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Pre-B cell to macrophage transdifferentiation without significant promoter DNA methylation changes.

Rodríguez-Ubreva J, Ciudad L, Gómez-Cabrero D, Parra M, Bussmann LH, di Tullio A, Kallin EM, Tegnér J, Graf T, Ballestar E - Nucleic Acids Res. (2011)

Bottom Line: Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types.We also demonstrated that C/EBPα and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes.Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role.

View Article: PubMed Central - PubMed

Affiliation: Chromatin and Disease Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08907 L'Hospitalet de Llobregat, Barcelona, Spain.

ABSTRACT
Transcription factor-induced lineage reprogramming or transdifferentiation experiments are essential for understanding the plasticity of differentiated cells. These experiments helped to define the specific role of transcription factors in conferring cell identity and played a key role in the development of the regenerative medicine field. We here investigated the acquisition of DNA methylation changes during C/EBPα-induced pre-B cell to macrophage transdifferentiation. Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types. In contrast, active and repressive histone modification marks changed according to the expression levels of these genes. We also demonstrated that C/EBPα and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes. Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role.

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Changes in the histone modification profile accompany changes in the expression status of key genes. Quantitative ChIP assays show changes in the levels of H3K9acK14ac and H3K4me3, as active marks, and H3K27me3, as a repressive mark, in both macrophage-specific genes (A) and B cell-specific genes (B). HAFTL pre-B cells (H) and RAW macrophages (R) are used as controls and C10 cells at 0 h and fully reprogrammed into functional macrophages (48 and 120 h). In all cases, data is normalized with respect to the values in HAFTL cells. On top of each gene, a scheme depicting the sequence covered for DNA methylation analysis (black bar) and ChIP analysis (grey bar), in relation with the transcription start site (arrow) is shown.
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gkr1015-F3: Changes in the histone modification profile accompany changes in the expression status of key genes. Quantitative ChIP assays show changes in the levels of H3K9acK14ac and H3K4me3, as active marks, and H3K27me3, as a repressive mark, in both macrophage-specific genes (A) and B cell-specific genes (B). HAFTL pre-B cells (H) and RAW macrophages (R) are used as controls and C10 cells at 0 h and fully reprogrammed into functional macrophages (48 and 120 h). In all cases, data is normalized with respect to the values in HAFTL cells. On top of each gene, a scheme depicting the sequence covered for DNA methylation analysis (black bar) and ChIP analysis (grey bar), in relation with the transcription start site (arrow) is shown.

Mentions: The lack of DNA methylation changes during B cell to macrophage transdifferentiation in genes that undergo expression changes suggests that alternative epigenetic factors, like histone modifications, have a stronger association with the expression status of these genes, at least in our model system. To address this issue we chose five macrophage-specific genes (Itgam, Sfpi1, Cd14 and Dock8) and four B cell-specific genes (Cd19, Pax5, Ebf1 and Bcl11a), for which we found differential promoter DNA methylation between HAFTL and RAW cells (Supplementary Table S1). These genes undergo changes in transcription as expected during transdifferentiation (Supplementary Figure S7; (10,11). We then performed ChIP assays for three histone H3 modifications well-characterized for their localization at promoters and functional association with gene expression status: histone H3 K4 trimethylation (H3K4me3) and acetylation (H3K9acK14ac), associated with active transcription, and histone H3 K27 trimethylation (H3K27me3), associated with gene repression (43–45). We analyzed the profile of these histone modification marks at the promoter of the two groups of genes studied in a region near the TSS overlapping with the region analyzed for DNA methylation (Figure 3, top).Figure 3.


Pre-B cell to macrophage transdifferentiation without significant promoter DNA methylation changes.

Rodríguez-Ubreva J, Ciudad L, Gómez-Cabrero D, Parra M, Bussmann LH, di Tullio A, Kallin EM, Tegnér J, Graf T, Ballestar E - Nucleic Acids Res. (2011)

Changes in the histone modification profile accompany changes in the expression status of key genes. Quantitative ChIP assays show changes in the levels of H3K9acK14ac and H3K4me3, as active marks, and H3K27me3, as a repressive mark, in both macrophage-specific genes (A) and B cell-specific genes (B). HAFTL pre-B cells (H) and RAW macrophages (R) are used as controls and C10 cells at 0 h and fully reprogrammed into functional macrophages (48 and 120 h). In all cases, data is normalized with respect to the values in HAFTL cells. On top of each gene, a scheme depicting the sequence covered for DNA methylation analysis (black bar) and ChIP analysis (grey bar), in relation with the transcription start site (arrow) is shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299990&req=5

gkr1015-F3: Changes in the histone modification profile accompany changes in the expression status of key genes. Quantitative ChIP assays show changes in the levels of H3K9acK14ac and H3K4me3, as active marks, and H3K27me3, as a repressive mark, in both macrophage-specific genes (A) and B cell-specific genes (B). HAFTL pre-B cells (H) and RAW macrophages (R) are used as controls and C10 cells at 0 h and fully reprogrammed into functional macrophages (48 and 120 h). In all cases, data is normalized with respect to the values in HAFTL cells. On top of each gene, a scheme depicting the sequence covered for DNA methylation analysis (black bar) and ChIP analysis (grey bar), in relation with the transcription start site (arrow) is shown.
Mentions: The lack of DNA methylation changes during B cell to macrophage transdifferentiation in genes that undergo expression changes suggests that alternative epigenetic factors, like histone modifications, have a stronger association with the expression status of these genes, at least in our model system. To address this issue we chose five macrophage-specific genes (Itgam, Sfpi1, Cd14 and Dock8) and four B cell-specific genes (Cd19, Pax5, Ebf1 and Bcl11a), for which we found differential promoter DNA methylation between HAFTL and RAW cells (Supplementary Table S1). These genes undergo changes in transcription as expected during transdifferentiation (Supplementary Figure S7; (10,11). We then performed ChIP assays for three histone H3 modifications well-characterized for their localization at promoters and functional association with gene expression status: histone H3 K4 trimethylation (H3K4me3) and acetylation (H3K9acK14ac), associated with active transcription, and histone H3 K27 trimethylation (H3K27me3), associated with gene repression (43–45). We analyzed the profile of these histone modification marks at the promoter of the two groups of genes studied in a region near the TSS overlapping with the region analyzed for DNA methylation (Figure 3, top).Figure 3.

Bottom Line: Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types.We also demonstrated that C/EBPα and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes.Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role.

View Article: PubMed Central - PubMed

Affiliation: Chromatin and Disease Group, Cancer Epigenetics and Biology Programme (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08907 L'Hospitalet de Llobregat, Barcelona, Spain.

ABSTRACT
Transcription factor-induced lineage reprogramming or transdifferentiation experiments are essential for understanding the plasticity of differentiated cells. These experiments helped to define the specific role of transcription factors in conferring cell identity and played a key role in the development of the regenerative medicine field. We here investigated the acquisition of DNA methylation changes during C/EBPα-induced pre-B cell to macrophage transdifferentiation. Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types. In contrast, active and repressive histone modification marks changed according to the expression levels of these genes. We also demonstrated that C/EBPα and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes. Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role.

Show MeSH