Hen1 is required for oocyte development and piRNA stability in zebrafish

Piwi-interacting RNAs (piRNAs) are germ line-specific small RNA molecules that have a function in genome defence and germ cell development. They associate with a specific class of Argonaute proteins, named Piwi, and function through an RNA interference-like mechanism. piRNAs carry a 2′-O-methyl modification at their 3′ end, which is added by the Hen1 enzyme. We show that zebrafish hen1 is specifically expressed in germ cells and is essential for maintaining a female germ line, whereas it is dispensable in the testis. Hen1 protein localizes to nuage through its C-terminal domain, but is not required for nuage formation. In hen1 mutant testes, piRNAs become uridylated and adenylated. Uridylation frequency is highest on retro-transposon-derived piRNAs and is accompanied by decreased piRNA levels and mild derepression of transposon transcripts. Altogether, our data suggest the existence of a uridylation-mediated 3′–5′ exonuclease activity acting on piRNAs in zebrafish germ cells, which is counteracted by nuage-bound Hen1 protein. This system discriminates between piRNA targets and is required for ovary development and fully efficient transposon silencing

PID-1 is a novel factor that operates during 21U-RNA biogenesis in Caenorhabditis elegans

The Piwi-piRNA pathway represents a small RNA-based mechanism responsible for the recognition and silencing of invading DNA. Biogenesis of piRNAs (21U-RNAs) is poorly understood. In Caenorhabditis elegans, the piRNA-binding Argonaute protein PRG-1 is the only known player acting downstream from precursor transcription. From a screen aimed at the isolation of piRNA-induced silencing-defective (Pid) mutations, we identified, among known Piwi pathway components, PID-1 as a novel player. PID-1 is a mostly cytoplasmic, germline-specific factor essential for 21U-RNA biogenesis, affecting an early step in the processing or transport of 21U precursor transcripts. We also show that maternal 21U-RNAs are essential to initiate silencing

Enhancers reside in a unique epigenetic environment during early zebrafish development

Background: Enhancers, not promoters, are the most dynamic in their DNA methylation status throughout development and differentiation. Generally speaking, enhancers that are primed to or actually drive gene expression are characterized by relatively low levels of DNA methylation (hypo-methylation), while inactive enhancers display hyper-methylation of the underlying DNA. The direct functional significance of the DNA methylation state of enhancers is, however, unclear for most loci. Results: In contrast to conventional epigenetic interactions at enhancers, we find that DNA methylation status and enhancer activity during early zebrafish development display very unusual correlation characteristics: hypo-methylation is a unique feature of primed enhancers whereas active enhancers are generally hyper-methylated. The hypo-methylated enhancers that we identify (hypo-enhancers) are enriched close to important transcription factors that act later in development. Interestingly, hypo-enhancers are de-methylated shortly before the midblastula transition and reside in a unique epigenetic environment. Finally, we demonstrate that hypo-enhancers do become active at later developmental stages and that they are physically associated with the transcriptional start site of target genes, irrespective of target gene activity. Conclusions: We demonstrate that early development in zebrafish embodies a time window characterized by non-canonical DNA methylation-enhancer relationships, including global DNA hypo-methylation of inactive enhancers and DNA hyper-methylation of active enhancers

Enhancers reside in a unique epigenetic environment during early zebrafish development

BACKGROUND: Enhancers, not promoters, are the most dynamic in their DNA methylation status throughout development and differentiation. Generally speaking, enhancers that are primed to or actually drive gene expression are characterized by relatively low levels of DNA methylation (hypo-methylation), while inactive enhancers display hyper-methylation of the underlying DNA. The direct functional significance of the DNA methylation state of enhancers is, however, unclear for most loci. RESULTS: In contrast to conventional epigenetic interactions at enhancers, we find that DNA methylation status and enhancer activity during early zebrafish development display very unusual correlation characteristics: hypo-methylation is a unique feature of primed enhancers whereas active enhancers are generally hyper-methylated. The hypo-methylated enhancers that we identify (hypo-enhancers) are enriched close to important transcription factors that act later in development. Interestingly, hypo-enhancers are de-methylated shortly before the midblastula transition and reside in a unique epigenetic environment. Finally, we demonstrate that hypo-enhancers do become active at later developmental stages and that they are physically associated with the transcriptional start site of target genes, irrespective of target gene activity. CONCLUSIONS: We demonstrate that early development in zebrafish embodies a time window characterized by non-canonical DNA methylation-enhancer relationships, including global DNA hypo-methylation of inactive enhancers and DNA hyper-methylation of active enhancers

HENN-1 is the <i>C. elegans</i> homolog of Hen1.

<p>(A) Left panel: protein gel stained with PageBlue shows purified GST, GST-HENN-1 and GST-HENN-1(D151N) proteins used to perform methyltransferase assays as shown in the right panel and B. Right panel: <i>in vitro</i> methyltransferase activity assay. RNA oligos were incubated with indicated proteins and 14C-labelled SAM. Reaction products were run on a 12% acryl-amide gel. (B) <i>In vitro</i> methyltransferase assay using different RNA substrates each differing in the identity of the most 3′ nucleotide. (C) Northern blot analysis using RNA from wild-type, <i>henn-1(pk2452)</i>, <i>henn-1(pk2295)</i> and <i>henn-1(pk2295); pgl-3:HENN-1::GFP</i> animals. Blots were probed for 21UR1 and 26G species siR26-263. Probing for <i>let-7</i> serves as loading and as oxidation-β-elimination control. (D) Response of wild type (N2), <i>henn-1(pk2295)</i> and <i>henn-1(pk2295); pgl-3:henn-1:GFP</i> to <i>dpy-13</i> RNAi. <i>Henn-1(pk2295)</i> sensitivity is significantly higher than both controls (two-tailed t-test, n = 5: p<0.05 for both). (E) Response of wild type (N2), <i>henn-1(pk2295)</i> and <i>henn-1(pk2295); pgl-3:HENN-1::GFP</i> to <i>pos-1</i> RNAi, delivered at three different dosages: undiluted (100%), diluted one to one (50%) and diluted one to four (25%). At 50% <i>pos-1</i> RNAi, <i>henn-1(pk2295); pgl-3:HENN-1::GFP</i> animals display significant rescue (p = 0.01) of the <i>henn-1(pk2295)</i> RNAi defect (p<0.0005). The p-values at 25% <i>pos-1</i> RNAi are p = 0.04 for both the <i>henn-1(pk2295)</i> RNAi defect and the rescue. P-values were calculated with a two-tailed t-test, n = 10.</p

<i>henn-1</i> affects 21UR1 sensor activity.

<p>(A) Activity of a transgene expressing GFP (21UR1 sensor), silenced by 21U species 21UR1 in wild-type, <i>prg-1(pk2298)</i> and <i>henn-1(pk2295)</i> mutant backgrounds. The gonads are outlined with a white dashed line. (B) Northern blot analysis of 21UR1 in young adult animals of the indicated genotypes. “Sensor” refers to the 21UR1 sensor also shown in panel A. Signal intensities are related to <i>let-7</i>, and the 21UR1:<i>let-7</i> ratio in N2 is set at one. The 21-mer and 20-mer signals of the 21UR1 probe have also been quantified separately. The signal intensity of the 20-mer relative to the total 21UR1 signal is presented. Repetition of this blot with two independent biological samples has shown that the apparent differences are not reproducible. The 22G-sensor blot shows the signals obtained after hybridizing probes homologous to the 21UR1 sensor.</p

Global effects of <i>henn-1</i> on 21U RNAs.

<p>(A) Bar diagram displaying the different annotated small RNA reads obtained after deep-sequencing. Reads from structural RNAs were removed before analysis. (B) The expression level of 21U RNAs in wild-type and <i>henn-1</i> mutant backgrounds. Total 21U reads are normalized to total miRNA reads. The differences between wild-type and <i>henn-1</i> mutant samples are significant (Chi-squared test; p<10⁻¹⁰). (C) Length distribution plot of 21U RNAs. (D) Scatter plot displaying the fraction of 20-mer species of individual 21U RNA loci that were represented by at least 250 raw reads (20+21-mers) in each of the libraries used for this analysis (<i>henn-1(pk2452)</i> and <i>henn-1(pk2295)</i>). (E) Scatter plot displaying individual 21U loci represented by at least 250 raw reads (20+21-mers) in each of the libraries used for this analysis (wild-type and <i>henn-1(pk2295)</i>). X-axis: fold loss of reads in the <i>henn-1(pk2295)</i> background relative to wild-type. Y-axis: fold increase of 20-mer species in the <i>henn-1(pk2295)</i> background relative to wild-type. (F) Bar diagram displaying the frequencies of non-templated base additions found on 21U reads, as a percentage of the total 21U read count.</p

Effects of <i>henn-1</i> 22G and 26G RNAs.

<p>(A) Length distribution plots of ‘siRNA’ category, containing both 22G and 26G RNAs, in diverse libraries. (B) Bar diagram displaying the frequencies of non-templated base additions found on 26G reads, as a percentage of the total 26G read count. P<0.0001 for <i>henn-1(pk2452)</i> and <i>henn-1(pk2295)</i> relative to wild-type (Chi-squared test). (C) Ratio of ALG-3/4 and ERGO-1 bound 26G RNAs as derived by previously described annotation (see main text), in diverse libraries. P-values of all differences <0.001 (Chi-squared test). (D) The 22G counts, in rpm, for all genes in total, ERGO-1, ALG-3/4 and CSR-1 target genes. 22G count for ‘Total’ was divided by 100 for better visualization.</p