Analysis of Transgenerational Epigenetic Inheritance in <em>C. elegans</em> Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through ...

University of Toronto

We describe the application of a microarray platform, which combines information from exon body and splice-junction probes, to perform a quantitative analysis of tissue-specific alternative splicing (AS) for thousands of exons in mammalian cells. Through this system, we have analyzed global features of AS in major mouse tissues. The results provide numerous inferences for the functions of tissue-specific AS, insights into how the evolutionary history of exons can impact on their inclusion levels, and also information on how global regulatory properties of AS define tissue type. Like global transcription profiles, global AS profiles reflect tissue identity. Interestingly, we find that transcription and AS act independently on different sets of genes in order to define tissue-specific expression profiles. These results demonstrate the utility of our quantitative microarray platform and data for revealing important global regulatory features of AS.

Revealing global regulatory features of mammalian alternative splicing using a quantitative microarray platform.

Sequence-based analyses have predicted that approximately 35% of mammalian alternative splicing (AS) events produce premature termination codon (PTC)-containing splice variants that are targeted by the process of nonsense-mediated mRNA decay (NMD). This led to speculation that AS may often regulate gene expression by activating NMD. Using AS microarrays, we show that PTC-containing splice variants are generally produced at uniformly low levels across diverse mammalian cells and tissues, independently of the action of NMD. Our results suggest that most PTC-introducing AS events are not under positive selection pressure and therefore may not contribute important functional roles.

Quantitative microarray profiling provides evidence against widespread coupling of alternative splicing with nonsense-mediated mRNA decay to control gene expression.

Most RNAs are processed from precursors by mechanisms that include covalent modifications, as well as the removal of flanking and intervening sequences. Traditional methods to detect RNA processing, such as Northern blotting, reverse-transcribed polymerase chain reaction and primer extension assays, are difficult to apply on a large scale. This chapter outlines several methods for analysis of the processing and modification of RNA using microarrays. These encompass protocols for the application of homemade microarrays and custom-designed commercial inkjet microarrays and are tailored for the large-scale analysis of processing of mRNA, including alternative splicing, as well as for the analysis of processing and modification of noncoding RNA. This chapter also describes practical aspects of microarray design, sample preparation, hybridization, and data analysis.

Microarray analysis of RNA processing and modification.

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Alternative splicing (AS) functions to expand proteomic complexity and plays numerous important roles in gene regulation. However, the extent to which AS coordinates functions in a cell and tissue type specific manner is not known. Moreover, the sequence code that underlies cell and tissue type specific regulation of AS is poorly understood.

Functional coordination of alternative splicing in the mammalian central nervous system.

&#xC9;pissage alternatif (AS) des fonctions d&#x26;#39;&#xE9;largir la complexit&#xE9; prot&#xE9;omique et joue de nombreux r&#xF4;les importants dans la r&#xE9;gulation des g&#xE8;nes. Toutefois, la mesure dans laquelle coordonne AS fonctions dans une cellule et d&#x26;#39;une mani&#xE8;re un type de tissu sp&#xE9;cifique n&#x26;#39;est pas connu. En outre, le code de la s&#xE9;quence sous-tend cette cellule et la r&#xE9;glementation de type sp&#xE9;cifique d&#x26;#39;un tissu de l&#x26;#39;AS est mal comprise.

Coordination fonctionnelle de l&#x26;#39;&#xE9;pissage alternatif dans le syst&#xE8;me nerveux central des mammif&#xE8;res

Alternatives Splei&szlig;en (AS) Funktionen zum Erweitern von Proteomic Komplexit&auml;t und spielt zahlreiche wichtige Rollen in der Genregulation. Das Ausma&szlig;, in welchem AS koordiniert jedoch Funktionen in einer Zelle und Gewebe-Typ, die spezifische Art und Weise nicht bekannt ist. Dar&uuml;ber hinaus zugrunde den Sequenz-Code Zellen und Gewebe-Typ, die spezifische Verordnung als schlecht verstanden wird.

Funktionale Koordination der alternatives Splei&szlig;en der S&auml;ugetier-Zentralnervensystem.

Splicing alternativo (AS) funzioni per espandere proteomic complessit&agrave; e svolge numerosi ruoli importanti nella regolazione genica. Tuttavia, la misura in cui AS coordina le funzioni in un tipo di cellule e tessuti modo specifico non &egrave; noto. Inoltre, il codice di sequenza che sottende il tipo delle cellule e del tessuto specifico regolamento di AS &egrave; capita male.

Coordinamento funzionale di splicing alternativo nel sistema nervoso centrale dei mammiferi.

&#x9078;&#x629E;&#x7684;&#x30B9;&#x30D7;&#x30E9;&#x30A4;&#x30B7;&#x30F3;&#x30B0;&#xFF08;AS&#xFF09;&#x95A2;&#x6570;&#x306F;&#x3001;&#x30D7;&#x30ED;&#x30C6;&#x30AA;&#x30DF;&#x30AF;&#x30B9;&#x306E;&#x8907;&#x96D1;&#x3055;&#x3092;&#x5C55;&#x958B;&#x3057;&#x3001;&#x907A;&#x4F1D;&#x5B50;&#x8ABF;&#x7BC0;&#x306B;&#x591A;&#x6570;&#x306E;&#x91CD;&#x8981;&#x306A;&#x5F79;&#x5272;&#x3092;&#x679C;&#x305F;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x7D30;&#x80DE;&#x3084;&#x7D44;&#x7E54;&#x306E;&#x30BF;&#x30A4;&#x30D7;&#x7279;&#x5B9A;&#x306E;&#x65B9;&#x6CD5;&#x3067;&#x6A5F;&#x80FD;&#x3092;&#x8ABF;&#x6574;&#x3059;&#x308B;&#x7A0B;&#x5EA6;&#x306F;&#x77E5;&#x3089;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3002; AS&#x306E;&#x57FA;&#x790E;&#x3068;&#x306A;&#x308B;&#x7D30;&#x80DE;&#x3084;&#x7D44;&#x7E54;&#x306E;&#x7A2E;&#x985E;&#x306B;&#x56FA;&#x6709;&#x306E;&#x898F;&#x5236;&#x304C;&#x5341;&#x5206;&#x306B;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x307E;&#x305F;&#x3001;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x306F;&#x3001;&#x30B3;&#x30FC;&#x30C9;&#x3067;&#x3059;&#x3002;

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&#xB300;&#xCCB4; &#xC811;&#xD569; (AS) proteomic &#xBCF5;&#xC7A1;&#xC131;&#xC744; &#xD655;&#xC7A5; &#xD558;&#xB294; &#xAE30;&#xB2A5; &#xBC0F; &#xC720;&#xC804;&#xC790; &#xC870;&#xC808;&#xC5D0; &#xC218;&#xB9CE;&#xC740; &#xC911;&#xC694; &#xD55C; &#xC5ED;&#xD560;&#xC744; &#xD55C;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xBC94;&#xC704;&#xB294; AS &#xD2B9;&#xC815; &#xBC29;&#xC2DD;&#xC73C;&#xB85C; &#xC54C; &#xC218; &#xC5C6;&#xB294; &#xC138;&#xD3EC; &#xBC0F; &#xC870;&#xC9C1;&#xC758; &#xC885;&#xB958;&#xC758; &#xD568;&#xC218;&#xB97C; &#xC870;&#xC815; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xC2DC;&#xD000;&#xC2A4; &#xCF54;&#xB4DC;&#xB294; &#xC138;&#xD3EC;&#xC640; &#xC870;&#xC9C1;&#xC758; &#xC885;&#xB958;&#xC758; &#xD2B9;&#xC815; &#xADDC;&#xC815;&#xC740; &#xC81C;&#xB300;&#xB85C; &#xC774;&#xD574;&#xC758; &#xAE30;&#xCD08;&#xAC00; &#xB418;.

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Splicing alternativo (AS) para ampliar las funciones de la complejidad prote&#xF3;mica y desempe&#xF1;a numerosas funciones importantes en la regulaci&#xF3;n g&#xE9;nica. Sin embargo, el grado en que AS coordina las funciones en una c&#xE9;lula y la forma del tejido tipo espec&#xED;fico no se conoce. Por otra parte, el c&#xF3;digo de secuencia que subyace en la c&#xE9;lula y la regulaci&#xF3;n del tejido tipo espec&#xED;fico de la EA es poco conocida.

Coordinaci&#xF3;n Funcional de splicing alternativo en el sistema nervioso central de mam&#xED;feros

During the past approximately 20 years, studies on alternative splicing (AS) have largely been directed at the identification and characterization of factors and mecha nisms responsible for the control of splice site selection, using model substrates and on a case by case basis. These studies have provided a wealth of information on the factors and interactions that control formation of the spliceosome. However, relatively little is known about the global regulatory properties of AS. Important questions that need to be addressed are: which exons are alternatively spliced and under which cellular contexts, what are the functional roles of AS events in different cellular contexts, and how are AS events controlled and coordinated with each other and with other levels of gene regulation to achieve cell- and development-specific functions. During the past several years, new technologies and experimental strategies have provided insight into these questions. For example, custom microarrays and data analysis tools are playing a prominent role in the discovery and analysis of splicing regulation. Moreover, several non-microarray-based technologies are emerging that will likely further fuel progress in this area. This review focuses on recent advances made in the development and application of high-throughput methods to study AS.

Technologies for the global discovery and analysis of alternative splicing.

Alternative splicing (AS) can regulate gene expression by introducing premature termination codons (PTCs) into spliced mRNA that subsequently elicit transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, the range of cellular functions controlled by this process and the factors required are poorly understood. By quantitative AS microarray profiling, we find that there are significant overlaps among the sets of PTC-introducing AS events affected by individual knockdown of the three core human NMD factors, Up-Frameshift 1 (UPF1), UPF2, and UPF3X/B. However, the levels of some PTC-containing splice variants are less or not detectably affected by the knockdown of UPF2 and/or UPF3X, compared with the knockdown of UPF1. The intron sequences flanking the affected alternative exons are often highly conserved, suggesting important regulatory roles for these AS events. The corresponding genes represent diverse cellular functions, and surprisingly, many encode core spliceosomal proteins and assembly factors. We further show that conserved, PTC-introducing AS events are enriched in genes that encode core spliceosomal proteins. Where tested, altering the expression levels of these core spliceosomal components affects the regulation of PTC-containing splice variants from the corresponding genes. Together, our results show that AS-coupled NMD can have different UPF factor requirements and is likely to regulate many general components of the spliceosome. The results further implicate general spliceosomal components in AS regulation.

Regulation of multiple core spliceosomal proteins by alternative splicing-coupled nonsense-mediated mRNA decay.

Alternative splicing (AS) plays a major role in the generation of proteomic diversity and in gene regulation. However, the role of the basal splicing machinery in regulating AS remains poorly understood. Here we show that the core snRNP (small nuclear ribonucleoprotein) protein SmB/B' self-regulates its expression by promoting the inclusion of a highly conserved alternative exon in its own pre-mRNA that targets the spliced transcript for nonsense-mediated mRNA decay (NMD). Depletion of SmB/B' in human cells results in reduced levels of snRNPs and a striking reduction in the inclusion levels of hundreds of additional alternative exons, with comparatively few effects on constitutive exon splicing levels. The affected alternative exons are enriched in genes encoding RNA processing and other RNA-binding factors, and a subset of these exons also regulate gene expression by activating NMD. Our results thus demonstrate a role for the core spliceosomal machinery in controlling an exon network that appears to modulate the levels of many RNA processing factors.

Regulation of alternative splicing by the core spliceosomal machinery.

Chromatin modifications, including methylation of histone H3 at lysine 27 (H3K27me) by the Polycomb group proteins, play a broadly conserved role in the maintenance of cell fate. Diverse chromatin organization modifier (chromo) domain proteins act as "readers" of histone methylation states. However, understanding the functional relationships among chromo domains and their roles in the inheritance of gene expression patterns remains challenging. Here, we identify two chromo-domain proteins, CEC-1 and CEC-6, as potential readers of H3K27me in , where they have divergent expression patterns and contribute to distinct phenotypes. Both  and  genetically interact with another chromo-domain gene, , a reader of H3K9 methylation. Combined loss of  and  leads to developmental defects in the adult that result in decreased fitness. Furthermore, loss of  and  surprisingly leads to a progressive loss of fertility across generations, a "mortal germline" phenotype. Our results provide evidence of functional compensation between H3K27me and H3K9me heterochromatin pathways, and show that histone methylation readers contribute to both somatic development and transgenerational fitness.

Multiple Histone Methyl-Lysine Readers Ensure Robust Development and Germline Immortality in .

Transgenerational epigenetic inheritance (TEI) is the inheritance of epigenetic information for two or more generations. In most cases, TEI is limited to a small number of generations (two to three). The short-term nature of TEI could be set by innate biochemical limitations to TEI or by genetically encoded systems that actively limit TEI. In , double-stranded RNA (dsRNA)-mediated gene silencing [RNAi (RNA interference)] can be inherited (termed RNAi inheritance or RNA-directed TEI). To identify systems that might actively limit RNA-directed TEI, we conducted a forward genetic screen for factors whose mutation enhanced RNAi inheritance. This screen identified the gene heritable enhancer of RNAi (), whose mutation causes RNAi inheritance to last longer (> 20 generations) than normal.  encodes a protein with a chromodomain, and a kinase homology domain that is expressed in germ cells and localizes to nuclei. In , a nuclear branch of the RNAi pathway [termed the nuclear RNAi or NRDE (nuclear RNA defective) pathway] promotes RNAi inheritance. We find that  animals have defects in spermatogenesis that are suppressible by mutations in the nuclear RNAi Argonaute (Ago) HRDE-1, suggesting that HERI-1 might normally act in sperm progenitor cells to limit nuclear RNAi and/or RNAi inheritance. Consistent with this idea, we find that the NRDE nuclear RNAi pathway is hyperresponsive to experimental RNAi treatments in  mutant animals. Interestingly, HERI-1 binds to genes targeted by RNAi, suggesting that HERI-1 may have a direct role in limiting nuclear RNAi and, therefore, RNAi inheritance. Finally, the recruitment of HERI-1 to chromatin depends upon the same factors that drive cotranscriptional gene silencing, suggesting that the generational perdurance of RNAi inheritance in  may be set by competing pro- and antisilencing outputs of the nuclear RNAi machinery.

Transgenerational Epigenetic Inheritance Is Negatively Regulated by the HERI-1 Chromodomain Protein.

Tools for -mediated single copy insertion (mosSCI) with excisable  or NeoR (G418) selection cassettes.

In most eukaryotes, the genome is packaged with histones and other proteins to form chromatin. One of the major mechanisms for chromatin regulation is through post-translational modification of histone proteins. Recognition of these modifications by effector proteins, often dubbed histone "readers," provides a link between the chromatin landscape and gene regulation. The diversity of histone reader proteins for each modification provides an added layer of regulatory complexity. In this review, we will focus on the roles of chromatin organization modifier (chromo) domain containing proteins in the model nematode, . An amenability to genetic and cell biological approaches, well-studied development and a short life cycle make  a powerful system to investigate the diversity of chromo domain protein functions in metazoans. We will highlight recent insights into the roles of chromo domain proteins in the regulation of heterochromatin and the spatial conformation of the genome as well as their functions in cell fate, fertility, small RNA pathways and transgenerational epigenetic inheritance. The spectrum of different chromatin readers may represent a layer of regulation that integrates chromatin landscape, genome organization and gene expression.

Emerging Roles for Chromo Domain Proteins in Genome Organization and Cell Fate in .

In the field of chromatin biology, a major goal of understanding the roles of histone post-translational modifications is to identify the proteins and domains that recognize these modifications. Synthetic histone peptides containing one or more modifications are a key tool to probe these interactions in pull-down assays with recombinant proteins or cell lysates. Building on these approaches, the binding specificity of a protein of interest can be screened against many histone peptides in parallel using a peptide array. In this protocol, we describe the expression and purification of a recombinant protein of interest in bacteria, followed by an assay for binding to histone post-translational modifications using a commercially available histone peptide array. The purification uses a versatile dual-tagging and cleavage strategy and equipment commonly available in a molecular biology laboratory. Graphic abstract: Overview of protocol for purifying recombinant protein and hybridizing to a histone peptide array.

Assessing the  Binding Specificity of Histone Modification Reader Proteins Using Histone Peptide Arrays.

RNA interference (RNAi) is a highly conserved gene regulatory phenomenon whereby Argonaute/small RNA (AGO/sRNA) complexes target transcripts by antisense complementarity to modulate gene expression. While initially appreciated as a cytoplasmic process, RNAi can also occur in the nucleus where AGO/sRNA complexes are recruited to nascent transcripts. Nuclear AGO/sRNA complexes recruit co-factors that regulate transcription by inhibiting RNA Polymerase II, modifying histones, compacting chromatin and, in some organisms, methylating DNA. C. elegans has a longstanding history in unveiling the mechanisms of RNAi and has become an outstanding model to delineate the mechanisms underlying nuclear RNAi. In this review we highlight recent discoveries in the field of nuclear RNAi in C. elegans and the roles of nuclear RNAi in the regulation of gene expression, chromatin organization, genome stability, and transgenerational epigenetic inheritance.

Mechanisms of epigenetic regulation by C. elegans nuclear RNA interference pathways.

Pathogens generate ubiquitous selective pressures and host-pathogen interactions alter social behaviours in many animals. However, very little is known about the neuronal mechanisms underlying pathogen-induced changes in social behaviour. Here we show that in adult Caenorhabditis elegans hermaphrodites, exposure to a bacterial pathogen (Pseudomonas aeruginosa) modulates sensory responses to pheromones by inducing the expression of the chemoreceptor STR-44 to promote mating. Under standard conditions, C. elegans hermaphrodites avoid a mixture of ascaroside pheromones to facilitate dispersal. We find that exposure to the pathogenic Pseudomonas bacteria enables pheromone responses in AWA sensory neurons, which mediate attractive chemotaxis, to suppress the avoidance. Pathogen exposure induces str-44 expression in AWA neurons, a process regulated by a transcription factor zip-5 that also displays a pathogen-induced increase in expression in AWA. STR-44 acts as a pheromone receptor and its function in AWA neurons is required for pathogen-induced AWA pheromone response and suppression of pheromone avoidance. Furthermore, we show that C. elegans hermaphrodites, which reproduce mainly through self-fertilization, increase the rate of mating with males after pathogen exposure and that this increase requires str-44 in AWA neurons. Thus, our results uncover a causal mechanism for pathogen-induced social behaviour plasticity, which can promote genetic diversity and facilitate adaptation of the host animals.

Arneet L. Saltzman

Department of Cell and Systems Biology

University of Toronto

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

Arneet L. Saltzman

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Cell and Systems Biology

University of Toronto

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

Department of Cell and Systems Biology