Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice

Abstract

Adult stem cells occur in niches that balance self-renewal with lineage selection and progression during tissue homeostasis. Following injury, culture or transplantation, stem cells outside their niche often display fate flexibility1,2,3,4. Here we show that super-enhancers5 underlie the identity, lineage commitment and plasticity of adult stem cells in vivo. Using hair follicle as a model, we map the global chromatin domains of hair follicle stem cells and their committed progenitors in their native microenvironments. We show that super-enhancers and their dense clusters (‘epicentres’) of transcription factor binding sites undergo remodelling upon lineage progression. New fate is acquired by decommissioning old and establishing new super-enhancers and/or epicentres, an auto-regulatory process that abates one master regulator subset while enhancing another. We further show that when outside their niche, either in vitro or in wound-repair, hair follicle stem cells dynamically remodel super-enhancers in response to changes in their microenvironment. Intriguingly, some key super-enhancers shift epicentres, enabling their genes to remain active and maintain a transitional state in an ever-changing transcriptional landscape. Finally, we identify SOX9 as a crucial chromatin rheostat of hair follicle stem cell super-enhancers, and provide functional evidence that super-enhancers are dynamic, dense transcription-factor-binding platforms which are acutely sensitive to pioneer master regulators whose levels define not only spatial and temporal features of lineage-status but also stemness, plasticity in transitional states and differentiation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Dynamic super-enhancer remodelling facilitates lineage progression.
Figure 2: Super-enhancer epicentres confer tissue, lineage and temporal specificity.
Figure 3: The sensitivity of super-enhancers to environmental changes allows hair follicle stem cell adaptation and plasticity.
Figure 4: SOX9 is a pioneer factor governing hair follicle stem cell fate and plasticity.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

ChIP-seq data are deposited in GEO under accession number GSE61316.

Change history

  • 20 May 2015

    Minor corrections were made to Fig. 3.

References

  1. 1

    Spradling, A., Drummond-Barbosa, D. & Kai, T. Stem cells find their niche. Nature 414, 98–104 (2001)

  2. 2

    Lopez-Garcia, C., Klein, A. M., Simons, B. D. & Winton, D. J. Intestinal stem cell replacement follows a pattern of neutral drift. Science 330, 822–825 (2010)

  3. 3

    van Es, J. H. et al. Dll1+ secretory progenitor cells revert to stem cells upon crypt damage. Nature Cell Biol. 14, 1099–1104 (2012)

  4. 4

    Blanpain, C. & Fuchs, E. Stem cell plasticity. Plasticity of epithelial stem cells in tissue regeneration. Science 344, 1242281 (2014)

  5. 5

    Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013)

  6. 6

    Hsu, Y. C., Li, L. & Fuchs, E. Transit-amplifying cells orchestrate stem cell activity and tissue regeneration. Cell 157, 935–949 (2014)

  7. 7

    Ezhkova, E. et al. EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair. Genes Dev. 25, 485–498 (2011)

  8. 8

    Lien, W. H. et al. Genome-wide maps of histone modifications unwind in vivo chromatin states of the hair follicle lineage. Cell Stem Cell 9, 219–232 (2011)

  9. 9

    Loven, J. et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153, 320–334 (2013)

  10. 10

    Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013)

  11. 11

    Gosselin, D. et al. Environment drives selection and function of enhancers controlling tissue-specific macrophage identities. Cell 159, 1327–1340 (2014)

  12. 12

    Lavin, Y. et al. Tissue-resident macrophage enhancer landscapes are shaped by the local microenvironment. Cell 159, 1312–1326 (2014)

  13. 13

    Liu, Z. et al. Enhancer activation requires trans-recruitment of a mega transcription factor complex. Cell 159, 358–373 (2014)

  14. 14

    Dowen, J. M. et al. Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes. Cell 159, 374–387 (2014)

  15. 15

    Folgueras, A. R. et al. Architectural niche organization by LHX2 is linked to hair follicle stem cell function. Cell Stem Cell 13, 314–327 (2013)

  16. 16

    Kadaja, M. et al. SOX9: a stem cell transcriptional regulator of secreted niche signaling factors. Genes Dev. 28, 328–341 (2014)

  17. 17

    Keyes, B. E. et al. Nfatc1 orchestrates aging in hair follicle stem cells. Proc Natl Acad Sci USA 110, E4950–E4959 (2013)

  18. 18

    Chang, C. Y. et al. NFIB is a governor of epithelial-melanocyte stem cell behaviour in a shared niche. Nature 495, 98–102 (2013)

  19. 19

    Lien, W. H. et al. In vivo transcriptional governance of hair follicle stem cells by canonical Wnt regulators. Nature Cell Biol. 16, 179–190 (2014)

  20. 20

    Siersbaek, R. et al. Transcription factor cooperativity in early adipogenic hotspots and super-enhancers. Cell Rep. 7, 1443–1455 (2014)

  21. 21

    Luyten, A., Zang, C., Liu, X. S. & Shivdasani, R. A. Active enhancers are delineated de novo during hematopoiesis, with limited lineage fidelity among specified primary blood cells. Genes Dev. 28, 1827–1839 (2014)

  22. 22

    Kim, T. H. et al. Broadly permissive intestinal chromatin underlies lateral inhibition and cell plasticity. Nature 506, 511–515 (2014)

  23. 23

    Beronja, S., Livshits, G., Williams, S. & Fuchs, E. Rapid functional dissection of genetic networks via tissue-specific transduction and RNAi in mouse embryos. Nature Med. 16, 821–827 (2010)

  24. 24

    Nowak, J. A., Polak, L., Pasolli, H. A. & Fuchs, E. Hair follicle stem cells are specified and function in early skin morphogenesis. Cell Stem Cell 3, 33–43 (2008)

  25. 25

    Ellis, T. et al. The transcriptional repressor CDP (Cutl1) is essential for epithelial cell differentiation of the lung and the hair follicle. Genes Dev. 15, 2307–2319 (2001)

  26. 26

    Blanpain, C., Lowry, W. E., Geoghegan, A., Polak, L. & Fuchs, E. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118, 635–648 (2004)

  27. 27

    Chen, T. et al. An RNA interference screen uncovers a new molecule in stem cell self-renewal and long-term regeneration. Nature 485, 104–108 (2012)

  28. 28

    Wu, X. et al. Skin stem cells orchestrate directional migration by regulating microtubule-ACF7 connections through GSK3β. Cell 144, 341–352 (2011)

  29. 29

    Tumbar, T. et al. Defining the epithelial stem cell niche in skin. Science 303, 359–363 (2004)

  30. 30

    Means, A. L., Xu, Y., Zhao, A., Ray, K. C. & Gu, G. A. CK19(CreERT) knockin mouse line allows for conditional DNA recombination in epithelial cells in multiple endodermal organs. Genesis 46, 318–323 (2008)

  31. 31

    Vasioukhin, V., Degenstein, L., Wise, B. & Fuchs, E. The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc. Natl Acad. Sci. USA 96, 8551–8556 (1999)

  32. 32

    Nowak, J. A. & Fuchs, E. Isolation and culture of epithelial stem cells. Methods Mol. Biol. 482, 215–232 (2009)

  33. 33

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

  34. 34

    Zhang, Y. et al. Model-based analysis of ChIP-seq (MACS). Genome Biol. 9, R137 (2008)

  35. 35

    McLean, C. Y. et al. GREAT improves functional interpretation of cis-regulatory regions. Nature Biotechnol. 28, 495–501 (2010)

  36. 36

    Ye, T. et al. seqMINER: an integrated ChIP-seq data interpretation platform. Nucleic Acids Res. 39, e35 (2011)

  37. 37

    Williams, S. E., Beronja, S., Pasolli, H. A. & Fuchs, E. Asymmetric cell divisions promote Notch-dependent epidermal differentiation. Nature 470, 353–358 (2011)

Download references

Acknowledgements

We thank S. Mazel, L. Li, S. Semova, and S. Tadesse for FACS sorting (Rockefeller University FACS facility); and C. Lai for assistance in high-throughput sequencing (Rockefeller University Genomics Resource Center). We thank E.F. laboratory members A. Aldeguer, S. Hacker, M. Sribour and J. Levorse for assistance in mouse research; I. Matos for advice on image acquisition; J. Racelis, S. Chai, and E. Wong for technical assistance; and Y. Ge, S. Naik, A. Kulukian, and N. Oshimori for discussions. R.C.A. was supported by the Anderson Cancer Center Graduate Student Fellowship. E.F. is an HHMI Investigator. This work was supported by grants from the National Institutes of Health (R01-AR31737 to E.F. and R21MH099452 to D.Z.).

Author information

R.C.A. and E.F. conceived the project and designed the experiments. R.C.A. and H.Y. performed the experiments, including FACS purification, ChIP-seq assays, data analysis and in vivo reporter assays. D.S.O. and L.P. carried out in utero lentiviral injections and wounding experiments with mice. S.B.L., M.N. and M.K. contributed to in vitro experiments. A.A., S.R. and D.Z. performed bioinformatics analyses. E.F. supervised the project. R.C.A. and E.F. wrote the manuscript.

Correspondence to Elaine Fuchs.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 FACS purification strategy to isolate hair follicle stem cells and TACs.

a, FACS purification of wild-type hair follicle stem cells for ChIP-seq according to established markers α6hi and CD34+26. Sca1 is used to remove basal epidermal cells. b, FACS purification of TACs from Krt14-H2B–GFP mice29. TACs are GFPloSca1α6lo/−CD34. c, Epifluorescence of Krt14-driven H2B–GFP. Hair follicle stem cells and epidermal cells are GFPhi, whereas TACs are GFPlo. d, q-PCR to verify the FACS sorting strategy and measure enrichment of cell-type-specific marker genes. Mean and standard deviation are shown (n = 3). P values from t-test: P < 0.05; P < 0.01; P < 0.001, relative to hair follicle stem cells.

Extended Data Figure 2 Enhancer distribution, size and gene assignment in hair follicle stem cells.

a, Distribution of H3K27ac occupancy at promoter and enhancers in hair follicle stem cells. b, Distribution of typical-enhancers and super-enhancers in hair follicle stem cells. c, Enhancer size distribution in hair follicle stem cells. d, Number of individual H3K27ac peaks per gene. Super-enhancers are clusters of H3K27ac peaks and mainly consist of ≥ 5 peaks per gene. e, f, Enhancer-gene assignments, exemplified by hair follicle stem cell super-enhancers Fzd6 and Btg2. FPKM, fragments per kilobase of transcript per million mapped reads (RNA-seq). g, Differential expression for genes driven by hair follicle stem cell super-enhancers and typical-enhancers. P values from t-test: P < 0.001. h, Density plot, contrasting expression levels of typical-enhancer versus super-enhancer associated hair follicle stem cell genes in hair follicle stem cells compared to epidermal progenitors. Note cell type-specific differences in expression for hair follicle stem cell genes controlled by super-enhancers but not typical-enhancers. i, Gene Ontology analysis of genes controlled by hair follicle stem cell enhancers. j, List of selected super-enhancer regulated hair follicle stem cell genes. SE, super-enhancer; TE, typical-enhancer.

Extended Data Figure 3 Hair follicle stem cell TFs are enriched within super-enhancers and cluster in epicentres.

a, b, Enrichment of hair follicle stem cell TFs within chromatin of super-enhancers, but not typical-enhancers. Comparisons were made with 377 randomly selected typical-enhancers and their flanking sequence extended 5′ and 3′ to match the average length of super-enhancers (average of 3 analyses is shown). Each ‘TF event’ (a) represents one hair follicle stem cell TF bound within a super-enhancer. ‘TF peaks’ (b) refers to the absolute amount of TFs occupying the super-enhancer. c, Heatmap showing ChIP-seq read densities (from −5 kb to +5 kb of peak centre) across H3K27ac peaks located in super-enhancers. Note that hair follicle stem cell TFs frequently bound densely together with strong H3K27ac peaks. d, Motif analysis of hair follicle stem cell super-enhancers for putative TF binding sites. e, Analysis of distance of H3K27ac peaks to their nearest transcription factor ChIP-seq peaks in hair follicle stem cells in vivo (distance of the two peak centres). Note that enrichment of TF binding occurs within 1-kb regions of H3K27ac peaks (‘epicentres’). f, Frequency and distribution of hair follicle stem cell super-enhancer epicentres. g, Rare ‘atypical’ enhancers co-bound by 7 hair follicle stem cell TFs are more highly expressed in hair follicle stem cells versus committed progenitors.

Extended Data Figure 4 Identification of super-enhancers in TACs.

a, Distribution of H3K27ac ChIP-seq signal across all enhancers in transit-amplifying cells (TACs) reveals 381 super-enhancers of little overlap with hair follicle stem cell super-enhancers. b, Tracking the status of TAC super-enhancers in hair follicle stem cells indicates striking enhancer remodelling upon lineage progression. Example shows the appearance of a de novo super-enhancer for the Dlx3/4 locus as hair follicle stem cells commit to a TAC fate. c, Examples of super-enhancer associated genes in TACs. Genes in green have a reported function in hair follicles.

Extended Data Figure 5 Super-enhancer reporters drive cell-type specific expression.

a, The lentiviral control (CTRL) reporter construct (containing no enhancer) is silent throughout all stages of the hair cycle, despite efficient infection (as evidenced by H2B–mRFP1). b, Immunofluorescence showing that Cxcl14-eGFP super-enhancer reporter activity co-localizes with Krt24+ hair follicle stem cells. DP, dermal papilla; Bu, Bulge. White dashed lines denote the epidermal–dermal border; solid lines delineate the DP. c, H3K27ac and MED1 ChIP-seq occupancy at the Cux1 locus in TACs. Red box shows the super-enhancer epicentre that was cloned for reporter assays. Note that epicentres bound by MED1 are sufficient to identify cell-stage specific loci, even without prior information about lineage-specific TFs. d, CUX1 expression pattern in hair follicles.

Extended Data Figure 6 Hair follicle stem cells adapt to microenvironmental changes by reversible remodelling of super-enhancers.

a, Absence of Cxcl14-SE-eGFP reporter activity in transduced cultured hair follicle stem cells. b, Transplanted cultured hair follicle stem cells establish de novo hair follicles and regain expression of hair follicle stem cell TFs. c, Note extensive hair follicle stem cell super-enhancer remodelling upon culture conditions. d, Hair follicle stem cells in vitro are molecularly distinct from activated hair follicle stem cells (aHFSC) in vivo. eh, H3K27ac levels at the Cxcl14, Sfrp1, Lhx2 and Ehf loci in hair follicle stem cells in vivo and in vitro. Note the dynamic regulation of super-enhancers and the resulting changes in gene expression. i, Selected list of super-enhancer associated genes in hair follicle stem cells in vitro. j, Note hair follicle stem cell super-enhancer plasticity in vitro and during wound repair: Fhl2 and Prrg4 display super-enhancer-mediated activity in vitro. Upon transplantation, hair follicle stem cells silence in vitro induced genes concomitant with hair follicle regeneration. However, during wounding, hair follicle stem cells (lineage marked with K19-CreER/R26YFP) regain expression of Fhl2 and Prrg4.

Extended Data Figure 7 Hair follicle stem cells activate different epicentres within super-enhancers to sustain expression of critical genes in different microenvironments.

a, b, H3K27ac and hair follicle stem cell TF ChIP-seq occupancies at the Macf1 and Rad51b loci in hair follicle stem cells in vivo and in vitro. Regions C, E and F mark epicentres active in vivo, richly bound by hair follicle stem cell TFs; adjacent regions D and G are novel epicentres active in vitro. Relative luciferase activities were driven by the 1–1.5 kb encompassing these epicentres. Mean and standard deviation are shown (n = 3). P values from t-test: P < 0.001. Functional validation of epicentre shifts in vivo. eGFP-reporter activity of in vitro epicentres is highly active in the epidermis, while physiological hair follicle stem cell epicentres are restricted to the hair follicle niche. c, Motif analysis of Macf1 epicentres (regions A and B, Fig. 3e) for putative TF binding sites. d, Number and distribution of hair follicle stem cell super-enhancer epicentres in vitro. e, Frequency of epicentre shifts in hair follicle stem cell super-enhancers (in vivo versus in vitro). Note that corresponding to the loss of hair follicle stem cell TFs in vitro, many super-enhancers display epicentre shifts to maintain expression of critical genes (for example, Macf1) in different microenvironments.

Extended Data Figure 8 Hair follicle stem cell TFs are reduced outside the niche but are sensitive to Sox9 levels.

a, SOX9 is expressed and displays nuclear localization in hair follicle stem cells in vitro. b, Colony formation assays on wild-type and Sox9-cKO hair follicle stem cells. Sox9fl/fl Rosa26YFPfl/+ hair follicle stem cells were seeded at 2,000 and 4,000 and transduced with lentiviral-Cre to achieve Sox9 ablation in vitro. All yellow and green colonies were not effectively targeted and are still SOX9+. All red colonies (SOX9-negative) aborted, as revealed by quantifications of colony numbers and sizes shown at right. c, Sox9-overexpression in cultured hair follicle stem cells. SOX9 induces the expression of Tle4, Tcf7l1, Tcf7l2 and Lhx2. d, e, Hair follicle stem cell TFs are expressed at substantially lower levels in basal epidermal progenitors in vivo or in cultured epidermal keratinocytes relative to hair follicle stem cells. f, Downregulation of hair follicle stem cell TFs in Sox9-cKO hair follicle stem cells in vivo before hair follicle stem cells are lost. g, Doxycycline-inducible overexpression of Lhx2 in cultured epidermal keratinocytes does not induce hair follicle stem cell TFs. For bg, mean and standard deviation are shown (n = 3). P values from t-test: P < 0.05; P < 0.01; P < 0.001; n.d., not detected; n.s., not significant.

Extended Data Figure 9 Sustained Sox9 expression in committed progenitors perturbs lineage progression.

a, Sustained Sox9 in adult mice (doxycycline for 3 weeks in adult mice, starting at P21) leads to de novo formation of minibulge-like structures along the ORS. b, Immunofluorescence showing that Lef1 (normally H3K27me3 repressed in hair follicle stem cells, but H3K27ac super-enhancer induced in TACs) remains repressed in mycSOX9+ hair follicles.

Supplementary information

Supplementary Table 1

This file comprises of 3 sheets as follows: sheet 1- ‘HFSC SE in vivo’ list of H3K27ac super-enhancers of hair follicle stem cells (HFSCs) in vivo: chromosomal coordinates and corresponding gene assignments. The table also contains the criteria for enhancer-gene assignments; sheet 2 - ‘TAC SE in vivo’ list of H3K27ac super-enhancers of transit-amplifying cells (TACs) in vivo: chromosomal coordinates and corresponding gene assignments. The table also contains the criteria for enhancer-gene assignments; and sheet 3 - ‘HFSC SE in vitro’ list of H3K27ac super-enhancers of hair follicle stem cells (HFSCs) in vitro: chromosomal coordinates and corresponding gene assignments. The table also contains the criteria for enhancer-gene assignments. (XLSX 127 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Adam, R., Yang, H., Rockowitz, S. et al. Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice. Nature 521, 366–370 (2015). https://doi.org/10.1038/nature14289

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.