Epidermal stem cells are defined by global histone modifications that are altered by Myc-induced differentiation

Abstract

Activation of Myc induces epidermal stem cells to exit their niche and differentiate into sebocytes and interfollicular epidermis, a process that is associated with widespread changes in gene transcription. We have identified chromatin modifications that are characteristic of epidermal stem cells and investigated the effects of Myc activation. Quiescent stem cells in the interfollicular epidermis and the hair follicle bulge had high levels of tri-methylated histone H3 at lysine 9 and H4 at lysine 20. Chromatin in both stem cell populations was hypoacteylated at histone H4 and lacked mono-methylation of histone H4 at lysine 20. Myc-induced exit from the stem cell niche correlated with increased acetylation at histone H4 and transiently increased mono-methylation at lysine 20. The latter was replaced by epigenetic modifications that are largely associated with chromatin silencing: di-methylation at histone H3 lysine 9 and histone H4 lysine 20. These modifications correlated with changes in the specific histone methyltransferases Set8 and Ash-1. The Myc-induced switch from mono- to di-methylated H4K20 required HDAC activity and was blocked by the HDAC inhibitor trichostatin A (TSA). TSA treatment induced a similar epidermal phenotype to activation of Myc, and activation of Myc in the presence of TSA resulted in massive stimulation of terminal differentiation. We conclude that Myc-induced chromatin modifications play a major role in Myc-induced exit from the stem cell compartment.

Figure 1. Histone modifications in basal layer of human interfollicular epidermis.

Double label immunofluorescence staining of whole mounts with antibodies to ß1 integrins (red) and (green) H3diK4 (A–C), H3diK9 (D–F), H3triK9 (G–I) and H4Ac (J–L), with DAPI nuclear counterstain (blue). C, F, I, L show merged images (left hand panels) and higher magnification views (right hand panels). Scale bars: 20 µm.

Figure 2. Variation in histone H3 methylation in mouse epidermis and effects of Myc activation.

Immunofluorescence staining for LRC (red) and (green) H3diK4 (A, G, J), H3diK9 (B, H, K) and H3triK9 (C, I, L) in whole mounts of wild type (A–C, G–I) and 4OHT treated K14MycER (J–L) epidermis. Position of hair follicle bulge is marked by line in A–C. Inserts in A–C show higher magnification views of bulge LRC. G–L show LRC in interfollicular epidermis. (D–F, M–O) Quantitation (±S.D.) of number of LRC per bulge (D–F) and interfollicular epidermis (M–O) that had high or low levels of staining (see Fig. S1) with antibodies to the H3 modifications indicated. Scale bars: 100 µm (A–C), 20 µm (G–L).

Figure 3. Variation in histone H4 acetylation and methylation in mouse epidermis and effects of Myc activation.

(A–C, E–G, I–K) Immunofluorescence staining of whole mounts of wild-type (A–C, F, G, K) and 4OHT treated K14MycER transgenic (E,I, J) epidermis with antibodies to (green) H4Ac (A–C, E), H4monoK20 (F, G, I, J) and H4triK20 (K). Double labelling for LRC (red) is shown in C, G, I, K. I and inserts in C, G, K show high magnification views of LRC. IFE: interfollicular epidermis; sebaceous gland (SG), bulge (Bg), bulb (Bb) are marked with arrows in A, E. Hair follicles and sebaceous glands are marked with dashed lines in A, B, E, F, J; LRC are marked with dashed lines in C, G, I, K; position of bulge is marked with line in C, G, K. D, H, L show quantitation (±S.D.) of number of LRC per bulge with high or low levels of the H4 modifications indicated. M–R: immunofluorescence labelling (green) with DAPI counterstain (blue) of wild-type (M–O) and 4OHT treated K14MycER (P–R) dorsal interfollicular epidermis using antibodies to H4monoK20 (M, P), H4diK20 (N, Q) and keratin 10 (O, R). Dashed lines indicate position of basement membrane. Scale bar: 50 µm

Figure 4. Effects of TSA treatment and Myc activation on histone H4 modification.

(A–D) whole mounts of wild-type (A, B) and K14MycER (C, D) epidermis treated with 4OHT alone (A, C) or in combination with TSA (B, D), labelled with antibody to H4monoK20 (green) and counterstained with DAPI (blue). Hair follicles and sebaceous glands (SG) are outlined with dashed lines. Bg: bulge; Bb: bulb (A–D). (E–P) Double immunofluorescence labelling of cultured human keratinocytes transduced with empty vector (k-pBabe; E, F, I, J, M, N) or MycER (k-MycER; G, H, K, L, O, P) and treated for 24 h with 4OHT alone (E, I, M, G, K, O) or 4OHT+TSA (F, J, N, H, L, P). Cells were stained with antibodies to H4monoK20 (red) and H4diK20 (green) with DAPI counterstain (blue). E–H are merged images of the panels below. (Q,R) Quantitation (±S.D.) of % cells per colony with high levels of H4monoK20 (Q) or H4diK20 (R). Cells were stimulated with 4OHT alone (4OHT) or 4OHT and TSA (TSA). P values (t-test) are *0.0046, **0.0505 (Q) and *0.0545, **0.0211, ***0.0192 (R). Scale bar: 100 µm (A–D), 20 µm (E–P).

Figure 5. Expression of histone methyl transferases in response to Myc and TSA.

(A) Western blots of keratinocytes infected with empty vector (K-pBabe) or MycER (K-MycER) and treated with 4OHT±TSA for the number of hours indicated. Blots were probed with antibodies to the proteins indicated. Tubulin (Tub) was used as a loading control. (B) Immunostaining for Ash-1 (red) with DAPI counterstain (blue). Scale bar: 10 µm.

Figure 6. TSA treatment induces similar changes in epidermal proliferation and differentiation to activation of Myc, and exacerbates the effects of Myc.

Wild-type (A, B, E, F, I, J, M, N, Q, R) and K14MycER transgenic (C, D, G, H, K, L, O, P, S, T) mice were treated with 4OHT alone (A, C, E, G, I, K, M, O, Q, S) or in combination with TSA (B, D, F, H, J, L, N, P, R, T). 4OHT treatment of wild-type epidermis served as a negative control. A–P are sections of back skin; Q–T are whole mounts of tail skin. Sections were stained with haematoxylin and eosin (A–H), anti-BrdU (brown, I–L) or anti Ki67 (brown, M–P). Whole mounts were stained for keratin 14 (green) with DAPI counterstain (blue). Arrows in B, C, F, G, H show thickening of interfollicular epidermis. Arrow in D shows detachment of epidermis from dermis. Arrowhead in F shows enlarged sebaceous gland. Arrowheads in J–L, N–P show different numbers of proliferating cells. SG: sebaceous glands. Scale bars: 100 µm

Figure 7. Model of histone modifications in different epidermal compartments and effect of Myc activation.

Red arrows indicate events stimulated by Myc. Stem cells and terminally differentiated cells are shown as having high levels of histone tri-methylation and low levels of histone acetylation, mono- and di-methylation. Myc increases global H4 acetylation and transient mono-methylation of H4K20, which is replaced di-methylation of H4K20, H3K4 and H3K9 at the expense of tri-methylation. HAT: histone acetyl transferase; HDAC: histone deacetyl transferase complex.