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Inactive DNMT3B splice variants modulate de novo DNA methylation.

Gordon CA, Hartono SR, Chédin F - PLoS ONE (2013)

Bottom Line: DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity.DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate.Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California Davis, Davis, California, United States of America.

ABSTRACT
Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.

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DNMT3B isoforms drive unique and distinct localization, DNA staining, and H3K9me3 patterns in human cells.FLAG-tagged DNMT3B2, DNMT3B3, and DNMT3B4 were transiently transfected into human HEK293 cells. 24 to 36 hours later, cells were fixed and stained with anti-FLAG (Sigma) and anti-H3K9me3 (Abcam) antibodies. Transfections were repeated at least in triplicate and cells were photographed with a fluorescence microscope. Representative DNMT3B (Globular, Diffuse, or Speckled: red), DNA (Condensed or Diffuse: blue), and H3K9me3 (Speckled, Spotty, or Faint: green) staining are depicted. Percentages of each type of staining versus DNMT3B isoform expressed are indicated below each image. The total number of independent cells analyzed: DNMT3B2, n = 237; DNMT3B3, n = 203; DNMT3B4, n = 215; none, n = 356.
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pone.0069486-g05: DNMT3B isoforms drive unique and distinct localization, DNA staining, and H3K9me3 patterns in human cells.FLAG-tagged DNMT3B2, DNMT3B3, and DNMT3B4 were transiently transfected into human HEK293 cells. 24 to 36 hours later, cells were fixed and stained with anti-FLAG (Sigma) and anti-H3K9me3 (Abcam) antibodies. Transfections were repeated at least in triplicate and cells were photographed with a fluorescence microscope. Representative DNMT3B (Globular, Diffuse, or Speckled: red), DNA (Condensed or Diffuse: blue), and H3K9me3 (Speckled, Spotty, or Faint: green) staining are depicted. Percentages of each type of staining versus DNMT3B isoform expressed are indicated below each image. The total number of independent cells analyzed: DNMT3B2, n = 237; DNMT3B3, n = 203; DNMT3B4, n = 215; none, n = 356.

Mentions: In order to gain further insights into how expression of DNMT3B3 and DNMT3B4 might exert their functions in vivo, we transiently overexpressed FLAG-tagged DNMT3B2, DNMT3B3, and DNMT3B4 in HEK293 cells, and compared their localization patterns using immunocytochemistry. Three different DNMT3B localization patterns were observed: globular, diffuse, and punctate (Figure 5). The majority of DNMT3B2 and DNMT3B3-expressing cells displayed a globular expression pattern, which was the least likely expression pattern for DNMT3B4-overexpressing cells (Figure 5). DNMT3B4, in contrast, displayed a punctate distribution pattern, which was not observed in any of the DNMT3B2 or DNMT3B3-expressing cells (Figure 5). Similar localization patterns were also observed in mouse NIH3T3 cells (Figure S6).


Inactive DNMT3B splice variants modulate de novo DNA methylation.

Gordon CA, Hartono SR, Chédin F - PLoS ONE (2013)

DNMT3B isoforms drive unique and distinct localization, DNA staining, and H3K9me3 patterns in human cells.FLAG-tagged DNMT3B2, DNMT3B3, and DNMT3B4 were transiently transfected into human HEK293 cells. 24 to 36 hours later, cells were fixed and stained with anti-FLAG (Sigma) and anti-H3K9me3 (Abcam) antibodies. Transfections were repeated at least in triplicate and cells were photographed with a fluorescence microscope. Representative DNMT3B (Globular, Diffuse, or Speckled: red), DNA (Condensed or Diffuse: blue), and H3K9me3 (Speckled, Spotty, or Faint: green) staining are depicted. Percentages of each type of staining versus DNMT3B isoform expressed are indicated below each image. The total number of independent cells analyzed: DNMT3B2, n = 237; DNMT3B3, n = 203; DNMT3B4, n = 215; none, n = 356.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3716610&req=5

pone.0069486-g05: DNMT3B isoforms drive unique and distinct localization, DNA staining, and H3K9me3 patterns in human cells.FLAG-tagged DNMT3B2, DNMT3B3, and DNMT3B4 were transiently transfected into human HEK293 cells. 24 to 36 hours later, cells were fixed and stained with anti-FLAG (Sigma) and anti-H3K9me3 (Abcam) antibodies. Transfections were repeated at least in triplicate and cells were photographed with a fluorescence microscope. Representative DNMT3B (Globular, Diffuse, or Speckled: red), DNA (Condensed or Diffuse: blue), and H3K9me3 (Speckled, Spotty, or Faint: green) staining are depicted. Percentages of each type of staining versus DNMT3B isoform expressed are indicated below each image. The total number of independent cells analyzed: DNMT3B2, n = 237; DNMT3B3, n = 203; DNMT3B4, n = 215; none, n = 356.
Mentions: In order to gain further insights into how expression of DNMT3B3 and DNMT3B4 might exert their functions in vivo, we transiently overexpressed FLAG-tagged DNMT3B2, DNMT3B3, and DNMT3B4 in HEK293 cells, and compared their localization patterns using immunocytochemistry. Three different DNMT3B localization patterns were observed: globular, diffuse, and punctate (Figure 5). The majority of DNMT3B2 and DNMT3B3-expressing cells displayed a globular expression pattern, which was the least likely expression pattern for DNMT3B4-overexpressing cells (Figure 5). DNMT3B4, in contrast, displayed a punctate distribution pattern, which was not observed in any of the DNMT3B2 or DNMT3B3-expressing cells (Figure 5). Similar localization patterns were also observed in mouse NIH3T3 cells (Figure S6).

Bottom Line: DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity.DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate.Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, University of California Davis, Davis, California, United States of America.

ABSTRACT
Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.

Show MeSH
Related in: MedlinePlus