8 research outputs found
Correction: RNAi-Dependent and Independent Control of LINE1 Accumulation and Mobility in Mouse Embryonic Stem Cells
<p>Correction: RNAi-Dependent and Independent Control of LINE1 Accumulation and Mobility in Mouse Embryonic Stem Cells</p
RNAi-Dependent and Independent Control of LINE1 Accumulation and Mobility in Mouse Embryonic Stem Cells
<div><p>In most mouse tissues, long-interspersed elements-1 (L1s) are silenced <i>via</i> methylation of their 5β²-untranslated regions (5β²-UTR). A gradual loss-of-methylation in pre-implantation embryos coincides with L1 retrotransposition in blastocysts, generating potentially harmful mutations. Here, we show that Dicer- and Ago2-dependent RNAi restricts L1 accumulation and retrotransposition in undifferentiated mouse embryonic stem cells (mESCs), derived from blastocysts. RNAi correlates with production of Dicer-dependent 22-nt small RNAs mapping to overlapping sense/antisense transcripts produced from the L1 5β²-UTR. However, RNA-surveillance pathways simultaneously degrade these transcripts and, consequently, confound the anti-L1 RNAi response. In <i>Dicer<sup>β/β</sup></i> mESC complementation experiments involving ectopic Dicer expression, L1 silencing was rescued in cells in which microRNAs remained strongly depleted. Furthermore, these cells proliferated and differentiated normally, unlike their non-complemented counterparts. These results shed new light on L1 biology, uncover defensive, in addition to regulatory roles for RNAi, and raise questions on the differentiation defects of <i>Dicer<sup>β/β</sup></i> mESCs.</p></div
L1 elements are up-regulated in <i>Dcr<sup>β/β</sup></i> mESCs.
<p>A. Sequencing reads, from WT mESCs, within the 19β32-nt range were aligned against the mouse genome (version mm9). The distinct sequences coverage (Reads per Million (RPM) normalized) is depicted for the full length L1Md_Tf L1 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003791#pgen.1003791-Chen1" target="_blank">[24]</a>. B. PCR-based genotyping of the <i>Dcr</i> deletion 24 h and 48 h post-tamoxifen (Tam) treatment. C. qRT-PCR analysis of miR-295 levels in the tamoxifen treated mESCs, as depicted in (B). D. L1_ORF2 mRNA accumulation detected by qRT-PCR before and after <i>Dcr</i> deletion. E. Western analysis of L1_ORF1 protein levels before and after <i>Dcr</i> deletion; CM: Coomassie staining of total protein. F. Semi-quantitative RT-PCR analysis of RNA levels from a single L1-Tf copy on chromosome 17 before and after <i>Dcr</i> deletion.</p
h<i>Dcr-c</i>omplemented <i>Dcr</i><sup>β/β</sup> ESCs differentiate normally despite accumulating 5β7% total miRNAs compared to WT.
<p>A. Visualization of Embryoid bodies from <i>Dcr</i><sup>Flx/Flx</sup>, <i>Dcr</i><sup>β/β</sup> and h<i>Dcr</i>-complemented <i>Dcr</i><sup>β/β</sup> mESCs after 1, 6 and 10 days of differentiation. B. Western analysis of OCT4 and endogenous AGO2 protein levels in the cells depicted in (A) before (d0) and after 10 days of differentiation (d10); CM: Coomassie staining of total protein. C. Same as (B) but in WT, <i>Xrn2</i>_KD and <i>Dgcr8</i>_KO mESCs.</p
L1 elements retrotranspose in <i>Dcr<sup>β/β</sup></i> mESCs.
<p>A. qPCR-based copy-number analysis of L1_Tf elements in <i>Dcr</i><sup>Flx/Flx</sup> P10 (5 replicates), P30 (2 replicates) and <i>Dcr</i><sup>β/β</sup> P30 (5 replicates) mESCs. **: p-value<0.01. B. qRT-PCR-based analysis of eGFP mRNA levels in WT and <i>Dcr</i><sup>β/β</sup> mESCs stably transformed with a WT human eGFP-tagged L1 transgene, 24 h post-transfection and 6 passages (P6) after puromycin selection. C. Integrated eGFP as a diagnostic of retrotransposition detected by PCR in the genomic DNA of <i>Dcr</i><sup>β/β</sup> mESCs carrying the human eGFP-tagged L1 transgene after 6 passages post-puromycin treatment.</p
Rescue of L1 silencing in <i>hDcr</i>-complemented <i>Dcr</i><sup>β/β</sup> mESCs.
<p>A. Western analysis of endogenous AGO1, AGO2 and L1_ORF1 protein levels in <i>Dcr</i><sup>Flx/Flx</sup>, <i>Dcr</i><sup>β/β</sup> and <i>Dgcr8</i>_KO ESCs; CM: Coomassie staining of total protein. B. Western analysis of endogenous AGO2 and L1_ORF1 protein levels in <i>Dcr</i><sup>Flx/Flx</sup>, <i>Dc</i>r<sup>β/β</sup> mESCs and one representative stable line of h<i>Dcr</i>-complemented <i>Dcr</i><sup>β/β</sup> mESC; CM: Coomassie staining of total protein. CβE. qRT-PCR analysis of L1_ORF2 mRNA levels (C), miR-295 and miR-16 levels (D), and Hmga2 and Btg2 mRNA levels (established targets of mmu-miR-196a and mmu-let-7a and mmu-miR-132, respectively) in the various cell lines depicted in (E).</p
L1 sRNAs are partially produced by DCR and 22-nt L1-sRNAs loaded into AGO2.
<p>A. Size distribution of sequencing reads mapping to L1_Tf elements in <i>Dcr</i><sup>β/β</sup> compared to WT mESCs. Note the deficit in 21β23-nt sRNAs in the former, and their enrichment in immunoprecipitated FHA-hAgo2, used in (B). B. Sequence coverages of L1_Tf elements from WT, <i>Dcr</i><sup>β/β</sup> and immunoprecipitated E14-FHA-hAgo2 mESCs, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003791#pgen-1003791-g001" target="_blank">Figure 1A</a>. * this peak is an artefact found in all libraries sequenced. C. qRT-PCR analysis of the most abundant sense and antisense L1_5β²-UTR-derived sRNAs and of two mESC miRNAs in immunoprecipitates of endogenous AGO1 and AGO2. Lower panel: control Western analysis of endogenous AGO1 and AGO2 levels after immunoprecipitation; CM: Coomassie staining of total protein. D. Snapshot representation of perfect, 22-nt-long sRNA duplexes with 2-nt 3β² overhangs (in red) mapped on the 5β²-UTR of a single L1-Tf element located on chromosome X. E. Relative proportions of all L1-Tf-derived sequences forming perfect duplexes with 2-nt overhangs in WT versus <i>Dcr<sup>β/β</sup></i> mESCs (left panel) and their enrichment in AGO2 immunoprecipitates (right panel). The numbers of 22-nt distinct sequences were normalized by the L1-Tf family coverage of RPM and expressed as proportion compare to WT.</p
Additional file 1: of HiC-Pro: an optimized and flexible pipeline for Hi-C data processing
The supplementary data file contains a description of the dataset used for this study as well as details about how the HiC-Pro, hiclib and HiCorrector software were used in practice. It also includes supplementary figures about the HiC-Pro results and output. (DOCX 1452 kb