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MIWI2 and MILI Have Differential Effects on piRNA Biogenesis and DNA Methylation

View Article: PubMed Central - PubMed

ABSTRACT

In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use Piwi-interacting RNA (piRNA) guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here, we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2 deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 is, suggesting that the proteins have independent roles in establishing DNA methylation patterns.

No MeSH data available.


Related in: MedlinePlus

The Effect of Miwi2 Knockout on piRNA Populations(A) The piRNA levels of young LINE1 families are decreased upon Miwi2 KO in prospermatogonia. The heatmap shows expression of piRNA targeting selected TE families in E16.5 prospermatogonia (gray) and the change in piRNA abundance (“total”) in Miwi2 mutants relative to Miwi2 Het mice. In addition, the heatmap shows the effect of Miwi2 deficiency on the fraction of antisense sequences (“as”) and fraction of secondary piRNA (“sec”, defined as reads that have adenine at position 10 and do not have uridine residue at position one). The differences between KOand Het mice are shown as log2 of ratios of values. Several L1 families show ∼2-fold decrease in piRNA abundance; however, the fractions of antisense and secondary piRNA from these families do not differ significantly when data from Miwi2-KO and Het mice are compared.(B) The distribution of piRNAs along L1-A body in E16.5 prospermatogonia. miRNA-normalized piRNA density shows that Miwi2 deficiency causes a reduction of both sense and antisense piRNA mapping to L1 consensus in E16.5 prospermatogonia. Separately shown are piRNA profiles on 5′ monomer sequences of two L1 families, L1-A and L1-T. Each bar represents a 5′ end of a piRNA mapping to either sense or antisense strand of the element. Arrows indicate the 5′ ends of sense and antisense ping-pong partners (piRNA on two opposite strands those 5′ ends are separated by 10 nt).(C) The effect of Miwi2 deficiency on the ping-pong piRNA biogenesis in E16.5 prospermatogonia. The E16.5 piRNA mapped to L1 and IAPEz consensuses with up to three mismatches were analyzed to find the signature of the ping-pong processing: complementary piRNA reads those 5′ ends are separated by 10 nt. The graph shows fraction of reads (y axis) whose 5′ ends overlap by a certain number of nucleotides (xaxis). The prominent peak at 10 nt that indicates ping-pong biogenesis is not significantly affected by Miwi2 deficiency.(D) The levels of L1 piRNAs are increased in Miwi2 mutants in P10 spermatogonia. The analysis and heatmap representation of piRNA at P10 spermatogonia are as described in (A). At P10, Miwi2 deficiency causes increase in total abundance of L1 piRNA as well as increase in the fraction of sense (decrease in antisense) piRNA relative to control.(E) The distribution of piRNAs along L1-Abody in P10 spermatogonia. miRNA-normalized piRNA densities show that Miwi2 deficiency causes increases in the levels of piRNA mapping to L1 consensus in P10 spermatogonia. More piRNAs are derived from 5′ end of L1-A in Miwi2-KO mice than in Het controls. Each bar on L1-A 5′ monomer represents a 5′ end of a piRNA mapping to either sense or antisense strand of the element. Arrows point to the 5′ ends of ping-pong partners, evident only in Mwi2-KO animals.(F) The effect of Miwi2 mutation on the ping-pong piRNA biogenesis in P10 spermatogonia. The analysis was performed as described in (C). The signature of the ping-pong processing, the fraction of piRNA pairs that overlap by 10nt, is increased in Mwi2KO mice relative to controls for L1-A, but notfortheIAPEz element.See also Figure S2.
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Figure 2: The Effect of Miwi2 Knockout on piRNA Populations(A) The piRNA levels of young LINE1 families are decreased upon Miwi2 KO in prospermatogonia. The heatmap shows expression of piRNA targeting selected TE families in E16.5 prospermatogonia (gray) and the change in piRNA abundance (“total”) in Miwi2 mutants relative to Miwi2 Het mice. In addition, the heatmap shows the effect of Miwi2 deficiency on the fraction of antisense sequences (“as”) and fraction of secondary piRNA (“sec”, defined as reads that have adenine at position 10 and do not have uridine residue at position one). The differences between KOand Het mice are shown as log2 of ratios of values. Several L1 families show ∼2-fold decrease in piRNA abundance; however, the fractions of antisense and secondary piRNA from these families do not differ significantly when data from Miwi2-KO and Het mice are compared.(B) The distribution of piRNAs along L1-A body in E16.5 prospermatogonia. miRNA-normalized piRNA density shows that Miwi2 deficiency causes a reduction of both sense and antisense piRNA mapping to L1 consensus in E16.5 prospermatogonia. Separately shown are piRNA profiles on 5′ monomer sequences of two L1 families, L1-A and L1-T. Each bar represents a 5′ end of a piRNA mapping to either sense or antisense strand of the element. Arrows indicate the 5′ ends of sense and antisense ping-pong partners (piRNA on two opposite strands those 5′ ends are separated by 10 nt).(C) The effect of Miwi2 deficiency on the ping-pong piRNA biogenesis in E16.5 prospermatogonia. The E16.5 piRNA mapped to L1 and IAPEz consensuses with up to three mismatches were analyzed to find the signature of the ping-pong processing: complementary piRNA reads those 5′ ends are separated by 10 nt. The graph shows fraction of reads (y axis) whose 5′ ends overlap by a certain number of nucleotides (xaxis). The prominent peak at 10 nt that indicates ping-pong biogenesis is not significantly affected by Miwi2 deficiency.(D) The levels of L1 piRNAs are increased in Miwi2 mutants in P10 spermatogonia. The analysis and heatmap representation of piRNA at P10 spermatogonia are as described in (A). At P10, Miwi2 deficiency causes increase in total abundance of L1 piRNA as well as increase in the fraction of sense (decrease in antisense) piRNA relative to control.(E) The distribution of piRNAs along L1-Abody in P10 spermatogonia. miRNA-normalized piRNA densities show that Miwi2 deficiency causes increases in the levels of piRNA mapping to L1 consensus in P10 spermatogonia. More piRNAs are derived from 5′ end of L1-A in Miwi2-KO mice than in Het controls. Each bar on L1-A 5′ monomer represents a 5′ end of a piRNA mapping to either sense or antisense strand of the element. Arrows point to the 5′ ends of ping-pong partners, evident only in Mwi2-KO animals.(F) The effect of Miwi2 mutation on the ping-pong piRNA biogenesis in P10 spermatogonia. The analysis was performed as described in (C). The signature of the ping-pong processing, the fraction of piRNA pairs that overlap by 10nt, is increased in Mwi2KO mice relative to controls for L1-A, but notfortheIAPEz element.See also Figure S2.

Mentions: Though total levels of piRNA were not significantly affected by Miwi2 deficiency, further analysis showed a pronounced effect of Miwi2 KO on piRNAs specific for certain TE families. PiRNAs against LINE1 families, such as L1-A, L1-T, and L1-Gf, were reduced approximately 2-fold (Figure 2A). The effect of Miwi2 on piRNA levels was specific for a few LINE1 families, as levels of piRNAs matching IAPEz and other ERV families were not decreased in Miwi2 mutant mice (Figures 2A and S2).For affected L1 families, levels of piRNAs distributed throughout the body of the transposable element were reduced in the Miwi2 KO mice compared to levels in wild-type controls (Figure 2B). The reductions in piRNAs targeting L1-A and L1-T 5′ monomers, which are repeated several times at the 5′ end of LINE and function as a promoter, were also significant (Figure 2B).


MIWI2 and MILI Have Differential Effects on piRNA Biogenesis and DNA Methylation
The Effect of Miwi2 Knockout on piRNA Populations(A) The piRNA levels of young LINE1 families are decreased upon Miwi2 KO in prospermatogonia. The heatmap shows expression of piRNA targeting selected TE families in E16.5 prospermatogonia (gray) and the change in piRNA abundance (“total”) in Miwi2 mutants relative to Miwi2 Het mice. In addition, the heatmap shows the effect of Miwi2 deficiency on the fraction of antisense sequences (“as”) and fraction of secondary piRNA (“sec”, defined as reads that have adenine at position 10 and do not have uridine residue at position one). The differences between KOand Het mice are shown as log2 of ratios of values. Several L1 families show ∼2-fold decrease in piRNA abundance; however, the fractions of antisense and secondary piRNA from these families do not differ significantly when data from Miwi2-KO and Het mice are compared.(B) The distribution of piRNAs along L1-A body in E16.5 prospermatogonia. miRNA-normalized piRNA density shows that Miwi2 deficiency causes a reduction of both sense and antisense piRNA mapping to L1 consensus in E16.5 prospermatogonia. Separately shown are piRNA profiles on 5′ monomer sequences of two L1 families, L1-A and L1-T. Each bar represents a 5′ end of a piRNA mapping to either sense or antisense strand of the element. Arrows indicate the 5′ ends of sense and antisense ping-pong partners (piRNA on two opposite strands those 5′ ends are separated by 10 nt).(C) The effect of Miwi2 deficiency on the ping-pong piRNA biogenesis in E16.5 prospermatogonia. The E16.5 piRNA mapped to L1 and IAPEz consensuses with up to three mismatches were analyzed to find the signature of the ping-pong processing: complementary piRNA reads those 5′ ends are separated by 10 nt. The graph shows fraction of reads (y axis) whose 5′ ends overlap by a certain number of nucleotides (xaxis). The prominent peak at 10 nt that indicates ping-pong biogenesis is not significantly affected by Miwi2 deficiency.(D) The levels of L1 piRNAs are increased in Miwi2 mutants in P10 spermatogonia. The analysis and heatmap representation of piRNA at P10 spermatogonia are as described in (A). At P10, Miwi2 deficiency causes increase in total abundance of L1 piRNA as well as increase in the fraction of sense (decrease in antisense) piRNA relative to control.(E) The distribution of piRNAs along L1-Abody in P10 spermatogonia. miRNA-normalized piRNA densities show that Miwi2 deficiency causes increases in the levels of piRNA mapping to L1 consensus in P10 spermatogonia. More piRNAs are derived from 5′ end of L1-A in Miwi2-KO mice than in Het controls. Each bar on L1-A 5′ monomer represents a 5′ end of a piRNA mapping to either sense or antisense strand of the element. Arrows point to the 5′ ends of ping-pong partners, evident only in Mwi2-KO animals.(F) The effect of Miwi2 mutation on the ping-pong piRNA biogenesis in P10 spermatogonia. The analysis was performed as described in (C). The signature of the ping-pong processing, the fraction of piRNA pairs that overlap by 10nt, is increased in Mwi2KO mice relative to controls for L1-A, but notfortheIAPEz element.See also Figure S2.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: The Effect of Miwi2 Knockout on piRNA Populations(A) The piRNA levels of young LINE1 families are decreased upon Miwi2 KO in prospermatogonia. The heatmap shows expression of piRNA targeting selected TE families in E16.5 prospermatogonia (gray) and the change in piRNA abundance (“total”) in Miwi2 mutants relative to Miwi2 Het mice. In addition, the heatmap shows the effect of Miwi2 deficiency on the fraction of antisense sequences (“as”) and fraction of secondary piRNA (“sec”, defined as reads that have adenine at position 10 and do not have uridine residue at position one). The differences between KOand Het mice are shown as log2 of ratios of values. Several L1 families show ∼2-fold decrease in piRNA abundance; however, the fractions of antisense and secondary piRNA from these families do not differ significantly when data from Miwi2-KO and Het mice are compared.(B) The distribution of piRNAs along L1-A body in E16.5 prospermatogonia. miRNA-normalized piRNA density shows that Miwi2 deficiency causes a reduction of both sense and antisense piRNA mapping to L1 consensus in E16.5 prospermatogonia. Separately shown are piRNA profiles on 5′ monomer sequences of two L1 families, L1-A and L1-T. Each bar represents a 5′ end of a piRNA mapping to either sense or antisense strand of the element. Arrows indicate the 5′ ends of sense and antisense ping-pong partners (piRNA on two opposite strands those 5′ ends are separated by 10 nt).(C) The effect of Miwi2 deficiency on the ping-pong piRNA biogenesis in E16.5 prospermatogonia. The E16.5 piRNA mapped to L1 and IAPEz consensuses with up to three mismatches were analyzed to find the signature of the ping-pong processing: complementary piRNA reads those 5′ ends are separated by 10 nt. The graph shows fraction of reads (y axis) whose 5′ ends overlap by a certain number of nucleotides (xaxis). The prominent peak at 10 nt that indicates ping-pong biogenesis is not significantly affected by Miwi2 deficiency.(D) The levels of L1 piRNAs are increased in Miwi2 mutants in P10 spermatogonia. The analysis and heatmap representation of piRNA at P10 spermatogonia are as described in (A). At P10, Miwi2 deficiency causes increase in total abundance of L1 piRNA as well as increase in the fraction of sense (decrease in antisense) piRNA relative to control.(E) The distribution of piRNAs along L1-Abody in P10 spermatogonia. miRNA-normalized piRNA densities show that Miwi2 deficiency causes increases in the levels of piRNA mapping to L1 consensus in P10 spermatogonia. More piRNAs are derived from 5′ end of L1-A in Miwi2-KO mice than in Het controls. Each bar on L1-A 5′ monomer represents a 5′ end of a piRNA mapping to either sense or antisense strand of the element. Arrows point to the 5′ ends of ping-pong partners, evident only in Mwi2-KO animals.(F) The effect of Miwi2 mutation on the ping-pong piRNA biogenesis in P10 spermatogonia. The analysis was performed as described in (C). The signature of the ping-pong processing, the fraction of piRNA pairs that overlap by 10nt, is increased in Mwi2KO mice relative to controls for L1-A, but notfortheIAPEz element.See also Figure S2.
Mentions: Though total levels of piRNA were not significantly affected by Miwi2 deficiency, further analysis showed a pronounced effect of Miwi2 KO on piRNAs specific for certain TE families. PiRNAs against LINE1 families, such as L1-A, L1-T, and L1-Gf, were reduced approximately 2-fold (Figure 2A). The effect of Miwi2 on piRNA levels was specific for a few LINE1 families, as levels of piRNAs matching IAPEz and other ERV families were not decreased in Miwi2 mutant mice (Figures 2A and S2).For affected L1 families, levels of piRNAs distributed throughout the body of the transposable element were reduced in the Miwi2 KO mice compared to levels in wild-type controls (Figure 2B). The reductions in piRNAs targeting L1-A and L1-T 5′ monomers, which are repeated several times at the 5′ end of LINE and function as a promoter, were also significant (Figure 2B).

View Article: PubMed Central - PubMed

ABSTRACT

In developing male germ cells, prospermatogonia, two Piwi proteins, MILI and MIWI2, use Piwi-interacting RNA (piRNA) guides to repress transposable element (TE) expression and ensure genome stability and proper gametogenesis. In addition to their roles in post-transcriptional TE repression, both proteins are required for DNA methylation of TE sequences. Here, we analyzed the effect of Miwi2 deficiency on piRNA biogenesis and transposon repression. Miwi2 deficiency had only a minor impact on piRNA biogenesis; however, the piRNA profile of Miwi2-knockout mice indicated overexpression of several LINE1 TE families that led to activation of the ping-pong piRNA cycle. Furthermore, we found that MILI and MIWI2 have distinct functions in TE repression in the nucleus. MILI is responsible for DNA methylation of a larger subset of TE families than MIWI2 is, suggesting that the proteins have independent roles in establishing DNA methylation patterns.

No MeSH data available.


Related in: MedlinePlus