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Large portions of our genome contain important regulatory functions which remain uncharacterized. Bioinformatic (e.g. conservation1) and biochemical (e.g. ChIP-seq2,3) approaches have provided significant inroads into identifying non-coding regions of the genome with potential regulatory functions. New studies and methods are necessary to characterize the functional activity of these putative regulatory regions and specificity in complex tissues such as the skin. The skin consists of multiple layers and cell types. In the skin, many of the cells express genes from three clustered loci, which contain keratin and barrier-function genes4-6. Coding and non-coding regions of these clusters are frequently implicated in genetic conditions. Like other clustered loci, these loci are highly conserved (Fig. 1) and are thought to retain this organization due to selective pressures to maintain regulatory networks necessary for co-expressed genes. Notably, differential expression of genes located in the three clusters often distinguish specific layers and cell types. Thus, regulatory domains within these clusters likely control spatial and cell-type specific gene expression. The methods to test the spatial activity of epithelial enhancers however remain limited.

In the definitive epidermis, at least four stages of development can be identified: basal, spinous, granular, and cornified envelope keratinocytes (Fig. 2). While during early development, these four stages appear in temporal order, emerging evidence from our lab and others suggest additional mechanisms play a role in the spatial identity of each layer. To address spatial regulation of keratin and barrier genes in the definitive epidermal tissue, we have identified a mouse model in which specific epithelial identities are absent from which P300 ChIP-seq was performed (Fig. 2). P300 sites which are lost in these mice are likely to correspond to regulatory regions important for layer-specific gene expression. Following identification of putative layer-specific enhancers, their activity was assayed through cell-based and a modified graft-based assay. Notably, this latter assay provides for a simple, high-throughput method to determine the spatial regulatory activity of epidermal enhancers.

GOALS

RESULTS (continued) RESULTS (continued)

REFERENCES

ACKNOWLEDGEMENTS

Division of Dermatology1, Department of Medicine2, Institute for Genomic Medicine3, Ludwig Cancer Institute4, Dept. of Cellular Molecular Medicine5, School of Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

Shantanu Kumar1-3, Suguna R. Krishnaswami1-3, Drew Grainger1-3, Christopher Adase1-3, Craig Ricker1-3, Y. Shen4,5, Bing Ren3,4,5, and Benjamin D. Yu1-3

A high-throughput approach to validate enhancers responsible for spatial patterning in the skin

RESULTS & CONCLUSION A major goal of this study was to systematically identify and validate regulatory domains within clustered loci which are active in the skin, keratin type I (KC1), keratin type II (KC2), and epidermal differentiation complex (EDC). Using a mouse model where intermediate cell types are lost, we observed a loss of ~50% of P300 sites within the KC2 locus (Fig 3). Regulatory changes occur at these sites, including H3K27ac, in mutant vs. wildtype epithelium and during normal embryonic development (Fig. 4). Experimental validation of enhancer activity showed ~80% of putative layer-specific mouse enhancers activate in response to differentiation in a human keratinocyte differentiation assay (Fig. 5). A graft-based assay was developed to reproduce the organization of epidermal development (Fig. 6) and to identify layer-specific activity of putative enhancers (Fig. 7). Overall, this approach allows for the rapid assessment of epidermal regulatory elements embedded within clustered loci.

Fig. 2. Spatial organization of the definitive epidermis (Left) Progenitors located in the stratum basale (sb) divide and give rise to post-mitotic daughter cells which move upward in the stratum spinousum (ss). Upon further differentiation, cells again move upward to the stratum granulosum (sg) layer and then the stratum corneum (sc). (Right) In hyperproliferative conditions such as psoriasis, intermediate cell types are often lost. This condition is also seen in children with congenital mutations in BRAF, e.g. cardiofaciocutaneous syndrome.

Fig. 6. Validation of epidermal engraftment model. (A) Epidermal cysts form in nu/nu mice injected with human neonatal keratinocytes. One week after injection, cysts are sectioned and stained (B) Staining of cysts for basal K14 (red) and spinous markers K10 (green) (C) Multiple epidermal stages spontaneously form in cyst model. Basal layer (KRT5/14pos), spinous (Krt1/10pos), granular (green FLGpos; not shown), final layer, cornifiied envelope (Lce3a/3bpos) in situ hybridization is shown.

Implanted keratinocytes generate layered cysts which recapitulate spatial pattern of epidermis

Fig. 1. Conservation within the keratin type II cluster (KC2). Strong conservation within intergenic regions (red) can be identified in visual-ization from ECR Browser (Ovcharenko 2004). Peaks demonstrate conservation over 50%.

Fig. 4. Increase H3K27ac at putative enhancers during appear-ance of differentiated layers in the E14.5 -> E17.5 epidermis. (A) Differentiated layers appear between E14.5 to E18.5. (B) Wildtype H3K4me1 changes at P300-sites between E14.5-17.5. (C) Wildtype H3K27ac increases between E14.5-17.5. Note: first two columns represent enhancer sites which do not change (positive controls).

Putative layer-specific enhancers identified by differential P300 occupancy

Putative layer-specific enhancers show increased H3K27 acetylation during development

1.  Ovcharenko et al (2004) Nucleic Acids Research 32: W280-W286 2.  Heintzman ND et al (2007) Distinct and predictive chromatin signatures

of transcriptional promoters and enhancers in the human genome. Nat Genet 39: 311–318.

3.  Visel A et al (2009) ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457: 854–858

4.  Bossuyt F (2012) Radiation and functional diversification of alpha keratins during early vertebrate evolution. Mol Bio Evol 29(3):995-1004

5.  Romano V (1997) Human type I cytokeratin genes are a compact cluster. Cytogenet Cell Genet 77(3-4):169-174

6.  Segre JA (2010) A milieu of regulatory elements in the epidermal differentiation complex syntenic block: implications for atopic dermatitis and psoriasis. Hum Mol Genet 19(8):1453-1460.

7.  Visel A et al (2007). VISTA Enhancer Browser-a database of tissue-specific human enhancers. Nucleic Acids Res 35:D88-92

INTRODUCTION RESULTS (continued)

Fig. 7. Enhancer drives spatial activation of GFP reporter consistent with granular layer-specific activity. (A) Low power view of epidermal cyst 1 week after injection in brightfield, positive fluorescence in unfixed tissue and merged image. (B) Immunofluorescent staining of cyst wall demonstrates spatial patterning of epidermis, basal (red, P63), spinous (red, KRT10), involucrin (red, IVL), and stage-specific activation of GFP (green, anti-GFP).

c = P300 loci present in both wt and mutant

Change in enrichment from E14.5 to E17.5 epidermis

spinous

spinous + granular

mature

basal

WT BRAFVE

H3K27Ac  LICR/ENCODE (H3K4me1)

mul

tiple

tiss

ues

(non

-epi

derm

al)

BrafVE

YU (P300, E17.5 epidermis)

• • • • • • • • • • • •

UCSC Tracks

wt

10 kb

Fig. 3. P300 ChIP-seq analysis of wildtype and BRAF epidermis and loss of putative enhancers in mutant epidermis. (A) Loss of intermediate stages of epidermal differentiation in K14-cre; BrafV600E E17.5 mice. Earlier to later steps in differentiation are ordered from bottom to top and detected by in situ hybridization. (B) Pooled mouse epidermis from four control and four mutant E17.5 embryos were used for P300 ChIP-seq. (C) Novel putative enhancers in mouse epidermis relative to non-skin enhancers; loss of specific P300-loci (red circles) in BrafV600E mice. .

60 µM calcium (undifferentiated)

1.5 mM calcium (differentiated)

Putative layer-specific enhancers activate reporter expression in upper layers of cysts

A

B

A B

A

B

A

B

C

A B

C

C

Fig. 5. Cell-based keratinocyte differentiation assay (A) Schematic of cell-based assay. High calcium condition induces differentiation of human keratinocytes in 3 days. (B) Generation of lentiviral reporters (enhancer + minimal Hsp68 promoter-GFP [Ref. 7]). Introduction of reporter in human keratinocyte and demonstration of activity in differentiation media vs. non-differentiating media. (Bottom panel, green) represents merge images of brightfield/GFP of loci 3-46. >80% of layer-specific enhancers activate during differentiation.

P63 (basal) GFP

KRT10 (spinous) GFP

IVL (granular) GFP

ACKNOWLEDGEMENTS This work was supported by generous grants from the NIH/NIAMS , California Institute for Regenerative Medicine RN2-00908, and institutional funds to B.D.Y..

H3K4me1

H3K27ac

% C

hang

e  

% C

hang

e  

normal hyperproliferative conditions

Control MuKrt71-200 MuKrt71-800 MuKrt5-792 MuIGRKrt5-402 MuKrt5-65 (1) (2) (3) (4) (7) min promoter

- +

phas

e di

ff

- + - +

Putative layer-specific enhancers drive reporter expression in cell-based differentiation assays

c c 1-40 putative layer-specific enhancers

c c 1-40 putative layer-specific enhancers

sb ss sg

200K – 1 million human keratinocytes 6-8 subcut sites 1 week

E8.5

E9.5

E14.5 E15.5 E16.5 E17.5