Jarid2 regulates mouse epidermal stem cell activation and differentiation
2011; Springer Nature; Volume: 30; Issue: 17 Linguagem: Inglês
10.1038/emboj.2011.265
ISSN1460-2075
AutoresStefania Mejetta, Lluís Morey, Gloria Pascual, Bernd Kuebler, Matthew R. Mysliwiec, Youngsook Lee, Ramin Shiekhattar, Luciano Di Croce, Salvador Aznar Benitah,
Tópico(s)Skin and Cellular Biology Research
ResumoArticle2 August 2011free access Jarid2 regulates mouse epidermal stem cell activation and differentiation Stefania Mejetta Stefania Mejetta Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Lluis Morey Lluis Morey Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Gloria Pascual Gloria Pascual Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Bernd Kuebler Bernd Kuebler Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Matthew R Mysliwiec Matthew R Mysliwiec University of Wisconsin Medical School, Madison, WI, USA Search for more papers by this author Youngsook Lee Youngsook Lee University of Wisconsin Medical School, Madison, WI, USA Search for more papers by this author Ramin Shiekhattar Ramin Shiekhattar The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Luciano Di Croce Luciano Di Croce Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Salvador Aznar Benitah Corresponding Author Salvador Aznar Benitah Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Stefania Mejetta Stefania Mejetta Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Lluis Morey Lluis Morey Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Gloria Pascual Gloria Pascual Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Bernd Kuebler Bernd Kuebler Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Matthew R Mysliwiec Matthew R Mysliwiec University of Wisconsin Medical School, Madison, WI, USA Search for more papers by this author Youngsook Lee Youngsook Lee University of Wisconsin Medical School, Madison, WI, USA Search for more papers by this author Ramin Shiekhattar Ramin Shiekhattar The Wistar Institute, Philadelphia, PA, USA Search for more papers by this author Luciano Di Croce Luciano Di Croce Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Salvador Aznar Benitah Corresponding Author Salvador Aznar Benitah Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain Search for more papers by this author Author Information Stefania Mejetta1, Lluis Morey1, Gloria Pascual1, Bernd Kuebler1, Matthew R Mysliwiec2, Youngsook Lee2, Ramin Shiekhattar3, Luciano Di Croce1,4 and Salvador Aznar Benitah 1,4 1Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, Barcelona, Spain 2University of Wisconsin Medical School, Madison, WI, USA 3The Wistar Institute, Philadelphia, PA, USA 4Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain *Corresponding author. Department of Differentiation and Cancer, Center for Genomic Regulation (CRG) and UPF, CRG and ICREA, Dr Aiguader 8, Barcelona 08003, Spain. Tel.: +34 93 316 0212; Fax: +34 93 316 0099; E-mail: [email protected] The EMBO Journal (2011)30:3635-3646https://doi.org/10.1038/emboj.2011.265 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Jarid2 is required for the genomic recruitment of the polycomb repressive complex-2 (PRC2) in embryonic stem cells. However, its specific role during late development and adult tissues remains largely uncharacterized. Here, we show that deletion of Jarid2 in mouse epidermis reduces the proliferation and potentiates the differentiation of postnatal epidermal progenitors, without affecting epidermal development. In neonatal epidermis, Jarid2 deficiency reduces H3K27 trimethylation, a chromatin repressive mark, in epidermal differentiation genes previously shown to be targets of the PRC2. However, in adult epidermis Jarid2 depletion does not affect interfollicular epidermal differentiation but results in delayed hair follicle (HF) cycling as a consequence of decreased proliferation of HF stem cells and their progeny. We conclude that Jarid2 is required for the scheduled proliferation of epidermal stem and progenitor cells necessary to maintain epidermal homeostasis. Introduction Polycomb (PcG) proteins are global transcriptional repressors that balance cell lineage choices during embryonic development (Surface et al, 2010). Misregulation of PcG proteins impinges on different aspects of tumour onset and progression of various types of human neoplasias (Sauvageau and Sauvageau, 2010). The two major polycomb complexes in mammals, polycomb repressive complex 1 (PRC1) and 2 (PRC2), interact with chromatin to sequentially establish distinct post-translational modifications of histone tails, which determine the degree of transcriptional repression of specific loci. The mammalian PRC2 complex is established by the interactions of the Ezh2 (or Ezh1), Eed, Suz12, and RBAP46/48 subunits (Cao et al, 2002; Czermin et al, 2002; Kuzmichev et al, 2002; Cao and Zhang, 2004; Kirmizis et al, 2004; Margueron et al, 2008; Shen et al, 2009). Accumulation at transcriptional start sites of the methyltransferase Ezh2 subunit, which trimethylates lysine 27 of histone H3 (to give H3K27me3), correlates with transcriptional repression. The PRC1 complex, which is comprising the CBX, RING1, BMI, and PH proteins (Levine et al, 2002), contributes to promoter silencing by catalysing the mono-ubiquitination of histone H2A on lysine 119. It has been shown that the chromodomains of the CBX subunits have an affinity for H3K27me3, which would allow for the simultaneous occupancy of PRC1 and PRC2, leading to further transcriptional repression (de Napoles et al, 2004; Wang et al, 2004; Kouzarides, 2007). Several publications have recently shown that Jarid2, the founding member of the Jumonji/JmjC domain-containing family of proteins, is necessary for the recruitment of PRC2 to promoters in ES cells (Peng et al, 2009; Shen et al, 2009; Landeira et al, 2010; Li et al, 2010; Pasini et al, 2010). However, whereas the role of Jarid2 in the genomic recruitment of PRC2 is undisputed, its impact on PRC2 activity is less clear (Landeira and Fisher, 2011; Herz and Shilatifard, 2010). In this sense, this interaction has been reported by some to inhibit (Peng et al, 2009; Shen et al, 2009), and by others to potentiate (Li et al, 2010; Pasini et al, 2010), the H3K27me3 methyltransferase activity of PRC2. In addition, although the reduced genomic recruitment of PRC2 in Jarid2-depleted ESCs translated into more-or-less pronounced changes in the transcript levels of polycomb target genes, it was accompanied by either no, or only subtle, changes in H3K27me3 enrichment at their promoter regions. Furthermore, Jarid2 also recruits PRC1 to the promoters of lineage-commitment genes and which are poised for expression during the initial steps of ESC differentiation (Landeira et al, 2010). Deletion of PRC2 subunits in ESCs causes profound changes in the expression of lineage-commitment genes, resulting in early lethality (Sauvageau and Sauvageau, 2010; Surface et al, 2010). However, less is known about the function of PRC2 in adult tissues. Deletion of Ezh2 causes defects in proliferation and differentiation of haematopoietic, pancreatic, and brain progenitors (Su et al, 2003; Chen et al, 2009; Majewski et al, 2010; Pereira et al, 2010). With respect to the epidermis, deletion of Ezh2 results in progenitors that have a reduced proliferative potential and undergo premature differentiation during embryonic development (Ezhkova et al, 2009). In addition, deletion of both Ezh2 and Ezh1 results in a progressive hair loss and in hyperproliferation of basal epidermal cells (Ezhkova et al, 2009, 2011). However, the mechanism by which PRC2 is recruited to specific loci in adult tissues, and which factors modulate its function, is still poorly understood. In addition, a role for Jarid2 plays in the genomic recruitment of PRC2 in adult tissues has not been described yet. Here, we have analysed the effects of deleting Jarid2 on late murine epidermal development, as well as on adult epidermal homeostasis. We have characterized the role of Jarid2 over murine epidermal and hair follicle (HF) embryonic development, postnatal morphogenesis, and adult homeostasis. In addition, we have studied whether epidermal deletion of Jarid2 had any effect over PRC2 activity. Results and discussion Epidermal deletion of Jarid2 does not affect embryonic development of the epidermis We conditionally deleted Jarid2 expression in murine epidermis by crossing Jarid2loxP/loxP mice with transgenic mice expressing Cre recombinase under the regulation of the basal layer keratinocyte-specific keratin-14 promoter (hereafter referred to as Jarid2 cKO mice; Mysliwiec et al, 2006). We verified by genomic PCR, RT–qPCR of Jarid2 transcript, and western immunoblotting that Jarid2 was efficiently deleted in newborn epidermal keratinocytes of Jarid2 cKO mice (Supplementary Figure S1A, S1C and S1D). In addition, we verified that Cre expression was homogenous throughout the entire epidermal compartment by crossing Jarid2 cKO mice with ROSA26-YFP transgenic mice (Supplementary Figure S1B). The keratin-14-Cre transgene drives Cre expression during development of embryonic ectoderm at post-coitum day 13.5 (E13.5), coinciding with the onset of embryonic epidermal stratification. Therefore, our mouse model allowed us to study whether Jarid2 deletion had any effect on epidermal development. Histological analysis of the epidermis of E14.5–E19.5 Jarid2wt/wt/K14Cre (wt mice), and Jarid2 cKO littermate embryos, did not reveal any changes in epidermal stratification, thickness, and overall morphology (Figure 1A). The expression of proliferation markers (Ki67), basal keratin-14 (K14), and terminal differentiation genes (loricrin) was indistinguishable between wt and Jarid2 cKO mice (Figure 1B). Figure 1.Epidermal deletion of Jarid2 does not affect embryonic development of the epidermis. (A) Haematoxylin–eosin staining revealed no abnormalities in epidermal embryonic development of Jarid2 cKO mice. At least three control (K14Cre/Jarid2wt/wt) and cKO mice were analysed for each time point shown. Scale bar: 100 μm. (B) Immunofluorescence of Ki67 and loricrin indicates that proliferation and differentiation are not modified during embryonic epidermal development upon Jarid2 deletion. Scale bar: 50 μm. (C) Higher magnification of skin sections shows a normal architecture for the embryonic hair follicles in absence of Jarid2. Scale bar: 50 μm. Download figure Download PowerPoint Embryonic HFs start to form from basal epidermal progenitors that invaginate around E15.5, to form hair placodes (Schneider et al, 2009). From E15.5 to birth, hair placodes grow inwards to give rise to embryonic HF structures, which contain embryonic stem cells that remain present throughout the entire life of the organism. No changes in the number, spacing, or morphology of the HF placodes were observed in Jarid2 cKO embryos, or in any of the subsequent HF embryonic morphogenesis stages from E15.5 until birth (Figure 1C). Additionally, HF placodes from wt and Jarid2 cKO embryos expressed similar levels of proliferation markers and basal keratin-14 (Figure 1B). Therefore, Jarid2 is dispensable for late embryonic epidermal development and HF morphogenesis. Interestingly, contrary to the expression pattern of Ezh2 in embryonic epidermis (Ezhkova et al, 2009), the expression of Jarid2 was the same in basal and suprabasal epidermal progenitors isolated from embryonic epidermis at E16.5 and E18.5 by FACS on the basis of their differential expression of integrin α6 (α6bright and α6dim, respectively; Supplementary Figure S2). Given that epidermal deletion of Ezh2 resulted in premature expression of terminal differentiation markers during embryonic epidermal development (Ezhkova et al, 2009), it is likely that the Ezh2-containing PRC2 complex regulates this process independently of Jarid2. Deletion of Jarid2 results in enhanced postnatal epidermal differentiation We next studied whether deletion of Jarid2 affected early postnatal epidermal morphogenesis. Expression of Jarid2 decreases as embryonic stem cells differentiate (Peng et al, 2009; Shen et al, 2009; Landeira et al, 2010; Li et al, 2010; Pasini et al, 2010). Intriguingly, although the expression of Jarid2 was similar between basal and suprabasal embryonic epidermal keratinocytes (Supplementary Figure S2), FACS sorting by α6 integrin expression revealed that basal epidermal progenitors isolated from neonatal P0 epidermis (with high α6; α6bright) had high Jarid2 transcript levels, while suprabasal differentiated cells (α6low) exhibited decreased Jarid2 transcript levels (Figure 2A). Figure 2.Jarid2 deletion results in enhanced postnatal epidermal differentiation. (A) Differential expression of Jarid2 in basal and suprabasal interfollicular epidermal cells, from P1 wild-type mice, that were FACS sorted on the basis of their cell surface levels of α6-integrin (bright, dim, and low). Data shown represent the mean average; vertical bars represent the s.e.m. N=12; *P< 0.05 and **P 2 wt and 2 cKO for time points. (C) The interfollicular epidermis of Jarid2 cKO mice contained a thicker filaggrin-positive layer than control mice. Scale bar: 50 μm. (D) Higher magnification of filaggrin staining of Jarid2 wt and Jarid2 cKO mice highlights the differences in the thickness of the granular layer. Scale bar: 25 μm. (E) Expression of filaggrin and loricrin is increased in newborn mouse keratinocytes isolated from Jarid2 cKO mice compared with control littermates. Keratin-14 and DAPI (green and blue fluorescence, respectively) are shown as counterstains. Pictures are representative of three independent experiments from three different litters of mice. Scale bar: 100 μm. (F) Western blot showing increased filaggrin expression in Jarid2 cKO newborn keratinocytes. Total H3 is used for normalization. (G) Deletion of Jarid2 reduced the clonogenic potential of newborn (P0) and adult (8-weeks old) primary mouse keratinocytes. Representative colonies from control and Jarid2 cKO mice are shown. Quantification of the colonies number per well is reported in the right. Values are presented as mean+s.e.m.; *P<0.05 and **P<0.01; N=3. (H) The interfollicular epidermis of Jarid2 cKO mice is less proliferative than that of control mice. Left panel: sorted α6-integrin bright cells from P1 Jarid2 cKO mice express lower transcript levels of Ki67 than their control littermates (n=14 wt, n=12 cKO; the vertical bar represents the s.e.m.; **P<0.05). Right panel: Quantification of Ki67+ cells in the interfollicular epidermis of Jarid2 wt and Jarid2 cKO newborn mice. N=4 mice for each genotype; vertical bar represents s.e.m.; **P<0.05. Download figure Download PowerPoint During the first 2 weeks after birth, strong proliferation is coupled with differentiation in the epidermis, to establish the definitive adult epidermal barrier. In addition, the first pelage emerges from the HF structures that formed during embryonic development (Schneider et al, 2009). A daily time course analysis from postnatal days 0 (P0) to 7 (P7) indicated that Jarid2 cKO mice had an expanded suprabasal granular layer and a thickened cornified envelope, compared with control littermates (Figure 2B). Expression of the interfollicular epidermis granular layer marker filaggrin was increased upon deletion of Jarid2, indicating an increased epidermal differentiation upon depletion of Jarid2 (Figure 2C and D). Enhanced expression of filaggrin was also observed by immunofluorescence and western immunoblotting in cultured primary mouse keratinocytes isolated from Jarid2 cKO P0 epidermis compared with control littermates (Figure 2E and F). The enhanced differentiation of the interfollicular epidermis was accompanied by a reduction in the proliferative potential of basal interfollicular progenitors. In this sense, both newborn and adult keratinocytes purified from Jarid2 cKO epidermis formed fewer proliferative clones as compared with wt cells (Figure 2G). Jarid2 cKO cells not only formed fewer colonies but also displayed a morphology characteristic of terminally differentiated abortive keratinocytes, as expected from the increase expression of differentiation markers (Figure 2G). Accordingly, we scored a statistically significant reduction in the number of proliferating basal interfollicular cells, as well as the transcript levels of the proliferation marker Ki67, in Jarid2 cKO epidermis compared with wt epidermis (Figure 2H). Although the interfollicular epidermis of neonatal mice was less proliferative upon depletion of Jarid2, both postnatal HF morphogenesis and pelage growth were unaffected in Jarid2 cKO mice (Figure 3A). Accordingly, proliferation along the outer root sheath and the matrix of wild-type and Jarid2 cKO HFs was undistinguishable (Figure 3B). Figure 3.Jarid2 does not influence postnatal hair follicle development. (A) Neonatal hair follicle morphogenesis is not altered upon the loss of Jarid2. Scale bar: 100 μm. (B) Ki67 expression (red fluorescence) was unchanged in the hair follicles of neonatal Jarid2 mice compared with control mice, but was slightly reduced in the basal IFE. Keratin-14 staining is shown as green fluorescence. Scale bar: 100 μm. Download figure Download PowerPoint Altogether, these results indicate that loss of Jarid2 reduces the proliferative potential of basal epidermal progenitors while enhancing their differentiation, whereas it has no effect on the formation of the first wave of postnatal pelage formation. Deletion of Jarid2 results in inefficient entry of HFs into anagen At postnatal days P8–P10, a population of quiescent epidermal stem cells is stably established at a permanent region of the HF, located just below the sebaceous glands, termed the bulge (Nowak et al, 2008). Bulge cells remain slow cycling throughout the entire lifetime of the organism, and only undergo a reduced number of proliferative rounds during each cycle of adult hair morphogenesis (Tiede et al, 2007). After the first bout of postnatal pelage formation, all the HFs synchronously undergo a second phase of HF growth (anagen) between P20 and P31, to establish the adult pelage (Blanpain et al, 2004; Morris et al, 2004; Tumbar et al, 2004; Schneider et al, 2009). Maintenance of the interfollicular epidermis does not depend on HF stem cells (Morris et al, 2004; Tumbar et al, 2004; Levy et al, 2005), but relies on continuous proliferation of basal interfollicular epidermal cells for its maintenance during adulthood (Clayton et al, 2007). We, therefore, next studied whether deletion of Jarid2 had (i) any impact on the maintenance of the quiescent bulge stem cell population; (ii) activation of bulge stem cells during synchronous HF growth; and (iii) homeostasis of basal interfollicular epidermal progenitors. Similar to the situation in the neonatal epidermis, expression of Jarid2 in adult P19 mouse skin was highest in basal undifferentiated epidermal progenitors (α6bright/CD34neg), and lower in the progressively differentiated keratinocytes (α6dim and α6low populations; Figure 4A). Expression of Jarid2 was also gradually reduced upon differentiation (as measured by the cell surface levels of integrin α6) of adult human epidermal keratinocytes, directly isolated from foreskin samples and maintained in culture (Supplementary Figure S3). Figure 4.Deletion of Jarid2 results in inefficient entry of hair follicles into anagen in adult epidermis. (A) Jarid2 expression was reduced in suprabasal interfollicular epidermal cells, compared with basal cells, in adult wild-type mice. Adult mouse keratinocytes were sorted from the backskin of P19 mice. Data are presented as mean+s.e.m.; N=3 pools of 4 mice each; *P<0.09 and **P<0.05. (B) Jarid2 cKO mice did not show significant changes in the proportion of bulge stem cells (α6bright/CD34+) with respect to control littermates. FACS profiles shown correspond to P31 mice. N=4 Jarid2 wt and 4 Jarid2 cKO for each time point analysed. (C) Deletion of Jarid2 resulted in a delay in the onset and progression of anagen between P21 and P31. Scale bar: 200 μm. N=8 controls and 7 cKO for each time point analysed. (D) Ki67 staining in P24 and P28, both control and Jarid2 cKO mice, indicates reduced proliferation in matrix, bulge, and ORS. Scale bar: 100 μm. The right panel shows whole-mount Ki67 immunostaining of tail epidermis of control and Jarid2 cKO P24 mice. Jarid2 cKO mice showed a significant reduction in the number of Ki67+ cells in the bulge, outer root sheath and matrix of late anagen (white arrow) and early anagen (light blue arrow) hair follicles. Scale bars: 100 and 25 μm, respectively. Quantification of the number of Ki67+ cells for either anagen (upper part) or telogen (lower graph) bulge is reported. Data are presented as average+s.e.m. **P<0.05; N=20 bulges. (E) Jarid2 deletion does not affect the number of bulge label-retaining cells (LRCs) pulsed chased for 8 weeks. Scale bars: 100 and 50 μm. Right panel: measurement of the number of BrdU+ bulge cells by FACS analysis in the epidermis of 8-week-old Jarid2 wt and Jarid2 cKO mice. Data are presented as average+s.e.m. n.s. indicates non-statistically significant. N=4 Jarid2 wt and 4 Jarid2 cKO. Download figure Download PowerPoint At adulthood, the percentage of bulge stem cells in the dorsal skin of Jarid2 cKO mice, as determined by their high expression of α6 integrin and CD34 (α6bright/CD34pos), was virtually identical to that of control mice, indicating that Jarid2 is not necessary to establish or to maintain HF bulge stem cells (Figure 4B). However, although the total number of bulge stem cells was unaffected in Jarid2 cKO mice, the mice displayed a significant delay in the onset and the progression of hair follicles into anagen from postnatal days P21 to P31, with respect to control littermates (Figure 4C). Accordingly, proliferation of hair germ cells, outer root sheath cells, and matrix cells, all of which are necessary to fuel HF anagen (Ito et al, 2004; Greco et al, 2009; Hsu et al, 2011), was significantly delayed in Jarid2 cKO mice between P21 and P31 as compared with control littermates (Figure 4D, only P24 and P28 time points are shown as representative). In addition, we observed a significant reduction in the number of Ki67+ proliferative cells in the bulge of HFs in full anagen (Figure 4D). In spite of these changes in proliferation, the HFs of Jarid2 cKO mice resumed their anagen cycle, resulting in normal, albeit delayed, hair growth. Although bulge proliferation was affected at late stages of anagen, a similar number of bulge cells were capable of stably accumulating BrdU, following an 8-week chase, in Jarid2 cKO and wt mice (Figure 4E). Jarid2 cKO HFs were more inefficient than wt ones in becoming active not only in physiological conditions, but also when ectopically stimulated enter anagen. In this sense, Jarid2 cKO bulge and hair matrix cells were less efficient in responding to topical administration of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which causes bulge hyperproliferation and ectopic anagen entry (Supplementary Figure S4A). Overall, Jarid2 cKO mice did end up responding to TPA treatment, displaying HFs in full anagen (Supplementary Figure S4B). At last, while the interfollicular epidermis of JaridKO mice was responsive to TPA, it displayed a thicker cornified layer than treated wild-type littermates and a higher expression of the differentiation marker loricrin, similar to the effect observed in the interfollicular epidermis of neonatal Jarid2 cKO mice (Supplementary Figure S4C). These results suggest that Jarid2 activity is essential in instances where strong proliferation is required (i.e., during postnatal morphogenesis, at the peak of anagen, and during TPA-induced hyperproliferation). However, deletion of Jarid2 does not impede either normal, or ectopically induced, entry of HFs into the anagen state, indicating that Jarid2 is required for the scheduled activation of HF bulge stem cells and their progeny, rather than being essential for their proliferation per se. Deletion of Jarid2 results in reduced PRC2 genomic occupancy, lower H3K27me3, and increased expression of PRC2-epidermal target genes in neonatal, but not adult epidermis Depletion of Jarid2 in embryonic stem cells significantly impairs binding of the PRC2 core complex subunits to their genomic targets (Herz and Shilatifard, 2010; Landeira and Fisher, 2011). However, the effect of Jarid2 on the histone H3K27 methyltransferase activity of PRC2, and its consequences for the expression of polycomb targets in ES cells or adult stem cells, is still open to debate. We first confirmed that endogenous Jarid2 interacted with endogenous PRC2 (Suz12 and Ezh2) in primary mouse keratinocytes (Figure 5A). We, therefore, next tested whether epidermal deletion of Jarid2: (i) affected genomic targeting of PRC2; (ii) modified the chromatin, by affecting the H3K27 trimethylation (H3K27me3) levels; and (iii) changed the expression of epidermal differentiation genes that have previously shown to be PRC2 targets in epidermal basal progenitors (Ezhkova et al, 2009). Figure 5.Jarid2 modulates PRC2 activity in newborn epidermal keratinocytes. (A) Endogenous Jarid2 co-immunoprecipitates with endogenous Ezh2 and Suz12 PRC2 in newborn mouse keratinocytes. (B) Jarid2 localizes to the promoters of PRC2 targets in newborn mouse keratinocytes, as shown by ChIP-qPCR. Data are presented as fold enrichment and are representative of three independent experiments. (C) ChIP analysis in newborn mouse keratinocytes isolated from Jarid2 cKO (n=15) and control mice (n=20) indicates that the occupancy of Suz12 in the promoters of epidermal differentiation and non-epidermal-specific PRC2 targets genes, is reduced in Jarid2 cKO mice. Data are presented as percentage of input. (D) The levels of H3K27me3 are reduced in the promoters of the same cohort of genes in Jarid2 cKO cells. Data are presented as fold enrichment normalized to total histone H3. (E) The levels of H3K27me3 in non-epidermal targets of PRC2 are reduced in Jarid2cKO cells. (F) The global level of H3K27me3 is slightly reduced in Jarid2 cKO mouse keratinocytes compared with the control cells. Total H3 was used as a control of equal loading. All ChIP data shown is statistically significant with a P-value <0.05. Download figure Download PowerPoint We first performed these analyses in keratinocytes from newborn mice, at which time Jarid2 cKO and control mice showed clear differences in interfollicular epidermal differentiation (Figure 2). Jarid2 localized to the proximal promoter region of several PRC2-target genes (both epidermal and non-epidermal specific), as determined by chromatin immunoprecipitation (ChIP) followed by quantitative PCR (Ezhkova et al, 2009; Figure 5B). We next tested whether deletion of Jarid2 would affect the occupancy of Suz12 (i.e., PRC2) and the levels of H3K27me3 in the promoter regions of epidermal differentiation genes, previously shown to be targets of PRC2 (Ezhkova et al, 2009). Jarid2 cKO neonatal keratinocytes had reduced levels of Suz12 and H3K27me3 in both epidermal differentiation genes and non-epidermal specific PRC2 targets (note that p16, Olig2, Neurog1, and Neurog2 are representative of the non-epidermal PRC2 targets; Figure 5C–E). The overall levels of H3K27me3 around the TSS of epidermal differentiation genes were considerably lower than in non-epidermal-specific PRC2 targets, in agreement with previous findings (Figure 5D; Ezhkova et al, 2009). In addition, Jarid2 cKO cells had lower levels of overall H3K27me3 than control cells (Figure 5F). This slight reduction, albeit significant, was similar to that observed upon deletion of Jarid2 both in the levels of H3K27me3 in some lineage-commitment genes, and total level of H3K27me3, in ES cells (Peng et al, 2009; Landeira et al, 2010; Li et al, 2010; Pasini et al, 2010). As expected from our in vivo and ChIP analyses, loss of Jarid2 correlated with an increased transcription of this same group of
Referência(s)