promoters regulating nuclear-encoded mitochondrial and other particular genes in at least some cells. Together, we propose that decoding a signature-based, expanded model of control at proximal promoter regions should lead to a better understanding of coordinated regulation of gene transcription

<i>Nxf1</i> Natural Variant E610G Is a Semi-dominant Suppressor of IAP-Induced RNA Processing Defects

<div><p>Endogenous retroviruses and retrotransposons contribute functional genetic variation in animal genomes. In mice, Intracisternal A Particles (IAPs) are a frequent source of both new mutations and polymorphism across laboratory strains. Intronic IAPs can induce alternative RNA processing choices, including alternative splicing. We previously showed IAP I∆1 subfamily insertional mutations are suppressed by a wild-derived allele of the major mRNA export factor, <i>Nxf1</i>. Here we show that a wider diversity of IAP insertions present in the mouse reference sequence induce insertion-dependent alternative processing that is suppressed by <i>Nxf1^CAST</i> alleles. These insertions typically show more modest gene expression changes than de novo mutations, suggesting selection or attenuation. Genome-wide splicing-sensitive microarrays and gene-focused assays confirm specificity of <i>Nxf1</i> genetic modifier activity for IAP insertion alleles. Strikingly, CRISPR/Cas9-mediated genome editing demonstrates that a single amino acid substitution in Nxf1, E610G, is sufficient to recreate a quantitative genetic modifier in a co-isogenic background.</p></div

Decoding a Signature-Based Model of Transcription Cofactor Recruitment Dictated by Cardinal Cis-Regulatory Elements in Proximal Promoter Regions

<div><p>Genome-wide maps of DNase I hypersensitive sites (DHSs) reveal that most human promoters contain perpetually active cis-regulatory elements between −150 bp and +50 bp (−150/+50 bp) relative to the transcription start site (TSS). Transcription factors (TFs) recruit cofactors (chromatin remodelers, histone/protein-modifying enzymes, and scaffold proteins) to these elements in order to organize the local chromatin structure and coordinate the balance of post-translational modifications nearby, contributing to the overall regulation of transcription. However, the rules of TF-mediated cofactor recruitment to the −150/+50 bp promoter regions remain poorly understood. Here, we provide evidence for a general model in which a series of cis-regulatory elements (here termed ‘cardinal’ motifs) prefer acting individually, rather than in fixed combinations, within the −150/+50 bp regions to recruit TFs that dictate cofactor signatures distinctive of specific promoter subsets. Subsequently, human promoters can be subclassified based on the presence of cardinal elements and their associated cofactor signatures. In this study, furthermore, we have focused on promoters containing the nuclear respiratory factor 1 (NRF1) motif as the cardinal cis-regulatory element and have identified the pervasive association of NRF1 with the cofactor lysine-specific demethylase 1 (LSD1/KDM1A). This signature might be distinctive of promoters regulating nuclear-encoded mitochondrial and other particular genes in at least some cells. Together, we propose that decoding a signature-based, expanded model of control at proximal promoter regions should lead to a better understanding of coordinated regulation of gene transcription.</p></div

Nxf1 alleles do not affect alternative processing at non-IAP sites.

<p><b>(A)</b> The ratio of alternative splice forms for 12 candidate genes was measured by in-gel fluorescence after PCR from congenic <i>Nxf1</i>^<i>CAST</i> or <i>Nxf1</i>^<i>B6</i> allele homozygotes. Box-and-whiskers plots show the ratio ratio between paired samples of alternate genotypes (C/B ratio). Ratios of individual sample pairs are plotted as points over each box. <b>(B)</b> Three samples of each genotype were hybridized to Affymetrix arrays to interrogate 13,002 alternative RNA events. Absolute value of the separation score (log₂ of the inclusion/exclusion rate for <i>Nxf1</i> C/B paired samples) between alleles is plotted by rank across all probe sets. Events that were confirmed in gene-specific assays on additional samples are indicated in red, events that were tested but not confirmed are in black. Positions of <i>Trpc6</i> and <i>Slc15a2</i> events tested in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005123#pgen.1005123.g002" target="_blank">Fig. 2</a> are indicated. <b>(C)</b> Ratios between paired samples, as in panel A, for alternative splicing events predicted by the arrays. Significant differences, plotted in red, are either events at IAP-containing introns or in genes within the Nxf1 congenic region on chromosome 19A. <b>(D)</b> Diagram of chromosome 19 shows the positions of genes with alternative outcomes in panel (C) relative to the congenic interval homozygous for CAST/EiJ alleles in all <i>Nxf1</i>^<i>CAST</i> samples (white), intervals between informative markers or heterozygous in some samples (gray), and homozygous for B6 alleles (black).</p

Suppression of distinct IAP structural classes by <i>Nxf1</i>^<i>CAST</i>.

<p>* Full list of elements by class is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005123#pgen.1005123.s001" target="_blank">S1 Table</a></p><p>** p-values from the Wilcoxon rank sum test for all paired measurements in the class</p><p>*** False Discovery Rate q-value from the Benjamini-Hochberg method.</p><p>¹ Deletions that remove at least half of the <i>pol</i> ORF, but less than half of the adjacent <i>prt</i> ORF.</p><p>Suppression of distinct IAP structural classes by <i>Nxf1</i>^<i>CAST</i>.</p

<i>Adamts13</i>^<i>S</i> is suppressed by congenic <i>Nxf1</i>^<i>CAST</i>.

<p><b>(A)</b> Alignment of Adamts13 protein domains to their corresponding exons in <i>Adamts13</i>^<i>L</i> and <i>Adamts13</i>^<i>S</i> shows loss of two thrombospondin (TSP) and two CUB domains from <i>Adamts13</i>^<i>S</i>, which terminates in an intronic IAP sequence. TM, transmembrane segment; PRO, protease domain. Locations of primers to detect exon 24 to exon 25 splicing around the IAP are indicated. <b>(B)</b> The <i>Adamts13</i>^<i>S</i> IAP shares greater sequence similarity with the <i>Atrn</i>^<i>mgL</i> full-length IAP than with IΔ1 elements, an example of which is shown. Percent nucleotide identity is shown for indicated segments. <b>(C)</b> Quantitative RT-PCR for exon 24-exon 25 splice junction relative to <i>Gapdh</i> in B6 x BALB/c F2 mice homozygous for both <i>Adamts13</i>^<i>S</i> and the indicated congenic allele of <i>Nxf1</i> shows substantial increase in the <i>Nxf1</i>^<i>CAST</i> congenic allele (p = 6.4x10^-5, Wilcoxon rank sum test). <b>(D)</b> Similar results were obtained for a smaller number of animals from the B6 congenic line (p = 1.1x10^-3, Wilcoxon rank sum test). Normalization to <i>Desmin</i> as a cell type-specific marker increased the separation of values. Each dot in the box plots represents the mean of technical replicates for one biological sample.</p

E610G variant accounts for <i>Modifier-of-vibrator</i> activity of <i>Nxf1</i>.

<p><b>(A)</b> The E610G edited allele decreased tremor severity. Tremor scores for <i>vibrator</i> mutant and littermate control animals of the indicated genotypes are plotted. Each point represents the mean observer score for one animal. P-values for the single hypothesis tests that one copy of the edited allele significantly reduced tremor relative to the unedited or pseudo-edited (ps.) B6 allele are shown. <b>(B)</b> The E610G edited allele increased genotype-dependent survival of <i>vibrator</i> animals. Kaplan-Meier plots show fraction alive over a ~3-month observation period. Heterozygotes for edited alleles are drawn in red, pseudo edited in blue. Censored animals are indicated with vertical lines. <b>(C)</b> Brain <i>Pitpna</i> gene expression from <i>vibrator</i> mutant alleles was increased by E610G alleles. Measurements were made from animals in (A) not used in (B). Relative quantities compared to three reference genes (<i>Gapdh</i>, <i>Sdha</i>, and <i>Ppia</i>) are plotted. Individual points represent the average of three replicate measurements per biological sample.</p

<i>Nxf1</i>^<i>CAST</i> congenic allele suppresses a diversity of IAP elements in the B6 genome.

<p><b>(A)</b> Schematic diagrams illustrate sequence compositions of IAP elements in this study. Deleted segments in each elements compared with full-length consensus are shown at 10% opacity. <b>(B)</b> For each structural class, genes whose expression level was tested are indicated. Genes measured with TaqMan assays are indicated by (TM); all others measured by real-time fluorescence stimulation with SYBR green. Gene symbols are color coded: grey for no significant differences between strains that have (Inserted) and strains that lack (Uninserted) each IAP, black for no evidence of suppression by <i>Nxf1</i> of an observed strain difference, brown for a non-significant trend toward suppression, and red for significant evidence of suppression by <i>Nxf1</i>^CAST. Strains that include (Inserted) or lack (Uninserted) each IAP are indicated: A, A/J; B, C57BL/6J (B6); L, BALB/cJ; S, 129S1/SvImJ. Relative expression ratios for each strain are expressed as Uninserted/Inserted (U/I). Relative expression ratios for <i>Nxf1</i> congenic animals homozygous for either <i>Nxf1</i>^<i>CAST</i> (C) or <i>Nxf1</i>^<i>B6</i> (B) allele are expressed as C/B, such that insertions suppressed by the <i>Nxf1</i>^<i>CAST</i> allele have values >1. P-values from a two-tailed Wilcoxon-Mann-Whitney test for strain difference (U/I, unpaired samples) or a one-tailed Wilcoxon signed rank test for <i>Nxf1</i>^<i>CAST</i>-dependent suppression (C/B, littermate paired samples) are given in parentheses. Three genes were also confirmed by fluorescence intensity of isoform-specific bands on agarose gels using 3-primer assays designed independently of the qRT-PCR assays.</p