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Gene expression profiling in Dis3l2-null and wild-type nephron progenitor cells

ABSTRACT: Purpose: Loss of DIS3L2 in humans is associated with congenital overgrowth in Perlman syndrome, as well as with the development of Wilms tumors. DIS3L2 is an exoribonuclease with a demonstrated preference for 3’-uridylated RNA substrates. However, its targets relevant to human disease are poorly understood. Our goal was to address transcriptomic changes in DIS3L2 deficient mouse nephron progenitor cells, a cell type of the developing kidney with demonstrated relevance to Wilms tumorigenesis. Methods: RNA-seq libraries were prepared using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina) and sequenced using 50 base pair single-end reads on an Illumia HiSeq2500 at the McDermott Center Next Generation Sequencing core at UT Southwestern. Samples were 2 biological replicates of wild-type and 3 of Dis3l2-null from embryos of the same litter. Results: Upregulated genes included replication-dependent histone mRNA and small non-coding Polymerase III transcripts, as expected from previous reports, as well as Igf2, a gene that has not previously been established to be regulated by DIS3L2 and whose upregulation is associated with overgrowth and Wilms tumor development. Conclusions: These data implicate Igf2 as a promising candidate for the overgrowth and Wilms tumorigenesis observed in DIS3L2-deficient disease states.

ORGANISM(S): Mus musculus

PROVIDER: GSE114673 | GEO | 2018/07/03

REPOSITORIES: GEO

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Project description:Mutations in the 3’-5’ exonuclease DIS3L2 are associated with Perlman syndrome and hypersusceptibility to Wilms’ tumorigenesis. Previously, we found that Dis3l2 specifically recognizes and degrades uridylated pre-let-7 microRNA. However, the widespread relevance of Dis3l2-mediated decay of uridylated substrates remains unknown. Here we applied an unbiased RNA immunoprecipitation strategy to identify Dis3l2 targets in mouse embryonic stem cells. The disease-associated long noncoding RNA (lncRNA) Rmrp, 7SL, as well as several other Pol III-transcribed noncoding RNAs (ncRNAs) were among the most highly enriched Dis3l2-bound RNAs. 3’-uridylated Rmrp, 7SL, and snRNA species were highly stabilized in the cytoplasm of Dis3l2-depleted cells. Deep sequencing analysis of Rmrp 3’ ends revealed extensive oligouridylation mainly on transcripts with imprecise ends. We implicate the TUTases Zcchc6/11 in the uridylation of these ncRNAs, and biochemical reconstitution assays demonstrate the sufficiency of TUTase-Dis3l2 for Rmrp decay. This establishes Dis3l2-Mediated Decay (DMD) as a quality control pathway that eliminates aberrant ncRNAs.obtaining over four billion bases of sequence from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of mouse embryonic stem cells, neural progenitor cells and embryonic fibroblasts. We find that lysine 4 and lysine 27 trimethylation effectively discriminates genes that are expressed, poised for expression, or stably repressed, and therefore reflect cell state and lineage potential. Lysine 36 trimethylation marks primary coding and non-coding transcripts, facilitating gene annotation. Trimethylation of lysine 9 and lysine 20 is detected at satellite, telomeric and active long-terminal repeats, and can spread into proximal unique sequences. Lysine 4 and lysine 9 trimethylation marks imprinting control regions. Finally, we show that chromatin state can be read in an allele-specific manner by using single nucleotide polymorphisms. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.

Project description:The exosome-independent exoribonuclease DIS3L2 is mutated in Perlman syndrome. Here we used extensive global transcriptomic and targeted biochemical analyses to identify novel DIS3L2 substrates in human cells. We show that DIS3L2 regulates pol II transcripts, comprising selected canonical and histone-coding mRNAs, and a novel FTL_short RNA from the ferritin mRNA 5' UTR. Importantly, DIS3L2 contributes to surveillance of pre-snRNAs during their cytoplasmic maturation. Among pol III transcripts, DIS3L2 particularly targets vault and Y RNAs and an Alu-like element BC200 RNA, but not Alu repeats, which are removed by exosome-associated DIS3. Using 3' RACE-Seq, we demonstrate that all novel DIS3L2 substrates are uridylated in vivo by TUT4/TUT7 poly(U) polymerases. Uridylation-dependent DIS3L2-mediated decay can be recapitulated in vitro, thus reinforcing the tight cooperation between DIS3L2 and TUTases. Together these results indicate that catalytically inactive DIS3L2, characteristic of Perlman syndrome, can lead to deregulation of its target RNAs to disturb transcriptome homeostasis. To investigate DIS3L2 functions genome-wide, total RNA samples were collected from model cell lines producing either WT or mut DIS3L2 three days after induction with doxycycline. The RNA samples were rRNA-depleted before preparation of strand-specific total RNA libraries according to the standard TruSeq (Illumina) protocol. TruSeq library preparation favours RNA molecules longer than 200 nt, and shorter transcripts are suboptimal for sequencing via this protocol. Thus, to obtain information about potential DIS3L2 RNA substrates with lengths between 20 and 220 nt, another RNA-Seq was carried out in parallel (with size selection through gel purification). The stable inducible HEK293 cell lines producing DIS3L2 variants were obtained using âpAL_01â and âpAL_02â plasmid constructs and the Flp-Inâ¢ T-RExâ¢ system according to the manufacturerâs guidelines. âpAL_01â and âpAL_02â plasmids are vectors for co-expression of recoded C-terminal FLAG-tagged DIS3L2 [wild type (WT) variant or its catalytic mutant counterpart (mut), respectively] and sh-miRNAs directed against endogenous DIS3L2 mRNA.

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Gene expression profiling in Dis3l2-null and wild-type nephron progenitor cells

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