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Overexpression of the Heterochromatinization Factor BAHD1 in HEK293 Cells Differentially Reshapes the DNA Methylome on Autosomes and X Chromosome.

Libertini E, Lebreton A, Lakisic G, Dillies MA, Beck S, Coppée JY, Cossart P, Bierne H - Front Genet (2015)

Bottom Line: We identified 91,358 regions that have different methylation patterns in HEK-BAHD1 compared to HEK-CT cells (termed "BAHD1-DMRs"), of which 83,850 mapped on autosomes and 7508 on the X chromosome (chrX).We further found that BAHD1-DMRs display a higher-order organization by being clustered within large chromosomal domains.Based on these results, we propose that BAHD1-mediated heterochromatin formation is linked to DNA methylation and may play a role in the spatial architecture of the genome.

View Article: PubMed Central - PubMed

Affiliation: Plate-forme Transcriptome et Epigénome, Département Génomes et Génétique, Institut Pasteur Paris, France ; Medical Genomics Group, UCL Cancer Institute, University College London London, UK.

ABSTRACT
BAH domain-containing protein 1 (BAHD1) is involved in heterochromatin formation and gene repression in human cells. BAHD1 also localizes to the inactive X chromosome (Xi), but the functional significance of this targeting is unknown. So far, research on this protein has been hampered by its low endogenous abundance and its role in epigenetic regulation remains poorly explored. In this work, we used whole-genome bisulfite sequencing (BS-seq) to compare the DNA methylation profile of HEK293 cells expressing low levels of BAHD1 (HEK-CT) to that of isogenic cells stably overexpressing BAHD1 (HEK-BAHD1). We show that increasing BAHD1 levels induces de novo DNA methylation on autosomes and a marked hypomethylation on the X chromosome (chrX). We identified 91,358 regions that have different methylation patterns in HEK-BAHD1 compared to HEK-CT cells (termed "BAHD1-DMRs"), of which 83,850 mapped on autosomes and 7508 on the X chromosome (chrX). Autosomal BAHD1-DMRs were predominantly hypermethylated and located to satellites, interspersed repeats, and intergenic regions. In contrast, BAHD1-DMRs on chrX were mainly hypomethylated and located to gene bodies and enhancers. We further found that BAHD1-DMRs display a higher-order organization by being clustered within large chromosomal domains. Half of these "BAHD1-Associated differentially methylated Domains" (BADs) overlapped with lamina-associated domains (LADs). Based on these results, we propose that BAHD1-mediated heterochromatin formation is linked to DNA methylation and may play a role in the spatial architecture of the genome.

No MeSH data available.


Related in: MedlinePlus

Constitutive expression of BAHD1 in HEK-BAHD1 cells. (A) RT-QPCR measurement of BAHD1 and IGF2 mRNA levels in HEK-BAHD1 relative to that in HEK-CT. Bars represent mean±SD of three replicates. (B) Immunoblot of chromatin extracts from HEK-CT and HEK-BAHD1 with BAHD1 or HDAC1 antibodies. Endogenous BAHD1 is undetectable in the control line. (C) Immunofluorescence studies of BAHD1 location in HEK-CT, HEK-BAHD1, or in HEK293 cells transfected with a plasmid expressing BAHD1-YFP. Scale bars, 5 μm. BAHD1 localizes to Xi in cells overexpressing BAHD1 from a chromosomal integration or from a plasmid (arrows). (D) Overlay image of an immunoFISH assay with anti-BAHD1 antibodies (green), combined with Xist RNA FISH (red), and staining of nuclei with DAPI (blue).
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Figure 1: Constitutive expression of BAHD1 in HEK-BAHD1 cells. (A) RT-QPCR measurement of BAHD1 and IGF2 mRNA levels in HEK-BAHD1 relative to that in HEK-CT. Bars represent mean±SD of three replicates. (B) Immunoblot of chromatin extracts from HEK-CT and HEK-BAHD1 with BAHD1 or HDAC1 antibodies. Endogenous BAHD1 is undetectable in the control line. (C) Immunofluorescence studies of BAHD1 location in HEK-CT, HEK-BAHD1, or in HEK293 cells transfected with a plasmid expressing BAHD1-YFP. Scale bars, 5 μm. BAHD1 localizes to Xi in cells overexpressing BAHD1 from a chromosomal integration or from a plasmid (arrows). (D) Overlay image of an immunoFISH assay with anti-BAHD1 antibodies (green), combined with Xist RNA FISH (red), and staining of nuclei with DAPI (blue).

Mentions: In order to investigate the effect of BAHD1 overexpression on the dynamics of DNA methylation, we generated a HEK293 cell line with stable expression of BAHD1 (referred to as HEK-BAHD1) by integration of a single copy of the BAHD1 coding sequence under the control of the human cytomegalovirus (CMV) promoter in the HEK293 genome. We also produced an isogenic control line (referred to as HEK-CT), by integration of the empty vector. The increase in BAHD1 mRNA levels by ~60 fold in HEK-BAHD1 cells (Figure 1A) enabled the detection of the BAHD1 protein in HEK-BAHD1 cells, in chromatin extracts (Figure 1B), as well as in nuclei by immunofluorescence microscopy, using BAHD1 antibodies (Figure 1C). In contrast, endogenous BAHD1 was undetectable in control HEK-CT chromatin (Figure 1B), consistent with the low expression of BAHD1 in HEK293 cells and many other cell lines (Bierne et al., 2009). We have previously shown that BAHD1 represses expression of the IGF2 gene in HEK293 cells transiently expressing BAHD1 from a plasmid (Bierne et al., 2009). In agreement with these data, IGF2 mRNA levels decreased by 6 fold in HEK-BAHD1 cells, when compared to control cells (Figure 1A). HEK293 are female cells with an atypical karyotype, with often two copies of the inactive X chromosome (Xi). The two Xi are visible as large heterochromatic bodies (Gilbert et al., 2000) to which BAHD1 is recruited (Bierne et al., 2009). Accordingly, we observed that BAHD1 was enriched at the Xi in HEK-BAHD1 cells, as shown by labeling of BAHD1 and XIST RNA (Figure 1D). The BAHD1 staining was less intense in HEK-BAHD1 cells than in cells transiently transfected with an YFP-BAHD1-expressing plasmid (Figure 1C). Thus, HEK-BAHD1 cells stably expressing BAHD1 can be used as a model system to study whether increasing BAHD1 cellular levels affect DNA methylation.


Overexpression of the Heterochromatinization Factor BAHD1 in HEK293 Cells Differentially Reshapes the DNA Methylome on Autosomes and X Chromosome.

Libertini E, Lebreton A, Lakisic G, Dillies MA, Beck S, Coppée JY, Cossart P, Bierne H - Front Genet (2015)

Constitutive expression of BAHD1 in HEK-BAHD1 cells. (A) RT-QPCR measurement of BAHD1 and IGF2 mRNA levels in HEK-BAHD1 relative to that in HEK-CT. Bars represent mean±SD of three replicates. (B) Immunoblot of chromatin extracts from HEK-CT and HEK-BAHD1 with BAHD1 or HDAC1 antibodies. Endogenous BAHD1 is undetectable in the control line. (C) Immunofluorescence studies of BAHD1 location in HEK-CT, HEK-BAHD1, or in HEK293 cells transfected with a plasmid expressing BAHD1-YFP. Scale bars, 5 μm. BAHD1 localizes to Xi in cells overexpressing BAHD1 from a chromosomal integration or from a plasmid (arrows). (D) Overlay image of an immunoFISH assay with anti-BAHD1 antibodies (green), combined with Xist RNA FISH (red), and staining of nuclei with DAPI (blue).
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Figure 1: Constitutive expression of BAHD1 in HEK-BAHD1 cells. (A) RT-QPCR measurement of BAHD1 and IGF2 mRNA levels in HEK-BAHD1 relative to that in HEK-CT. Bars represent mean±SD of three replicates. (B) Immunoblot of chromatin extracts from HEK-CT and HEK-BAHD1 with BAHD1 or HDAC1 antibodies. Endogenous BAHD1 is undetectable in the control line. (C) Immunofluorescence studies of BAHD1 location in HEK-CT, HEK-BAHD1, or in HEK293 cells transfected with a plasmid expressing BAHD1-YFP. Scale bars, 5 μm. BAHD1 localizes to Xi in cells overexpressing BAHD1 from a chromosomal integration or from a plasmid (arrows). (D) Overlay image of an immunoFISH assay with anti-BAHD1 antibodies (green), combined with Xist RNA FISH (red), and staining of nuclei with DAPI (blue).
Mentions: In order to investigate the effect of BAHD1 overexpression on the dynamics of DNA methylation, we generated a HEK293 cell line with stable expression of BAHD1 (referred to as HEK-BAHD1) by integration of a single copy of the BAHD1 coding sequence under the control of the human cytomegalovirus (CMV) promoter in the HEK293 genome. We also produced an isogenic control line (referred to as HEK-CT), by integration of the empty vector. The increase in BAHD1 mRNA levels by ~60 fold in HEK-BAHD1 cells (Figure 1A) enabled the detection of the BAHD1 protein in HEK-BAHD1 cells, in chromatin extracts (Figure 1B), as well as in nuclei by immunofluorescence microscopy, using BAHD1 antibodies (Figure 1C). In contrast, endogenous BAHD1 was undetectable in control HEK-CT chromatin (Figure 1B), consistent with the low expression of BAHD1 in HEK293 cells and many other cell lines (Bierne et al., 2009). We have previously shown that BAHD1 represses expression of the IGF2 gene in HEK293 cells transiently expressing BAHD1 from a plasmid (Bierne et al., 2009). In agreement with these data, IGF2 mRNA levels decreased by 6 fold in HEK-BAHD1 cells, when compared to control cells (Figure 1A). HEK293 are female cells with an atypical karyotype, with often two copies of the inactive X chromosome (Xi). The two Xi are visible as large heterochromatic bodies (Gilbert et al., 2000) to which BAHD1 is recruited (Bierne et al., 2009). Accordingly, we observed that BAHD1 was enriched at the Xi in HEK-BAHD1 cells, as shown by labeling of BAHD1 and XIST RNA (Figure 1D). The BAHD1 staining was less intense in HEK-BAHD1 cells than in cells transiently transfected with an YFP-BAHD1-expressing plasmid (Figure 1C). Thus, HEK-BAHD1 cells stably expressing BAHD1 can be used as a model system to study whether increasing BAHD1 cellular levels affect DNA methylation.

Bottom Line: We identified 91,358 regions that have different methylation patterns in HEK-BAHD1 compared to HEK-CT cells (termed "BAHD1-DMRs"), of which 83,850 mapped on autosomes and 7508 on the X chromosome (chrX).We further found that BAHD1-DMRs display a higher-order organization by being clustered within large chromosomal domains.Based on these results, we propose that BAHD1-mediated heterochromatin formation is linked to DNA methylation and may play a role in the spatial architecture of the genome.

View Article: PubMed Central - PubMed

Affiliation: Plate-forme Transcriptome et Epigénome, Département Génomes et Génétique, Institut Pasteur Paris, France ; Medical Genomics Group, UCL Cancer Institute, University College London London, UK.

ABSTRACT
BAH domain-containing protein 1 (BAHD1) is involved in heterochromatin formation and gene repression in human cells. BAHD1 also localizes to the inactive X chromosome (Xi), but the functional significance of this targeting is unknown. So far, research on this protein has been hampered by its low endogenous abundance and its role in epigenetic regulation remains poorly explored. In this work, we used whole-genome bisulfite sequencing (BS-seq) to compare the DNA methylation profile of HEK293 cells expressing low levels of BAHD1 (HEK-CT) to that of isogenic cells stably overexpressing BAHD1 (HEK-BAHD1). We show that increasing BAHD1 levels induces de novo DNA methylation on autosomes and a marked hypomethylation on the X chromosome (chrX). We identified 91,358 regions that have different methylation patterns in HEK-BAHD1 compared to HEK-CT cells (termed "BAHD1-DMRs"), of which 83,850 mapped on autosomes and 7508 on the X chromosome (chrX). Autosomal BAHD1-DMRs were predominantly hypermethylated and located to satellites, interspersed repeats, and intergenic regions. In contrast, BAHD1-DMRs on chrX were mainly hypomethylated and located to gene bodies and enhancers. We further found that BAHD1-DMRs display a higher-order organization by being clustered within large chromosomal domains. Half of these "BAHD1-Associated differentially methylated Domains" (BADs) overlapped with lamina-associated domains (LADs). Based on these results, we propose that BAHD1-mediated heterochromatin formation is linked to DNA methylation and may play a role in the spatial architecture of the genome.

No MeSH data available.


Related in: MedlinePlus