This study aims to characterize regulatory elements that control spatial gene expression in the skin. The researchers used P300 ChIP-seq in mouse skin to identify putative layer-specific enhancers. They developed assays to test enhancer activity, including a cell-based differentiation assay and a graft-based assay using human keratinocytes. Around 80% of putative enhancers activated reporter expression in differentiating keratinocytes in the cell-based assay. The graft-based assay showed layer-specific reporter expression driven by putative enhancers, recapitulating the spatial patterning of skin. This approach allows rapid validation of epidermal enhancers responsible for spatial gene regulation.

1. RESEARCH POSTER PRESENTATION DESIGN © 2012
www.PosterPresentations.com
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)
multipletissues
(non-epidermal)
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
%Change
%Change
normal hyperproliferative conditions
Control MuKrt71-200 MuKrt71-800 MuKrt5-792 MuIGRKrt5-402 MuKrt5-65
(1) (2) (3) (4) (7)min promoter
-
+
phasediff
-
+
-
+
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