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Human imprinted retrogenes exhibit non-canonical imprint chromatin signatures and reside in non-imprinted host genes.

Monk D, Arnaud P, Frost JM, Wood AJ, Cowley M, Martin-Trujillo A, Guillaumet-Adkins A, Iglesias Platas I, Camprubi C, Bourc'his D, Feil R, Moore GE, Oakey RJ - Nucleic Acids Res. (2011)

Bottom Line: Imprinted retrotransposed genes share a common genomic organization including a promoter-associated differentially methylated region (DMR) and a position within the intron of a multi-exonic 'host' gene.To address the mechanisms governing imprinted retrogene expression, histone modifications were assayed at the DMRs.Two human retrogenes showed monoallelic enrichment of active, but not of repressive marks suggesting a partial uncoupling of the relationship between DNA methylation and repressive histone methylation, possibly due to the smaller size and lower CpG density of these DMRs.

View Article: PubMed Central - PubMed

Affiliation: Imprinting and Cancer Group, Cancer Epigenetics and Biology Program, Bellvitge Institute for Biomedical Research (IDIBELL), L'Hospitalet de Llobregat, 08907, Barcelona, Spain. dmonk@iconcologia.net

ABSTRACT
Imprinted retrotransposed genes share a common genomic organization including a promoter-associated differentially methylated region (DMR) and a position within the intron of a multi-exonic 'host' gene. In the mouse, at least one transcript of the host gene is also subject to genomic imprinting. Human retrogene orthologues are imprinted and we reveal that human host genes are not imprinted. This coincides with genomic rearrangements that occurred during primate evolution, which increase the separation between the retrogene DMRs and the host genes. To address the mechanisms governing imprinted retrogene expression, histone modifications were assayed at the DMRs. For the mouse retrogenes, the active mark H3K4me2 was associated with the unmethylated paternal allele, while the methylated maternal allele was enriched in repressive marks including H3K9me3 and H4K20me3. Two human retrogenes showed monoallelic enrichment of active, but not of repressive marks suggesting a partial uncoupling of the relationship between DNA methylation and repressive histone methylation, possibly due to the smaller size and lower CpG density of these DMRs. Finally, we show that the genes immediately flanking the host genes in mouse and human are biallelically expressed in a range of tissues, suggesting that these loci are distinct from large imprinted clusters.

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(A) Methylation-immunoprecipitation was performed on placental DNA using anti 5mC antibody. The efficiency of IP was assessed by PCR specific for the methylated SERPIN-B5 promoter and the unmethylated UBE2B promoter. The precipitations were subsequent used to assess the methylation at the human MCTS2, INPP5F_V2 and NAP1L5 DMRs. (B) Using the same PCR primer combinations, allelic-ChIP was performed on human placental cell lines (for clarity, only ChIP-bound fractions are shown). (C) qPCR on ChIP-bound material from the TCL1 cell immunoprecipitations. Levels of precipitation are compared to the SNURF DMR (red line, equal to one).
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Figure 4: (A) Methylation-immunoprecipitation was performed on placental DNA using anti 5mC antibody. The efficiency of IP was assessed by PCR specific for the methylated SERPIN-B5 promoter and the unmethylated UBE2B promoter. The precipitations were subsequent used to assess the methylation at the human MCTS2, INPP5F_V2 and NAP1L5 DMRs. (B) Using the same PCR primer combinations, allelic-ChIP was performed on human placental cell lines (for clarity, only ChIP-bound fractions are shown). (C) qPCR on ChIP-bound material from the TCL1 cell immunoprecipitations. Levels of precipitation are compared to the SNURF DMR (red line, equal to one).

Mentions: Extensive genotyping of the human NAP1L5, MCTS2 and INPP5F_V2 DMRs revealed that SNPs in these regulatory regions are rare. However, we were able to identify heterozygous samples that allowed us to discriminate between alleles. The SNP rs2972011 is located ∼200 bp from the TSS of NAP1L5, whereas rs7907781 and rs1115713 are ∼600 bp and ∼50 bp from the TSS of INPP5F_V2 and MCTS2, respectively. To ensure that these SNPs mapped within the DMRs, we performed DNA methylation immunoprecipitation (meDIP) using antisera directed against 5-methylcytosine. This was due to the difficultly in amplifying bisulphite converted DNA in the vicinity of SNPs rs7907781 and rs1115713. For all three regions we observed monoallelic enrichment in heterozygous placental DNA samples, and where informative, the DNA methylation was detected on the maternal allele (Figure 4A). Using these same amplification conditions, we performed ChIP on native chromatin isolated from adult peripheral blood leukocytes and from two human placental cell lines, TCL1 and TCL2 (23) for the NAP1L5 and MCTS2 DMRs. Unfortunately, no heterozygous cell lines could be found that were informative for INPP5F_V2, despite genotyping of over 140 leukocyte samples and normal tissue cell lines. Similar to the mouse, we observe strong monoallelic enrichment for H3K4me2 at the NAP1L5 and MCTS2 DMRs, but since no parental DNA samples were available, allelic origin could not be assigned. Unexpectedly, we did not observe allelic precipitation for any of the repressive histone marks at these DMRs, despite strong allelic enrichment at the SNURF/SNRPN, H19 and MEST DMRs (Figure 4B; data not shown). To confirm that the histone modifications were present at the NAP1L5 and MCTS2 DMRs, we performed quantitative ChIP analysis on the placental cell line TCL1 (Figure 4C). The precipitation values obtained were normalized to those for the SNURF/SNRPN DMR, which revealed that H3K4me2 is more abundant at the MCTS2 and NAP1L5 promoters, whereas, the repressive histone modifications were precipitated several fold less. Interrogation of human histone maps [http://dir.nhlbi.nih.gov/papers/lmi/epigenomes/hgtcell.aspx, and (24)] confirmed the absence of significant enrichment for these repressive marks at 1–2 nucleosomes resolution (the approximate size of the MCTS2 and NAP1L5 DMRs), despite strong H3K4me2 enrichment. Together, these data suggest that these repressive marks are not present, or very low, in these two regions.Figure 4.


Human imprinted retrogenes exhibit non-canonical imprint chromatin signatures and reside in non-imprinted host genes.

Monk D, Arnaud P, Frost JM, Wood AJ, Cowley M, Martin-Trujillo A, Guillaumet-Adkins A, Iglesias Platas I, Camprubi C, Bourc'his D, Feil R, Moore GE, Oakey RJ - Nucleic Acids Res. (2011)

(A) Methylation-immunoprecipitation was performed on placental DNA using anti 5mC antibody. The efficiency of IP was assessed by PCR specific for the methylated SERPIN-B5 promoter and the unmethylated UBE2B promoter. The precipitations were subsequent used to assess the methylation at the human MCTS2, INPP5F_V2 and NAP1L5 DMRs. (B) Using the same PCR primer combinations, allelic-ChIP was performed on human placental cell lines (for clarity, only ChIP-bound fractions are shown). (C) qPCR on ChIP-bound material from the TCL1 cell immunoprecipitations. Levels of precipitation are compared to the SNURF DMR (red line, equal to one).
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Related In: Results  -  Collection

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Figure 4: (A) Methylation-immunoprecipitation was performed on placental DNA using anti 5mC antibody. The efficiency of IP was assessed by PCR specific for the methylated SERPIN-B5 promoter and the unmethylated UBE2B promoter. The precipitations were subsequent used to assess the methylation at the human MCTS2, INPP5F_V2 and NAP1L5 DMRs. (B) Using the same PCR primer combinations, allelic-ChIP was performed on human placental cell lines (for clarity, only ChIP-bound fractions are shown). (C) qPCR on ChIP-bound material from the TCL1 cell immunoprecipitations. Levels of precipitation are compared to the SNURF DMR (red line, equal to one).
Mentions: Extensive genotyping of the human NAP1L5, MCTS2 and INPP5F_V2 DMRs revealed that SNPs in these regulatory regions are rare. However, we were able to identify heterozygous samples that allowed us to discriminate between alleles. The SNP rs2972011 is located ∼200 bp from the TSS of NAP1L5, whereas rs7907781 and rs1115713 are ∼600 bp and ∼50 bp from the TSS of INPP5F_V2 and MCTS2, respectively. To ensure that these SNPs mapped within the DMRs, we performed DNA methylation immunoprecipitation (meDIP) using antisera directed against 5-methylcytosine. This was due to the difficultly in amplifying bisulphite converted DNA in the vicinity of SNPs rs7907781 and rs1115713. For all three regions we observed monoallelic enrichment in heterozygous placental DNA samples, and where informative, the DNA methylation was detected on the maternal allele (Figure 4A). Using these same amplification conditions, we performed ChIP on native chromatin isolated from adult peripheral blood leukocytes and from two human placental cell lines, TCL1 and TCL2 (23) for the NAP1L5 and MCTS2 DMRs. Unfortunately, no heterozygous cell lines could be found that were informative for INPP5F_V2, despite genotyping of over 140 leukocyte samples and normal tissue cell lines. Similar to the mouse, we observe strong monoallelic enrichment for H3K4me2 at the NAP1L5 and MCTS2 DMRs, but since no parental DNA samples were available, allelic origin could not be assigned. Unexpectedly, we did not observe allelic precipitation for any of the repressive histone marks at these DMRs, despite strong allelic enrichment at the SNURF/SNRPN, H19 and MEST DMRs (Figure 4B; data not shown). To confirm that the histone modifications were present at the NAP1L5 and MCTS2 DMRs, we performed quantitative ChIP analysis on the placental cell line TCL1 (Figure 4C). The precipitation values obtained were normalized to those for the SNURF/SNRPN DMR, which revealed that H3K4me2 is more abundant at the MCTS2 and NAP1L5 promoters, whereas, the repressive histone modifications were precipitated several fold less. Interrogation of human histone maps [http://dir.nhlbi.nih.gov/papers/lmi/epigenomes/hgtcell.aspx, and (24)] confirmed the absence of significant enrichment for these repressive marks at 1–2 nucleosomes resolution (the approximate size of the MCTS2 and NAP1L5 DMRs), despite strong H3K4me2 enrichment. Together, these data suggest that these repressive marks are not present, or very low, in these two regions.Figure 4.

Bottom Line: Imprinted retrotransposed genes share a common genomic organization including a promoter-associated differentially methylated region (DMR) and a position within the intron of a multi-exonic 'host' gene.To address the mechanisms governing imprinted retrogene expression, histone modifications were assayed at the DMRs.Two human retrogenes showed monoallelic enrichment of active, but not of repressive marks suggesting a partial uncoupling of the relationship between DNA methylation and repressive histone methylation, possibly due to the smaller size and lower CpG density of these DMRs.

View Article: PubMed Central - PubMed

Affiliation: Imprinting and Cancer Group, Cancer Epigenetics and Biology Program, Bellvitge Institute for Biomedical Research (IDIBELL), L'Hospitalet de Llobregat, 08907, Barcelona, Spain. dmonk@iconcologia.net

ABSTRACT
Imprinted retrotransposed genes share a common genomic organization including a promoter-associated differentially methylated region (DMR) and a position within the intron of a multi-exonic 'host' gene. In the mouse, at least one transcript of the host gene is also subject to genomic imprinting. Human retrogene orthologues are imprinted and we reveal that human host genes are not imprinted. This coincides with genomic rearrangements that occurred during primate evolution, which increase the separation between the retrogene DMRs and the host genes. To address the mechanisms governing imprinted retrogene expression, histone modifications were assayed at the DMRs. For the mouse retrogenes, the active mark H3K4me2 was associated with the unmethylated paternal allele, while the methylated maternal allele was enriched in repressive marks including H3K9me3 and H4K20me3. Two human retrogenes showed monoallelic enrichment of active, but not of repressive marks suggesting a partial uncoupling of the relationship between DNA methylation and repressive histone methylation, possibly due to the smaller size and lower CpG density of these DMRs. Finally, we show that the genes immediately flanking the host genes in mouse and human are biallelically expressed in a range of tissues, suggesting that these loci are distinct from large imprinted clusters.

Show MeSH
Related in: MedlinePlus