Transcriptome-pathology correlation identifies interplay between TDP-43 and the expression of its kinase CK1E in sporadic ALS.

Sporadic amyotrophic lateral sclerosis (sALS) is the most common form of ALS, however, the molecular mechanisms underlying cellular damage and motor neuron degeneration remain elusive. To identify molecular signatures of sALS we performed genome-wide expression profiling in laser capture microdissection-enriched surviving motor neurons (MNs) from lumbar spinal cords of sALS patients with rostral onset and caudal progression. After correcting for immunological background, we discover a highly specific gene expression signature for sALS that is associated with phosphorylated TDP-43 (pTDP-43) pathology. Transcriptome-pathology correlation identified casein kinase 1ε (CSNK1E) mRNA as tightly correlated to levels of pTDP-43 in sALS patients. Enhanced crosslinking and immunoprecipitation in human sALS patient- and healthy control-derived frontal cortex, revealed that TDP-43 binds directly to and regulates the expression of CSNK1E mRNA. Additionally, we were able to show that pTDP-43 itself binds RNA. CK1E, the protein product of CSNK1E, in turn interacts with TDP-43 and promotes cytoplasmic accumulation of pTDP-43 in human stem-cell-derived MNs. Pathological TDP-43 phosphorylation is therefore, reciprocally regulated by CK1E activity and TDP-43 RNA binding. Our framework of transcriptome-pathology correlations identifies candidate genes with relevance to novel mechanisms of neurodegeneration

Interlaboratory analytical validation of a Next-generation sequencing strategy for clonotypic assessment and minimal residual disease monitoring in multiple myeloma

[Context]: Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited. [Objective]: To standardize the performance of the LymphoTrack next-generation sequencing (NGS) assays (Invivoscribe), targeting clonal immunoglobulin rearrangements, in order to reproduce the detection of tumor clonotypes and MRD quantitation in myeloma. [Design]: The quantification ability of the assay was evaluated through serial dilution experiments. Paired samples from 101 patients were tested by LymphoTrack, using Sanger sequencing and EuroFlow's next-generation flow (NGF) assay as validated references for diagnostic and follow-up evaluation, respectively. MRD studies using LymphoTrack were performed in parallel at 2 laboratories to evaluate reproducibility. [Results]: Sensitivity was set as 1.3 tumor cells per total number of input cells. Clonality was confirmed in 99% and 100% of cases with Sanger and NGS, respectively, showing great concordance (97.9%), although several samples had minor discordances in the nucleotide sequence of rearrangements. Parallel NGS was performed in 82 follow-up cases, achieving a median sensitivity of 0.001%, while for NGF, median sensitivity was 0.0002%. Reproducibility of LymphoTrack-based MRD studies (85.4%) and correlation with NGF (R2 > 0.800) were high. Bland-Altman tests showed highly significant levels of agreement between flow and sequencing. [Conclusions]: Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures. Multiple myeloma (MM) is a plasma-cell dyscrasia characterized by the accumulation of plasma cells in the bone marrow that produces an excess of clonal immunoglobulins (M-protein or monoclonal component).1 New treatment approaches have increased the number of patients achieving complete response (CR),2–5 progressively improving progression-free and overall survival rates in the last 10 years.6–11 Nonetheless, the presence of low levels of drug-resistant cells (known as minimal residual disease, MRD)12–14 that remain undetected by conventional serologic and morphologic methods explains frequent relapses with this disease, which is still considered an incurable illness.Minimal residual disease is currently considered one of the most informative prognostic parameters, since those patients with undetectable disease have shown prolonged survival rates as compared with MRD-positive patients,15–17 and this difference is still significant even when patients achieving only stringent complete response (sCR) are taken into account.18 The International Myeloma Working Group (IMWG) defined MRD positivity as the persistence of clonal malignant plasma cells assessed with a sensitivity of at least 10−5 (1 malignant cell per hundred thousand normal cells)19 ; therefore, MRD should be monitored with only highly sensitive methods. To date, 3 different approaches have been tested for MRD monitoring in hematologic malignancies: immunophenotypic (multiparametric flow cytometry [MFC]),20 molecular (quantitative polymerase chain reaction [PCR], next-generation sequencing [NGS], digital PCR),21–23 and imaging tools (positron emission tomography–computed tomography; magnetic resonance imaging).24,25 However, in MM standardization has been achieved only for MFC26 and NGS.27,28 As a result, the IMWG recommended the use of highly sensitive, standardized flow and sequencing approaches,19 including EuroFlow's next-generation flow (NGF)29 and Adaptive Biotechnologies' ClonoSEQ solutions (Adaptive Biotechnologies, Seattle, Washington). NGF is a 2-tube, 8-color flow assay that allows the simultaneous analysis of 10 million cells, providing a sensitivity of around 2·10−6.This work was partially supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness PI15/01956, CIBERONC-CB16/12/00233, and “Una manera de hacer Europa” (Innocampus; CEI-2010-1-0010). García-Álvarez, Prieto-Conde, and Jiménez were supported by the Fundación Española de Hematología y Hemoterapia (FEHH, cofunded by Fundación Cris in the latter case), Medina by the European Social Fund through the University of Salamanca and the ISCIII (FI19/00320), and Sarasquete by the ISCIII (CPII18/00028). All Spanish funding is cosponsored by the European Union FEDER program

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

High-throughput genomic assays : applications and analysis of DSL technology and next- generation sequencing

Determining DNA sequence has been a principle tool for several methods in biology research. From whole genome sequencing to RNA expression assays to several types of immunoprecipitation experiments, sequencing DNA has been a staple detection technique. Recent advances in sequencing and detection of DNA has revealed many new possibilities, and problems, with regards to data analysis. Here, I present a study analyzing a novel detection technology and sequencing method. These are both important contributions for not only providing new insights for utilizing a more sensitive detection technique, but also creating a method which enables any researcher to quickly analyze the unheralded amount of sequence data now being produced, soon be available to everyone. Chapter 2 focuses on the current uses and analysis of a novel DNA detection technique called DNA Selection and Ligation (DSL). By taking advantage of the more sensitive and specific DSL strategy, any assay that is dependent on DNA detection is improved. In this chapter, I show how DSL can be used to modify the standard chromatin immunoprecipitation (ChIP)- on-chip assay (termed ChIP-DSL), in both the promoter- specific and tiling cases. Additionally, I show how the ChIP-DSL method gives promising results for a high- throughput version of the chromatin conformation capture (3C) assay, which is used to measure if two regions of DNA are interacting. Chapter 3 concentrates on next-generation sequencing, more specifically on the Illumina Genome Analyzer (GA). After describing the details of how the sequencing is performed and analyzed, I discuss the current flaw in these datasets, and propose a solution to the problem, the Genome Ontology. I then give several examples where the Genome Ontology is helpful in extracting knowledge from these incredibly large datasets. Chapter 4 describes numerous future directions of my own work as well as several of my observations that resulted from working with these next-generation sequencing dataset

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

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