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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

BAHD1-induced differential methylation among chromosomes and genomic elements. (A) Global difference in the percentage of CpG methylation level between HEK-CT and HEK-BAHD1 DNA in autosomes and chrX (methylation level = 100 * total mC reads/all reads). Results are mean±SD of two biological replicates. (B) Number of BAHD1-specific DMRs in autosomes and chrX of HEK-BAHD1 cells relative to that in HEK-CT cells. Hypermethylated DMRs: red. Hypomethylated DMRs: blue. (C) Enrichment of BAHD1-DMRs relative to autosome size; chrX is shown apart. (D) Number of BAHD1-DMRs in genomic elements. (E,F) Relative enrichment in genomic elements of BAHD1-hyper-DMRs on autosomes (E) or BAHD1-hypo-DMRs on chrX (F). The proportion of DMRs was normalized by the length and abundance of the queried element in the genome. Regions significantly over-represented in differentially methylated regions are indicated (***p < 0.001). (G,H) Number of BAHD1-specific peaks in HPT-BAHD1 NChIP replicates in chromosomes (G) or genomic elements (H).
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Figure 2: BAHD1-induced differential methylation among chromosomes and genomic elements. (A) Global difference in the percentage of CpG methylation level between HEK-CT and HEK-BAHD1 DNA in autosomes and chrX (methylation level = 100 * total mC reads/all reads). Results are mean±SD of two biological replicates. (B) Number of BAHD1-specific DMRs in autosomes and chrX of HEK-BAHD1 cells relative to that in HEK-CT cells. Hypermethylated DMRs: red. Hypomethylated DMRs: blue. (C) Enrichment of BAHD1-DMRs relative to autosome size; chrX is shown apart. (D) Number of BAHD1-DMRs in genomic elements. (E,F) Relative enrichment in genomic elements of BAHD1-hyper-DMRs on autosomes (E) or BAHD1-hypo-DMRs on chrX (F). The proportion of DMRs was normalized by the length and abundance of the queried element in the genome. Regions significantly over-represented in differentially methylated regions are indicated (***p < 0.001). (G,H) Number of BAHD1-specific peaks in HPT-BAHD1 NChIP replicates in chromosomes (G) or genomic elements (H).

Mentions: In order to obtain high-resolution DNA methylation profiling, we performed whole-genome BS-seq (MethylC-seq/BS-seq Lister et al., 2009) of two HEK-BAHD1 replicates and of the HEK-CT control DNA, generating complete reference methylomes of these cell lines at single base resolution (see Materials and Methods and Supplementary Figure S1A for detailed analytical procedures). We verified that our BS-seq results were in agreement with partial methylomes previously released for HEK293 cells in ENCODE RRBS and HEK293 chromosome 21 datasets (Zhang et al., 2009; ENCODE Project Consortium, 2012; Supplementary Figure S2). Comparison of these methylomes highlighted a significant gain of methylation (on average ~2 ± 0.66%) in autosomes of HEK-BAHD1 cells, when compared to autosomes of HEK-CT cells, corresponding to methylation of ~478,000 CpG. In contrast, the average methylation level on chrX decreased by ~4 ± 0.05% (Figure 2A; Table 1). Differential analysis of HEK-CT and HEK-BAHD1 methylome replicates (Fisher's exact test, p < 0.1, BH-corrected, minimum methylation difference 25%) identified 91,358 regions of 300 bp that became reproducibly differentially methylated in the two biological replicates of the HEK-BAHD1 DNA, when compared to the isogenic control. 83,850 of these differentially methylated regions (herein referred to as “BAHD1-DMRs”) mapped on autosomes and 7508 on chrX (Figure 2B; Table 2). Relative to chromosome size, the highest enrichment of BAHD1-DMRs was on chrX (Figure 2C; Table 2). In agreement with global methylation levels, 89.5% of BAHD1-specific DMRs identified on autosomes showed a gain of methylation (referred to as “hyper-DMRs”), whereas 81.8% DMRs on chrX showed a loss of methylation (“hypo-DMRs”). These results indicate that up-regulation of BAHD1 induces de novo cytosine methylation on autosomes and loss of methylation on chrX.


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)

BAHD1-induced differential methylation among chromosomes and genomic elements. (A) Global difference in the percentage of CpG methylation level between HEK-CT and HEK-BAHD1 DNA in autosomes and chrX (methylation level = 100 * total mC reads/all reads). Results are mean±SD of two biological replicates. (B) Number of BAHD1-specific DMRs in autosomes and chrX of HEK-BAHD1 cells relative to that in HEK-CT cells. Hypermethylated DMRs: red. Hypomethylated DMRs: blue. (C) Enrichment of BAHD1-DMRs relative to autosome size; chrX is shown apart. (D) Number of BAHD1-DMRs in genomic elements. (E,F) Relative enrichment in genomic elements of BAHD1-hyper-DMRs on autosomes (E) or BAHD1-hypo-DMRs on chrX (F). The proportion of DMRs was normalized by the length and abundance of the queried element in the genome. Regions significantly over-represented in differentially methylated regions are indicated (***p < 0.001). (G,H) Number of BAHD1-specific peaks in HPT-BAHD1 NChIP replicates in chromosomes (G) or genomic elements (H).
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Figure 2: BAHD1-induced differential methylation among chromosomes and genomic elements. (A) Global difference in the percentage of CpG methylation level between HEK-CT and HEK-BAHD1 DNA in autosomes and chrX (methylation level = 100 * total mC reads/all reads). Results are mean±SD of two biological replicates. (B) Number of BAHD1-specific DMRs in autosomes and chrX of HEK-BAHD1 cells relative to that in HEK-CT cells. Hypermethylated DMRs: red. Hypomethylated DMRs: blue. (C) Enrichment of BAHD1-DMRs relative to autosome size; chrX is shown apart. (D) Number of BAHD1-DMRs in genomic elements. (E,F) Relative enrichment in genomic elements of BAHD1-hyper-DMRs on autosomes (E) or BAHD1-hypo-DMRs on chrX (F). The proportion of DMRs was normalized by the length and abundance of the queried element in the genome. Regions significantly over-represented in differentially methylated regions are indicated (***p < 0.001). (G,H) Number of BAHD1-specific peaks in HPT-BAHD1 NChIP replicates in chromosomes (G) or genomic elements (H).
Mentions: In order to obtain high-resolution DNA methylation profiling, we performed whole-genome BS-seq (MethylC-seq/BS-seq Lister et al., 2009) of two HEK-BAHD1 replicates and of the HEK-CT control DNA, generating complete reference methylomes of these cell lines at single base resolution (see Materials and Methods and Supplementary Figure S1A for detailed analytical procedures). We verified that our BS-seq results were in agreement with partial methylomes previously released for HEK293 cells in ENCODE RRBS and HEK293 chromosome 21 datasets (Zhang et al., 2009; ENCODE Project Consortium, 2012; Supplementary Figure S2). Comparison of these methylomes highlighted a significant gain of methylation (on average ~2 ± 0.66%) in autosomes of HEK-BAHD1 cells, when compared to autosomes of HEK-CT cells, corresponding to methylation of ~478,000 CpG. In contrast, the average methylation level on chrX decreased by ~4 ± 0.05% (Figure 2A; Table 1). Differential analysis of HEK-CT and HEK-BAHD1 methylome replicates (Fisher's exact test, p < 0.1, BH-corrected, minimum methylation difference 25%) identified 91,358 regions of 300 bp that became reproducibly differentially methylated in the two biological replicates of the HEK-BAHD1 DNA, when compared to the isogenic control. 83,850 of these differentially methylated regions (herein referred to as “BAHD1-DMRs”) mapped on autosomes and 7508 on chrX (Figure 2B; Table 2). Relative to chromosome size, the highest enrichment of BAHD1-DMRs was on chrX (Figure 2C; Table 2). In agreement with global methylation levels, 89.5% of BAHD1-specific DMRs identified on autosomes showed a gain of methylation (referred to as “hyper-DMRs”), whereas 81.8% DMRs on chrX showed a loss of methylation (“hypo-DMRs”). These results indicate that up-regulation of BAHD1 induces de novo cytosine methylation on autosomes and loss of methylation on chrX.

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