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DNMT1 maintains progenitor function in self-renewing somatic tissue.

Sen GL, Reuter JA, Webster DE, Zhu L, Khavari PA - Nature (2010)

Bottom Line: DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation.Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation.These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

View Article: PubMed Central - PubMed

Affiliation: Programs in Epithelial Biology and Cancer Biology and the Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.

ABSTRACT
Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

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DNMT1 loss triggers differentiation and failure of self-renewala, Keratinocytes expressing shRNAs for DNMT1 (DNMT1i) or control (CTL) were used to regenerate human epidermis on immune deficient mice. K1 (green) marks differentiated epidermal layers; human species-specific antibody against Desmoglein 3 (DSG3:orange). White arrows denote areas of ectopic basal layer differentiation. Scale bar=50μm, dotted line=basement membrane. b, Graft survival (n=9 grafted mice/group). c, Human epidermal progenitor competition assay. Marked cells with altered gene function are mixed with unmarked controls to regenerate epidermis. Maintenance of marked undifferentiated progenitor cells in the basal layer (orange) as well as in differentiating suprabasal layers (green in control; yellow in marked) over time. The proportion of marked cells expressing control shRNAs remains constant [top panel] whereas the proportion of marked cells depleted of genes required for progenitor persistence decreases over time [bottom panel]. d, Human epidermal progenitor tracking in vivo. shRNA-receiving cells (top panels=control, bottom panels=DNMT1i) marked with HA tag (orange) in tissue mosaic with non-marked cells. White arrow denotes ectopic K1 differentiation protein expression in the basal layer of a marked DNMT1i cell. Scale bar=50μm. e, Quantitation of HA marked basal layer cells. 500 basal nuclei were counted for each time point (n=9 grafted mice/group);error bars=s.d.
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Figure 2: DNMT1 loss triggers differentiation and failure of self-renewala, Keratinocytes expressing shRNAs for DNMT1 (DNMT1i) or control (CTL) were used to regenerate human epidermis on immune deficient mice. K1 (green) marks differentiated epidermal layers; human species-specific antibody against Desmoglein 3 (DSG3:orange). White arrows denote areas of ectopic basal layer differentiation. Scale bar=50μm, dotted line=basement membrane. b, Graft survival (n=9 grafted mice/group). c, Human epidermal progenitor competition assay. Marked cells with altered gene function are mixed with unmarked controls to regenerate epidermis. Maintenance of marked undifferentiated progenitor cells in the basal layer (orange) as well as in differentiating suprabasal layers (green in control; yellow in marked) over time. The proportion of marked cells expressing control shRNAs remains constant [top panel] whereas the proportion of marked cells depleted of genes required for progenitor persistence decreases over time [bottom panel]. d, Human epidermal progenitor tracking in vivo. shRNA-receiving cells (top panels=control, bottom panels=DNMT1i) marked with HA tag (orange) in tissue mosaic with non-marked cells. White arrow denotes ectopic K1 differentiation protein expression in the basal layer of a marked DNMT1i cell. Scale bar=50μm. e, Quantitation of HA marked basal layer cells. 500 basal nuclei were counted for each time point (n=9 grafted mice/group);error bars=s.d.

Mentions: To study effects of DNMT1 loss on epidermal progenitor function in vivo, short hairpin RNA constructs targeting DNMT1 (DNMT1i) were generated (Supplementary Fig. 1). DNMT1i and control knockdown cells were used to regenerate human skin on immune-deficient mice by an approach recapitulating features of intact tissue, including epidermal self-renewal kinetics17,18. By two weeks, DNMT1i epidermis was markedly hypoplastic (Fig. 2a) without increased cell death (Supplementary Fig. 2), and only 34% of DNMT1-deficient epidermal tissues survived to week 3 compared to 100% for controls (Fig. 2a,b). DNMT1i tissue displayed ectopic expression of differentiation genes, such as keratin 1 (K1), in the normally undifferentiated basal layer (Fig. 2a), indicating premature differentiation occurs in the absence of DNMT1. Granular layer proteins were not seen in the basal layer of DNMT1i epidermis, possibly because of DNMT1i cell loss from this setting prior to their expression (data not shown). Consistent with premature entry into the post-mitotic differentiation pathway, DNMT1 depletion also diminished cell proliferation by week 3 to <15% of control (Supplementary Fig. 3a, b). These results indicate that epidermal DNMT1 loss leads to premature differentiation within the progenitor-containing compartment and loss of tissue self-renewal.


DNMT1 maintains progenitor function in self-renewing somatic tissue.

Sen GL, Reuter JA, Webster DE, Zhu L, Khavari PA - Nature (2010)

DNMT1 loss triggers differentiation and failure of self-renewala, Keratinocytes expressing shRNAs for DNMT1 (DNMT1i) or control (CTL) were used to regenerate human epidermis on immune deficient mice. K1 (green) marks differentiated epidermal layers; human species-specific antibody against Desmoglein 3 (DSG3:orange). White arrows denote areas of ectopic basal layer differentiation. Scale bar=50μm, dotted line=basement membrane. b, Graft survival (n=9 grafted mice/group). c, Human epidermal progenitor competition assay. Marked cells with altered gene function are mixed with unmarked controls to regenerate epidermis. Maintenance of marked undifferentiated progenitor cells in the basal layer (orange) as well as in differentiating suprabasal layers (green in control; yellow in marked) over time. The proportion of marked cells expressing control shRNAs remains constant [top panel] whereas the proportion of marked cells depleted of genes required for progenitor persistence decreases over time [bottom panel]. d, Human epidermal progenitor tracking in vivo. shRNA-receiving cells (top panels=control, bottom panels=DNMT1i) marked with HA tag (orange) in tissue mosaic with non-marked cells. White arrow denotes ectopic K1 differentiation protein expression in the basal layer of a marked DNMT1i cell. Scale bar=50μm. e, Quantitation of HA marked basal layer cells. 500 basal nuclei were counted for each time point (n=9 grafted mice/group);error bars=s.d.
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Figure 2: DNMT1 loss triggers differentiation and failure of self-renewala, Keratinocytes expressing shRNAs for DNMT1 (DNMT1i) or control (CTL) were used to regenerate human epidermis on immune deficient mice. K1 (green) marks differentiated epidermal layers; human species-specific antibody against Desmoglein 3 (DSG3:orange). White arrows denote areas of ectopic basal layer differentiation. Scale bar=50μm, dotted line=basement membrane. b, Graft survival (n=9 grafted mice/group). c, Human epidermal progenitor competition assay. Marked cells with altered gene function are mixed with unmarked controls to regenerate epidermis. Maintenance of marked undifferentiated progenitor cells in the basal layer (orange) as well as in differentiating suprabasal layers (green in control; yellow in marked) over time. The proportion of marked cells expressing control shRNAs remains constant [top panel] whereas the proportion of marked cells depleted of genes required for progenitor persistence decreases over time [bottom panel]. d, Human epidermal progenitor tracking in vivo. shRNA-receiving cells (top panels=control, bottom panels=DNMT1i) marked with HA tag (orange) in tissue mosaic with non-marked cells. White arrow denotes ectopic K1 differentiation protein expression in the basal layer of a marked DNMT1i cell. Scale bar=50μm. e, Quantitation of HA marked basal layer cells. 500 basal nuclei were counted for each time point (n=9 grafted mice/group);error bars=s.d.
Mentions: To study effects of DNMT1 loss on epidermal progenitor function in vivo, short hairpin RNA constructs targeting DNMT1 (DNMT1i) were generated (Supplementary Fig. 1). DNMT1i and control knockdown cells were used to regenerate human skin on immune-deficient mice by an approach recapitulating features of intact tissue, including epidermal self-renewal kinetics17,18. By two weeks, DNMT1i epidermis was markedly hypoplastic (Fig. 2a) without increased cell death (Supplementary Fig. 2), and only 34% of DNMT1-deficient epidermal tissues survived to week 3 compared to 100% for controls (Fig. 2a,b). DNMT1i tissue displayed ectopic expression of differentiation genes, such as keratin 1 (K1), in the normally undifferentiated basal layer (Fig. 2a), indicating premature differentiation occurs in the absence of DNMT1. Granular layer proteins were not seen in the basal layer of DNMT1i epidermis, possibly because of DNMT1i cell loss from this setting prior to their expression (data not shown). Consistent with premature entry into the post-mitotic differentiation pathway, DNMT1 depletion also diminished cell proliferation by week 3 to <15% of control (Supplementary Fig. 3a, b). These results indicate that epidermal DNMT1 loss leads to premature differentiation within the progenitor-containing compartment and loss of tissue self-renewal.

Bottom Line: DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation.Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation.These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

View Article: PubMed Central - PubMed

Affiliation: Programs in Epithelial Biology and Cancer Biology and the Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California 94305, USA.

ABSTRACT
Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

Show MeSH
Related in: MedlinePlus