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Raf1 Is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification.

ABSTRACT: Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro. DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alignment and mutation of signature WDxR motifs in Raf1 indicate that it is a DCAF for CLRC. We demonstrate that Raf1 promotes H3K9 methylation and siRNA amplification via two distinct, separable functions. The association of the DCAF Raf1 with Cul4-Rik1 is critical for H3K9 methylation, but dispensable for processing of centromeric transcripts into siRNAs. Thus the association of a DCAF, Raf1, with its adaptor, Rik1, is required for histone methylation and to allow RNAi to signal to chromatin.

SUBMITTER: Buscaino A

PROVIDER: S-EPMC3271066 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Raf1 Is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification.

Buscaino Alessia A White Sharon A SA Houston Douglas R DR Lejeune Erwan E Simmer Femke F de Lima Alves Flavia F Diyora Piyush T PT Urano Takeshi T Bayne Elizabeth H EH Rappsilber Juri J Allshire Robin C RC

PLoS genetics 20120202 2

Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro. DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alig ...[more]

PMID: 22319459

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Project description:BackgroundThe WD40-repeat containing proteins, including DDB1-CUL4-associated factors (DCAFs), are abundant and conserved proteins that play important roles in different cellular processes including spermatogenesis. DCAFs are subset of WD40 family proteins that contain WDxR motif and have been proposed to function as substrate receptor for Cullin4-RING-based E3 ubiquitin ligase complexes to recruit diverse proteins for ubiquitination, a vital process in spermatogenesis. Large number of WD40 genes has been identified in different species including mouse and human. However, a systematic expression profiling of WD40 genes in different tissues of mouse and human has not been investigated. We hypothesize that large number of WD40 genes may express highly or specifically in the testis, where their expression is uniquely regulated during testis development and spermatogenesis. Therefore, the objective of this study is to mine and characterize expression patterns of WD40 genes in different tissues of mouse and human with particular emphasis on DCAF genes expressions during mouse testicular development.ResultsPublically available RNA sequencing (RNA seq) data mining identified 347 and 349 WD40 genes in mouse and human, respectively. Hierarchical clustering and heat map analyses of RNA seq datasets revealed differential expression patterns of WD40 genes with around 60-73% of the genes were highly or specifically expressed in testis. Similarly, around 74-83% of DCAF genes were predominantly or specifically expressed in testis. Moreover, WD40 genes showed distinct expression patterns during embryonic and postnatal testis development in mice. Finally, different germ cell populations of testis showed specific patterns of WD40 genes expression. Predicted gene ontology analyses revealed more than 80% of these proteins are implicated in cellular, metabolic, biological regulation and cell localization processes.ConclusionsWe have identified large number of WD40 family genes that are highly or specifically expressed in the testes of mouse and human. Moreover, WD40 genes have distinct expression patterns during embryonic and postnatal development of the testis in mice. Further, different germ cell populations within the testis showed specific patterns of WD40 genes expression. These results provide foundation for further research towards understanding the functional genomics and molecular mechanisms of mammalian testis development and spermatogenesis.

Project description:Pancreatic cancer (PC) is the fourth leading cause of cancer death with an overall 5-year survival rate of < 5%, a statistic that has changed little in almost 50 years. A deeper understanding of the underlying molecular pathophysiology is expected to advance the urgent need to develop novel therapeutic and early detection strategies for this disease. Genomic characterisation of PC has previously relied on targeted PCR based exome sequencing of small cohorts of mixed primary and metastatic lesions propagated as xenografts or cell lines (Jones et al, Science 321:1801-1806), leaving the true mutational spectrum of the clinical disease largely unresolved. Here we use exome sequencing (https://www.ebi.ac.uk/ega/studies/EGAS00001000154) and copy number analysis (not submitted) to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (Stage I and II) pancreatic adenocarcinoma. Detailed analysis of 99 informative tumours identified 1982 non-silent mutations and 1628 significant CNV events, and defined 439 significantly mutated genes based on stringent Significant Mutated Gene or GISTIC analysis. Integration with functional data from in vitro shRNA and in vivo Sleeping Beauty-mediated somatic mutagenesis screens provided supportive evidence for 184 of these as candidate driver mutations. Pathway based analysis recapitulated clustering of mutations in core signalling pathways in PC, and identified multiple new components in each, particularly in DNA damage repair mechanisms (ATM, TOP2A, TLM, RPA1). We also identified frequent somatic aberrations in genes involved in novel mechanisms including chromatin modification (SWI/SNF complex members, SETD2, EPC1), and axon guidance (Semaphorin, Slit, Netrin and Ephrin signalling), extending the number of core perturbed pathways in PC. Aberrant expression of axon guidance genes co- segregated with poor patient survival, and in animal models was associated with disease development and progression, further implicating perturbation of the axon guidance pathway as a novel mechanism important in PC. This dataset includes gene expression data from 90 primary tumour samples, 88 of which were used in this manuscript for survival analysis. Much of this data is also available through the International Cancer Genome Consortium (ICGC) Data Portal (http://dcc/icgc.org), under the project code: "Pancreatic Cancer (QCMG, AU)". Access to the strictly restricted clinical data must be made through the ICGC Data Access Compliance Office (http://www.icgc.org/daco). This dataset contains expression array data from 90 primary pancreatic ductal adenocarcinoma samples. One sample is present with two biological replicates, all others have 1 biological replicate.

Dataset Information

Raf1 Is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification.

Publications

Raf1 Is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification.

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