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Signalling couples hair follicle stem cell quiescence with reduced histone H3 K4/K9/K27me3 for proper tissue homeostasis.

Lee J, Kang S, Lilja KC, Colletier KJ, Scheitz CJ, Zhang YV, Tumbar T - Nat Commun (2016)

Bottom Line: The level of marks over specific gene promoters did not correlate to mRNA level changes in quiescent HFSCs.Furthermore, removal of proliferation factors and addition of BMP4 reduced histone methylases and increased demethylases mRNAs in cultured skin epithelial cells.We conclude that signalling couples hair follicle stem cell quiescence with reduced H3 K4/K9/K27me3 levels for proper tissue homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
Mechanisms of plasticity to acquire different cell fates are critical for adult stem cell (SC) potential, yet are poorly understood. Reduced global histone methylation is an epigenetic state known to mediate plasticity in cultured embryonic SCs and T-cell progenitors. Here we find histone H3 K4/K9/K27me3 levels actively reduced in adult mouse skin and hair follicle stem cells (HFSCs) during G0 quiescence. The level of marks over specific gene promoters did not correlate to mRNA level changes in quiescent HFSCs. Skin hypomethylation during quiescence was necessary for subsequent progression of hair homeostasis (cycle). Inhibiting BMP signal, a known HFSC anti-proliferative factor, elevated HFSC methylation in vivo during quiescence prior to proliferation onset. Furthermore, removal of proliferation factors and addition of BMP4 reduced histone methylases and increased demethylases mRNAs in cultured skin epithelial cells. We conclude that signalling couples hair follicle stem cell quiescence with reduced H3 K4/K9/K27me3 levels for proper tissue homeostasis.

No MeSH data available.


Related in: MedlinePlus

The level of histone H3 K4/K9/K27me3 in the skin is important for hair homeostasis.(a) Experimental scheme of using demethylase inhibitors (DIs). (b,c) Western blot (b) and quantification of band intensity (c) of histone methylation from whole skin chromatin extracts of control and DI-treated mice. Band intensities were normalized to H3 signal. (d) Immunofluorescence staining of control and DI-treated (scheme 1 of (a)) hair follicle sections labelled with H3K9me3 (red) and CD34 (green). Scale bar, 20 μm. (e) Fluorescence intensity of all three marks from immunostainings were quantified for each bulge cell (CD34+). Student's t-test was used to determine statistical significance between control and DI-treated mice for each mark. (f) Immunofluorescence staining with a proliferation marker Ki67 of control and DI-treated (scheme 2 of a) skin sections. Scale bar, 20 μm. (g) The number of Ki67+ cells in f in each hair follicle were counted, and they showed reduced proliferation in DI-treated mice compared with control. The differences across the conditions were significant according to a Student's t-test. (h) Hair follicles of control and DI-treated mice were categorized in their hair cycle stages. Notice a dramatic increase in the number of telogen (Telo) hair follicles and reduction in anagen (Ana) I and II in DI-treated mice. (i) Mice were shaved, treated with DI in catagen (Cata; PD35–42; a, scheme 3), and followed long-term to monitor the hair cycle re-entry. Notice that mice in the control group show hair growth, whereas mice in DI-treated group show no growth. Avg, average.
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f7: The level of histone H3 K4/K9/K27me3 in the skin is important for hair homeostasis.(a) Experimental scheme of using demethylase inhibitors (DIs). (b,c) Western blot (b) and quantification of band intensity (c) of histone methylation from whole skin chromatin extracts of control and DI-treated mice. Band intensities were normalized to H3 signal. (d) Immunofluorescence staining of control and DI-treated (scheme 1 of (a)) hair follicle sections labelled with H3K9me3 (red) and CD34 (green). Scale bar, 20 μm. (e) Fluorescence intensity of all three marks from immunostainings were quantified for each bulge cell (CD34+). Student's t-test was used to determine statistical significance between control and DI-treated mice for each mark. (f) Immunofluorescence staining with a proliferation marker Ki67 of control and DI-treated (scheme 2 of a) skin sections. Scale bar, 20 μm. (g) The number of Ki67+ cells in f in each hair follicle were counted, and they showed reduced proliferation in DI-treated mice compared with control. The differences across the conditions were significant according to a Student's t-test. (h) Hair follicles of control and DI-treated mice were categorized in their hair cycle stages. Notice a dramatic increase in the number of telogen (Telo) hair follicles and reduction in anagen (Ana) I and II in DI-treated mice. (i) Mice were shaved, treated with DI in catagen (Cata; PD35–42; a, scheme 3), and followed long-term to monitor the hair cycle re-entry. Notice that mice in the control group show hair growth, whereas mice in DI-treated group show no growth. Avg, average.

Mentions: Next, we examined the potential implication of histone demethylases in erasure of histone H3 K4/K9/K27me3 marks in HFSCs at catagen, and probed the functional significance of these levels. We employed demethylase chemical inhibitors (DIs) known to be specific for H3 K4me3, K9me3 (ref. 28) and K27me3 (ref. 29), shown by biochemical and structural studies to have potent and specific activities towards these modifications. We applied a cocktail of DI onto mouse back skin from anagen (PD32) up to different time points, and assessed the proliferation status of hair follicle in the subsequent hair cycle stage at early anagen (Fig. 7a). We used mice from a 1:1 mixed (CD1 × Fvb) genetic background, which we previously found to have a relatively shorter second telogen and more consistent early onset of the second adult anagen2730, than other common mouse strains. Western blot analysis of histones extracted from total mouse skin DI-treated for 7 days (PD32–39; Fig. 7a, scheme 1) showed increased histone methylation levels (Fig. 7b,c) confirming the activity of DI in vivo. Similarly, CD34+ bulge cells also showed increased methylation levels by immunofluorescence and its quantification (Fig. 7d,e). When we interrogated timing of early anagen onset at the expected later stages (∼PD56–62), the mice treated with DI (Fig. 7a, scheme 2), showed a delay relative to untreated (no DI) mice both by morphology and by number of proliferative hair germ cells (Fig. 7f–h). Finally, mice treated with DI in catagen (PD35–42; Fig. 7a, scheme 3) failed to grow new hair in the subsequent hair cycle when monitored long-term up to PD98, whereas control mice clearly showed hair shaft growth throughout substantial portions of the shaved region (Fig. 7i). Taken together, these results demonstrate the importance of reducing H3 K4/K9/K27 trimethylation levels in the skin during quiescence for proper subsequent hair follicle homeostasis.


Signalling couples hair follicle stem cell quiescence with reduced histone H3 K4/K9/K27me3 for proper tissue homeostasis.

Lee J, Kang S, Lilja KC, Colletier KJ, Scheitz CJ, Zhang YV, Tumbar T - Nat Commun (2016)

The level of histone H3 K4/K9/K27me3 in the skin is important for hair homeostasis.(a) Experimental scheme of using demethylase inhibitors (DIs). (b,c) Western blot (b) and quantification of band intensity (c) of histone methylation from whole skin chromatin extracts of control and DI-treated mice. Band intensities were normalized to H3 signal. (d) Immunofluorescence staining of control and DI-treated (scheme 1 of (a)) hair follicle sections labelled with H3K9me3 (red) and CD34 (green). Scale bar, 20 μm. (e) Fluorescence intensity of all three marks from immunostainings were quantified for each bulge cell (CD34+). Student's t-test was used to determine statistical significance between control and DI-treated mice for each mark. (f) Immunofluorescence staining with a proliferation marker Ki67 of control and DI-treated (scheme 2 of a) skin sections. Scale bar, 20 μm. (g) The number of Ki67+ cells in f in each hair follicle were counted, and they showed reduced proliferation in DI-treated mice compared with control. The differences across the conditions were significant according to a Student's t-test. (h) Hair follicles of control and DI-treated mice were categorized in their hair cycle stages. Notice a dramatic increase in the number of telogen (Telo) hair follicles and reduction in anagen (Ana) I and II in DI-treated mice. (i) Mice were shaved, treated with DI in catagen (Cata; PD35–42; a, scheme 3), and followed long-term to monitor the hair cycle re-entry. Notice that mice in the control group show hair growth, whereas mice in DI-treated group show no growth. Avg, average.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f7: The level of histone H3 K4/K9/K27me3 in the skin is important for hair homeostasis.(a) Experimental scheme of using demethylase inhibitors (DIs). (b,c) Western blot (b) and quantification of band intensity (c) of histone methylation from whole skin chromatin extracts of control and DI-treated mice. Band intensities were normalized to H3 signal. (d) Immunofluorescence staining of control and DI-treated (scheme 1 of (a)) hair follicle sections labelled with H3K9me3 (red) and CD34 (green). Scale bar, 20 μm. (e) Fluorescence intensity of all three marks from immunostainings were quantified for each bulge cell (CD34+). Student's t-test was used to determine statistical significance between control and DI-treated mice for each mark. (f) Immunofluorescence staining with a proliferation marker Ki67 of control and DI-treated (scheme 2 of a) skin sections. Scale bar, 20 μm. (g) The number of Ki67+ cells in f in each hair follicle were counted, and they showed reduced proliferation in DI-treated mice compared with control. The differences across the conditions were significant according to a Student's t-test. (h) Hair follicles of control and DI-treated mice were categorized in their hair cycle stages. Notice a dramatic increase in the number of telogen (Telo) hair follicles and reduction in anagen (Ana) I and II in DI-treated mice. (i) Mice were shaved, treated with DI in catagen (Cata; PD35–42; a, scheme 3), and followed long-term to monitor the hair cycle re-entry. Notice that mice in the control group show hair growth, whereas mice in DI-treated group show no growth. Avg, average.
Mentions: Next, we examined the potential implication of histone demethylases in erasure of histone H3 K4/K9/K27me3 marks in HFSCs at catagen, and probed the functional significance of these levels. We employed demethylase chemical inhibitors (DIs) known to be specific for H3 K4me3, K9me3 (ref. 28) and K27me3 (ref. 29), shown by biochemical and structural studies to have potent and specific activities towards these modifications. We applied a cocktail of DI onto mouse back skin from anagen (PD32) up to different time points, and assessed the proliferation status of hair follicle in the subsequent hair cycle stage at early anagen (Fig. 7a). We used mice from a 1:1 mixed (CD1 × Fvb) genetic background, which we previously found to have a relatively shorter second telogen and more consistent early onset of the second adult anagen2730, than other common mouse strains. Western blot analysis of histones extracted from total mouse skin DI-treated for 7 days (PD32–39; Fig. 7a, scheme 1) showed increased histone methylation levels (Fig. 7b,c) confirming the activity of DI in vivo. Similarly, CD34+ bulge cells also showed increased methylation levels by immunofluorescence and its quantification (Fig. 7d,e). When we interrogated timing of early anagen onset at the expected later stages (∼PD56–62), the mice treated with DI (Fig. 7a, scheme 2), showed a delay relative to untreated (no DI) mice both by morphology and by number of proliferative hair germ cells (Fig. 7f–h). Finally, mice treated with DI in catagen (PD35–42; Fig. 7a, scheme 3) failed to grow new hair in the subsequent hair cycle when monitored long-term up to PD98, whereas control mice clearly showed hair shaft growth throughout substantial portions of the shaved region (Fig. 7i). Taken together, these results demonstrate the importance of reducing H3 K4/K9/K27 trimethylation levels in the skin during quiescence for proper subsequent hair follicle homeostasis.

Bottom Line: The level of marks over specific gene promoters did not correlate to mRNA level changes in quiescent HFSCs.Furthermore, removal of proliferation factors and addition of BMP4 reduced histone methylases and increased demethylases mRNAs in cultured skin epithelial cells.We conclude that signalling couples hair follicle stem cell quiescence with reduced H3 K4/K9/K27me3 levels for proper tissue homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.

ABSTRACT
Mechanisms of plasticity to acquire different cell fates are critical for adult stem cell (SC) potential, yet are poorly understood. Reduced global histone methylation is an epigenetic state known to mediate plasticity in cultured embryonic SCs and T-cell progenitors. Here we find histone H3 K4/K9/K27me3 levels actively reduced in adult mouse skin and hair follicle stem cells (HFSCs) during G0 quiescence. The level of marks over specific gene promoters did not correlate to mRNA level changes in quiescent HFSCs. Skin hypomethylation during quiescence was necessary for subsequent progression of hair homeostasis (cycle). Inhibiting BMP signal, a known HFSC anti-proliferative factor, elevated HFSC methylation in vivo during quiescence prior to proliferation onset. Furthermore, removal of proliferation factors and addition of BMP4 reduced histone methylases and increased demethylases mRNAs in cultured skin epithelial cells. We conclude that signalling couples hair follicle stem cell quiescence with reduced H3 K4/K9/K27me3 levels for proper tissue homeostasis.

No MeSH data available.


Related in: MedlinePlus