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Small RNA expression from the human macrosatellite DXZ4.

Pohlers M, Calabrese JM, Magnuson T - G3 (Bethesda) (2014)

Bottom Line: DXZ4 was found to express a wide range of small RNAs potentially representing several classes of small RNAs.A subpopulation of these RNAs is bound by Argonaute.We hypothesize that the RNAs are involved in Argonaute-dependent methylation of DXZ4 DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, the Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, North Carolina 27599 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599.

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AGO associated with DXZ4 chromatin. (A) Schematic representation of a DXZ4 monomer showing fragments amplified to analyze coprecipitated DXZ4 DNA. Locations of RNAs and histone modifications were adopted from Chadwick (2008). (B) Chromatin immunoprecipitation (ChIP) in HEK293T cells using a pan-AGO antibody. Coprecipitated DNA was quantified by real-time polymerase chain reaction. Relative DXZ4 enrichment over host matched IgG was normalized to the repetitive 5S rDNA locus (n = 4–5). Double asterisks indicate significant enrichment (P < 0.05) calculated by using the t-test: DXZ4-C P-value = 0.029, DXZ4-D P-value = 0.006. (C) Alignment of DXZ4 matching small RNA reads including alignment to non-DXZ4 genomic loci with the same or higher stringency. Data obtained from merged high-throughput sequencing data from K562, HEK293, MCF10a, H1, and IMR-90 cell lines (n = 154).
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fig3: AGO associated with DXZ4 chromatin. (A) Schematic representation of a DXZ4 monomer showing fragments amplified to analyze coprecipitated DXZ4 DNA. Locations of RNAs and histone modifications were adopted from Chadwick (2008). (B) Chromatin immunoprecipitation (ChIP) in HEK293T cells using a pan-AGO antibody. Coprecipitated DNA was quantified by real-time polymerase chain reaction. Relative DXZ4 enrichment over host matched IgG was normalized to the repetitive 5S rDNA locus (n = 4–5). Double asterisks indicate significant enrichment (P < 0.05) calculated by using the t-test: DXZ4-C P-value = 0.029, DXZ4-D P-value = 0.006. (C) Alignment of DXZ4 matching small RNA reads including alignment to non-DXZ4 genomic loci with the same or higher stringency. Data obtained from merged high-throughput sequencing data from K562, HEK293, MCF10a, H1, and IMR-90 cell lines (n = 154).

Mentions: Small RNAs associate with Argonaute proteins to guide them to nascent transcripts in a sequence-specific manner (Grewal and Jia 2007). Based on our finding that several AGO proteins bound a substantial number of DXZ4 small RNAs (Figure 1E), we asked whether AGO directly associates with DXZ4. To address this question, we performed ChIP using a pan-AGO antibody specific for AGO-1 through AGO-4 in HEK293T cells (Nelson et al. 2007; Schwartz et al. 2008). Coprecipitated DNA was detected in immunoprecipitated chromatin by quantitative PCR and normalized relative to a species-matched IgG control. DXZ4 was analyzed using a set of PCR fragments dispersed across a DXZ4 monomer (Figure 3A). We used the 5S rDNA repeat cluster, which only showed a modest enrichment compared with IgG as a negative control to copy number-match the DXZ4 array and further normalized DXZ4 enrichments relative to it. DXZ4 amplification between base pairs 820 and 1600 of the monomer was significantly enriched relative to the IgG control (Figure 3B).


Small RNA expression from the human macrosatellite DXZ4.

Pohlers M, Calabrese JM, Magnuson T - G3 (Bethesda) (2014)

AGO associated with DXZ4 chromatin. (A) Schematic representation of a DXZ4 monomer showing fragments amplified to analyze coprecipitated DXZ4 DNA. Locations of RNAs and histone modifications were adopted from Chadwick (2008). (B) Chromatin immunoprecipitation (ChIP) in HEK293T cells using a pan-AGO antibody. Coprecipitated DNA was quantified by real-time polymerase chain reaction. Relative DXZ4 enrichment over host matched IgG was normalized to the repetitive 5S rDNA locus (n = 4–5). Double asterisks indicate significant enrichment (P < 0.05) calculated by using the t-test: DXZ4-C P-value = 0.029, DXZ4-D P-value = 0.006. (C) Alignment of DXZ4 matching small RNA reads including alignment to non-DXZ4 genomic loci with the same or higher stringency. Data obtained from merged high-throughput sequencing data from K562, HEK293, MCF10a, H1, and IMR-90 cell lines (n = 154).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199704&req=5

fig3: AGO associated with DXZ4 chromatin. (A) Schematic representation of a DXZ4 monomer showing fragments amplified to analyze coprecipitated DXZ4 DNA. Locations of RNAs and histone modifications were adopted from Chadwick (2008). (B) Chromatin immunoprecipitation (ChIP) in HEK293T cells using a pan-AGO antibody. Coprecipitated DNA was quantified by real-time polymerase chain reaction. Relative DXZ4 enrichment over host matched IgG was normalized to the repetitive 5S rDNA locus (n = 4–5). Double asterisks indicate significant enrichment (P < 0.05) calculated by using the t-test: DXZ4-C P-value = 0.029, DXZ4-D P-value = 0.006. (C) Alignment of DXZ4 matching small RNA reads including alignment to non-DXZ4 genomic loci with the same or higher stringency. Data obtained from merged high-throughput sequencing data from K562, HEK293, MCF10a, H1, and IMR-90 cell lines (n = 154).
Mentions: Small RNAs associate with Argonaute proteins to guide them to nascent transcripts in a sequence-specific manner (Grewal and Jia 2007). Based on our finding that several AGO proteins bound a substantial number of DXZ4 small RNAs (Figure 1E), we asked whether AGO directly associates with DXZ4. To address this question, we performed ChIP using a pan-AGO antibody specific for AGO-1 through AGO-4 in HEK293T cells (Nelson et al. 2007; Schwartz et al. 2008). Coprecipitated DNA was detected in immunoprecipitated chromatin by quantitative PCR and normalized relative to a species-matched IgG control. DXZ4 was analyzed using a set of PCR fragments dispersed across a DXZ4 monomer (Figure 3A). We used the 5S rDNA repeat cluster, which only showed a modest enrichment compared with IgG as a negative control to copy number-match the DXZ4 array and further normalized DXZ4 enrichments relative to it. DXZ4 amplification between base pairs 820 and 1600 of the monomer was significantly enriched relative to the IgG control (Figure 3B).

Bottom Line: DXZ4 was found to express a wide range of small RNAs potentially representing several classes of small RNAs.A subpopulation of these RNAs is bound by Argonaute.We hypothesize that the RNAs are involved in Argonaute-dependent methylation of DXZ4 DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, the Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, North Carolina 27599 Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599.

Show MeSH