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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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Transdifferentiation of B cells to macrophages occurs without DNA methylation changes in key genes. (A) Selected examples of bisulfite genomic sequencing at the promoter CpG islands and CpG island shores of two macrophage-specific genes in parental HAFTL B cells, RAW macrophages, and C/EBPαER-expressing B C10 cells (derived from HAFTL) at 0 h (after C/EBPα induction) and reprogrammed into functional macrophages (48 and 120 h). Percentage of DNA methylation is represented by red bars and CpG site number included in the analysis is indicated at the bottom. The two analyzed regions are CpG island (right panel, black bar) and CpG island shore (left panel, grey bar). (B) Selected examples of bisulfite genomic sequencing at the promoter CpG islands and CpG island shores of two B cell-specific genes in parental HAFTL and RAW cells and C10 cells at 0 h and fully reprogrammed into functional macrophages (48 and 120 h). The two analyzed regions are CpG islands (right panel, black bar) and CpG island shores (left panel, grey bar). (C) Heatmap of DNA methylation data for 13 macrophage-specific genes (top) and 11 B cell-specific genes (bottom) in HAFTL and RAW cells and C10 cells at three times during β-estradiol-induced reprogramming (0, 48 and 120 h). Heatmap of DNA methylation data including the four macrophage-specific genes that are differentially methylated between primary B cells and macrophages are also included at the bottom. The number of CpG sites analyzed for each gene is indicated on the right of the heatmap. Values are in Supplementary Table S2. (D) qRT-PCR kinetics of expression of DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b) and activation induced deaminase (Aid). H and R, respectively, designate HAFTL and RAW cells. (E) qPCR analysis of MBD-bound (methylated) fractions obtained from MethylCAP experiments with HAFTL and RAW cells and C10 cells at 0, 48 and 120 h for two macrophage-specific and two B-cell-specific genes.
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gkr1015-F1: Transdifferentiation of B cells to macrophages occurs without DNA methylation changes in key genes. (A) Selected examples of bisulfite genomic sequencing at the promoter CpG islands and CpG island shores of two macrophage-specific genes in parental HAFTL B cells, RAW macrophages, and C/EBPαER-expressing B C10 cells (derived from HAFTL) at 0 h (after C/EBPα induction) and reprogrammed into functional macrophages (48 and 120 h). Percentage of DNA methylation is represented by red bars and CpG site number included in the analysis is indicated at the bottom. The two analyzed regions are CpG island (right panel, black bar) and CpG island shore (left panel, grey bar). (B) Selected examples of bisulfite genomic sequencing at the promoter CpG islands and CpG island shores of two B cell-specific genes in parental HAFTL and RAW cells and C10 cells at 0 h and fully reprogrammed into functional macrophages (48 and 120 h). The two analyzed regions are CpG islands (right panel, black bar) and CpG island shores (left panel, grey bar). (C) Heatmap of DNA methylation data for 13 macrophage-specific genes (top) and 11 B cell-specific genes (bottom) in HAFTL and RAW cells and C10 cells at three times during β-estradiol-induced reprogramming (0, 48 and 120 h). Heatmap of DNA methylation data including the four macrophage-specific genes that are differentially methylated between primary B cells and macrophages are also included at the bottom. The number of CpG sites analyzed for each gene is indicated on the right of the heatmap. Values are in Supplementary Table S2. (D) qRT-PCR kinetics of expression of DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b) and activation induced deaminase (Aid). H and R, respectively, designate HAFTL and RAW cells. (E) qPCR analysis of MBD-bound (methylated) fractions obtained from MethylCAP experiments with HAFTL and RAW cells and C10 cells at 0, 48 and 120 h for two macrophage-specific and two B-cell-specific genes.

Mentions: To explore DNA methylation changes during B cell to macrophage transdifferentiation we first analyzed C/EBPαER-expressing B C10 cells at three times during reprogramming, by focusing on the 24 genes that display differential DNA methylation between HAFTL B cells and RAW macrophages (Supplementary Tables S1 and Figure S1). We tested cells at 0 h, immediately after the addition of β-estradiol; at 48 h, when the phenotype has already become that of a macrophage-like cell and reversion does not occur following β-estradiol depletion; and at 120 h, when the macrophage-like phenotype has had time to fully stabilize. Unexpectedly, the DNA methylation patterns for these genes in C10 cells did not change during transdifferentiation and was identical for the 24 genes analyzed in C10-derived functional macrophages and uninduced C10 cells or HAFTL B cells (Figure 1A–C). Thus, macrophage-associated genes remained methylated in C10-derived macrophages and conserved an identical methylation pattern to that observed in HAFTL and unreprogrammed C10 B cells (Figure 1A and C). Conversely, B cell-associated genes that are not expressed either in RAW cells or in reprogrammed cells, were only methylated in RAW cells, and reprogrammed macrophages retained the unmethylated status present in HAFTL B cells (Figure 1B and C). Similar results were obtained in transdifferentiation experiments with primary cells since there were no changes in DNA methylation during reprogramming for the few candidate genes displaying differences between the two parental primary cell types (Figure 1C and Supplementary Figure S2B).Figure 1.


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)

Transdifferentiation of B cells to macrophages occurs without DNA methylation changes in key genes. (A) Selected examples of bisulfite genomic sequencing at the promoter CpG islands and CpG island shores of two macrophage-specific genes in parental HAFTL B cells, RAW macrophages, and C/EBPαER-expressing B C10 cells (derived from HAFTL) at 0 h (after C/EBPα induction) and reprogrammed into functional macrophages (48 and 120 h). Percentage of DNA methylation is represented by red bars and CpG site number included in the analysis is indicated at the bottom. The two analyzed regions are CpG island (right panel, black bar) and CpG island shore (left panel, grey bar). (B) Selected examples of bisulfite genomic sequencing at the promoter CpG islands and CpG island shores of two B cell-specific genes in parental HAFTL and RAW cells and C10 cells at 0 h and fully reprogrammed into functional macrophages (48 and 120 h). The two analyzed regions are CpG islands (right panel, black bar) and CpG island shores (left panel, grey bar). (C) Heatmap of DNA methylation data for 13 macrophage-specific genes (top) and 11 B cell-specific genes (bottom) in HAFTL and RAW cells and C10 cells at three times during β-estradiol-induced reprogramming (0, 48 and 120 h). Heatmap of DNA methylation data including the four macrophage-specific genes that are differentially methylated between primary B cells and macrophages are also included at the bottom. The number of CpG sites analyzed for each gene is indicated on the right of the heatmap. Values are in Supplementary Table S2. (D) qRT-PCR kinetics of expression of DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b) and activation induced deaminase (Aid). H and R, respectively, designate HAFTL and RAW cells. (E) qPCR analysis of MBD-bound (methylated) fractions obtained from MethylCAP experiments with HAFTL and RAW cells and C10 cells at 0, 48 and 120 h for two macrophage-specific and two B-cell-specific genes.
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gkr1015-F1: Transdifferentiation of B cells to macrophages occurs without DNA methylation changes in key genes. (A) Selected examples of bisulfite genomic sequencing at the promoter CpG islands and CpG island shores of two macrophage-specific genes in parental HAFTL B cells, RAW macrophages, and C/EBPαER-expressing B C10 cells (derived from HAFTL) at 0 h (after C/EBPα induction) and reprogrammed into functional macrophages (48 and 120 h). Percentage of DNA methylation is represented by red bars and CpG site number included in the analysis is indicated at the bottom. The two analyzed regions are CpG island (right panel, black bar) and CpG island shore (left panel, grey bar). (B) Selected examples of bisulfite genomic sequencing at the promoter CpG islands and CpG island shores of two B cell-specific genes in parental HAFTL and RAW cells and C10 cells at 0 h and fully reprogrammed into functional macrophages (48 and 120 h). The two analyzed regions are CpG islands (right panel, black bar) and CpG island shores (left panel, grey bar). (C) Heatmap of DNA methylation data for 13 macrophage-specific genes (top) and 11 B cell-specific genes (bottom) in HAFTL and RAW cells and C10 cells at three times during β-estradiol-induced reprogramming (0, 48 and 120 h). Heatmap of DNA methylation data including the four macrophage-specific genes that are differentially methylated between primary B cells and macrophages are also included at the bottom. The number of CpG sites analyzed for each gene is indicated on the right of the heatmap. Values are in Supplementary Table S2. (D) qRT-PCR kinetics of expression of DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b) and activation induced deaminase (Aid). H and R, respectively, designate HAFTL and RAW cells. (E) qPCR analysis of MBD-bound (methylated) fractions obtained from MethylCAP experiments with HAFTL and RAW cells and C10 cells at 0, 48 and 120 h for two macrophage-specific and two B-cell-specific genes.
Mentions: To explore DNA methylation changes during B cell to macrophage transdifferentiation we first analyzed C/EBPαER-expressing B C10 cells at three times during reprogramming, by focusing on the 24 genes that display differential DNA methylation between HAFTL B cells and RAW macrophages (Supplementary Tables S1 and Figure S1). We tested cells at 0 h, immediately after the addition of β-estradiol; at 48 h, when the phenotype has already become that of a macrophage-like cell and reversion does not occur following β-estradiol depletion; and at 120 h, when the macrophage-like phenotype has had time to fully stabilize. Unexpectedly, the DNA methylation patterns for these genes in C10 cells did not change during transdifferentiation and was identical for the 24 genes analyzed in C10-derived functional macrophages and uninduced C10 cells or HAFTL B cells (Figure 1A–C). Thus, macrophage-associated genes remained methylated in C10-derived macrophages and conserved an identical methylation pattern to that observed in HAFTL and unreprogrammed C10 B cells (Figure 1A and C). Conversely, B cell-associated genes that are not expressed either in RAW cells or in reprogrammed cells, were only methylated in RAW cells, and reprogrammed macrophages retained the unmethylated status present in HAFTL B cells (Figure 1B and C). Similar results were obtained in transdifferentiation experiments with primary cells since there were no changes in DNA methylation during reprogramming for the few candidate genes displaying differences between the two parental primary cell types (Figure 1C and Supplementary Figure S2B).Figure 1.

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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