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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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Combination of ChIP analysis and bisulfite sequencing of B cell-specific and macrophage genes shows uncoupling of histone modification marks and DNA methylation status for reprogrammed C10 cells. (A) Scheme depicting the rationale for ChIP-BS experiments, where DNA methylation status of the DNA fraction bound to a specific histone modification or transcription factors is determined. (B) Five heatmaps show the DNA methylation status (as assessed by bisulfite genomic sequencing of multiple clones) of five macrophage-specific genes associated with H3K9acK14ac, H3K4me3, C/EBPα and RNA pol II in C10 reprogrammed macrophages (at 48 and 120 h) and RAW macrophages. (C) An example of the results obtained from ChIP-BS experiments where the profiles of the Cebpa promoter CpG island associated with H3K9acK14ac, H3K4me3, C/EBPα and RNA pol II are compared for reprogrammed macrophages and RAW macrophages. (D) Schematic representation focusing on the different epigenetic profiles observed for the two different sets of macrophages: those resulting from C/EBPα-dependent transdifferentiation of C10 cells and RAW cells. Histone octamers are represented by grey circles. DNA is represented as a red line in which only methylated CpG dinucleotides are shown (as red circles). Histone tails are lines protruding from octamers, where H3K9acK14ac, and H3K4me3 are indicated. Loss of these two marks is indicated by absence of the protruding lines. The arrows indicate transcription of macrophage-specific genes.
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gkr1015-F5: Combination of ChIP analysis and bisulfite sequencing of B cell-specific and macrophage genes shows uncoupling of histone modification marks and DNA methylation status for reprogrammed C10 cells. (A) Scheme depicting the rationale for ChIP-BS experiments, where DNA methylation status of the DNA fraction bound to a specific histone modification or transcription factors is determined. (B) Five heatmaps show the DNA methylation status (as assessed by bisulfite genomic sequencing of multiple clones) of five macrophage-specific genes associated with H3K9acK14ac, H3K4me3, C/EBPα and RNA pol II in C10 reprogrammed macrophages (at 48 and 120 h) and RAW macrophages. (C) An example of the results obtained from ChIP-BS experiments where the profiles of the Cebpa promoter CpG island associated with H3K9acK14ac, H3K4me3, C/EBPα and RNA pol II are compared for reprogrammed macrophages and RAW macrophages. (D) Schematic representation focusing on the different epigenetic profiles observed for the two different sets of macrophages: those resulting from C/EBPα-dependent transdifferentiation of C10 cells and RAW cells. Histone octamers are represented by grey circles. DNA is represented as a red line in which only methylated CpG dinucleotides are shown (as red circles). Histone tails are lines protruding from octamers, where H3K9acK14ac, and H3K4me3 are indicated. Loss of these two marks is indicated by absence of the protruding lines. The arrows indicate transcription of macrophage-specific genes.

Mentions: The above results suggest that C/EBPα and RNA Pol II associate the promoters of macrophages-specific genes without sensing or inducing a modification of the DNA methylation status. They also suggest that during C/EBPα-mediated B cell to macrophage transdifferentiation the DNA methylation status of the genes whose expression changes are at least partially uncoupled from their histone modification status. We therefore decided to assess whether histone H3 K9/K14 acetylation and K4 trimethylation, as well as C/EBPα and RNA Pol II binding, were associated with macrophage-specific genes that remain methylated state in reprogrammed C10 macrophages. To this end, we performed bisulfite sequencing of DNA isolated from chromatin immunoprecipitation experiments using H3K4me3, H3K9acK14ac and C/EBPα antibodies (Figure 5A). Comparison of the DNA methylation status of the DNA associated with H3K4me3, H3K9acK14ac, C/EBPα and RNA Pol II in four macrophage-associated genes (Itgam, Cebpa, Cd14 and Dock8) in both reprogrammed macrophages and RAW cells showed that the DNA methylation status of these genes and the two histone H3 modifications and C/EBPα and Pol II binding are uncoupled (Figure 5B). The absence of methylation changes at any CpG sites in the DNA fraction associated with C/EBPα, RNA Pol II or H3K4me3, H3K9acK14ac was confirmed by looking at the methylation profiles throughout the entire sequence (an example is shown in Figure 5C).Figure 5.


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)

Combination of ChIP analysis and bisulfite sequencing of B cell-specific and macrophage genes shows uncoupling of histone modification marks and DNA methylation status for reprogrammed C10 cells. (A) Scheme depicting the rationale for ChIP-BS experiments, where DNA methylation status of the DNA fraction bound to a specific histone modification or transcription factors is determined. (B) Five heatmaps show the DNA methylation status (as assessed by bisulfite genomic sequencing of multiple clones) of five macrophage-specific genes associated with H3K9acK14ac, H3K4me3, C/EBPα and RNA pol II in C10 reprogrammed macrophages (at 48 and 120 h) and RAW macrophages. (C) An example of the results obtained from ChIP-BS experiments where the profiles of the Cebpa promoter CpG island associated with H3K9acK14ac, H3K4me3, C/EBPα and RNA pol II are compared for reprogrammed macrophages and RAW macrophages. (D) Schematic representation focusing on the different epigenetic profiles observed for the two different sets of macrophages: those resulting from C/EBPα-dependent transdifferentiation of C10 cells and RAW cells. Histone octamers are represented by grey circles. DNA is represented as a red line in which only methylated CpG dinucleotides are shown (as red circles). Histone tails are lines protruding from octamers, where H3K9acK14ac, and H3K4me3 are indicated. Loss of these two marks is indicated by absence of the protruding lines. The arrows indicate transcription of macrophage-specific genes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3299990&req=5

gkr1015-F5: Combination of ChIP analysis and bisulfite sequencing of B cell-specific and macrophage genes shows uncoupling of histone modification marks and DNA methylation status for reprogrammed C10 cells. (A) Scheme depicting the rationale for ChIP-BS experiments, where DNA methylation status of the DNA fraction bound to a specific histone modification or transcription factors is determined. (B) Five heatmaps show the DNA methylation status (as assessed by bisulfite genomic sequencing of multiple clones) of five macrophage-specific genes associated with H3K9acK14ac, H3K4me3, C/EBPα and RNA pol II in C10 reprogrammed macrophages (at 48 and 120 h) and RAW macrophages. (C) An example of the results obtained from ChIP-BS experiments where the profiles of the Cebpa promoter CpG island associated with H3K9acK14ac, H3K4me3, C/EBPα and RNA pol II are compared for reprogrammed macrophages and RAW macrophages. (D) Schematic representation focusing on the different epigenetic profiles observed for the two different sets of macrophages: those resulting from C/EBPα-dependent transdifferentiation of C10 cells and RAW cells. Histone octamers are represented by grey circles. DNA is represented as a red line in which only methylated CpG dinucleotides are shown (as red circles). Histone tails are lines protruding from octamers, where H3K9acK14ac, and H3K4me3 are indicated. Loss of these two marks is indicated by absence of the protruding lines. The arrows indicate transcription of macrophage-specific genes.
Mentions: The above results suggest that C/EBPα and RNA Pol II associate the promoters of macrophages-specific genes without sensing or inducing a modification of the DNA methylation status. They also suggest that during C/EBPα-mediated B cell to macrophage transdifferentiation the DNA methylation status of the genes whose expression changes are at least partially uncoupled from their histone modification status. We therefore decided to assess whether histone H3 K9/K14 acetylation and K4 trimethylation, as well as C/EBPα and RNA Pol II binding, were associated with macrophage-specific genes that remain methylated state in reprogrammed C10 macrophages. To this end, we performed bisulfite sequencing of DNA isolated from chromatin immunoprecipitation experiments using H3K4me3, H3K9acK14ac and C/EBPα antibodies (Figure 5A). Comparison of the DNA methylation status of the DNA associated with H3K4me3, H3K9acK14ac, C/EBPα and RNA Pol II in four macrophage-associated genes (Itgam, Cebpa, Cd14 and Dock8) in both reprogrammed macrophages and RAW cells showed that the DNA methylation status of these genes and the two histone H3 modifications and C/EBPα and Pol II binding are uncoupled (Figure 5B). The absence of methylation changes at any CpG sites in the DNA fraction associated with C/EBPα, RNA Pol II or H3K4me3, H3K9acK14ac was confirmed by looking at the methylation profiles throughout the entire sequence (an example is shown in Figure 5C).Figure 5.

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
Related in: MedlinePlus