0

200
400
600
800
1,000
1,200
1,400
1,600
All SCDM
genes
SCDM not
MS
(subset of
previous)
All MS
genes
MS not
SCDM
(subset of
previous)
Genes that
are MS &
SCDM
N
u
m
b
e
r
o
f
g
e
n
e
s
present
absent




References
Anderson, GE. The Life History and Genetics of Coprinus lagopus. Cardiff, UK: Harris Biological Supplies Ltd. (1971).
Burns C, Stajich JE, Rechtsteiner A, Casselton L, Hanlon SE, Wilke SK, Savytskyy OP, Gathman AC, Lilly WW, Lieb
JD, Zolan ME, Pukkila PJ. Analysis of the Basidiomycete Coprinopsis cinerea reveals conservation of the core
meiotic expression program over half a billion years of evolution. PLoS Genetics 6 (9) e1001135 (2010).
Giresi, PG and Lieb, JD. Isolation of active regulatory elements from eukaryotic chromatin using FAIRE
(Formaldehyde Assisted Isolation of Regulatory Elements). Methods 48; 233239 (2009).
Rashid NU, Giresi PG, Ibrahim JG, Sun W, Lieb JD. ZINBA integrates local covariates with DNA-seq data to identify
broad and narrow regions of enrichment, even within amplified genomic regions. Genome Biol. Jul 25;12(7):R67
(2011).

FAIRE Sample Preparation
and Analysis Methods
Gel shows the DNA shearing at various time points prior to
sequencing library prep. The circled lanes were used for FAIRE
and INPUT samples
FAIRE sample INPUT sample
FAIRE, or Formaldehyde Assisted Isolation of
Regulatory Elements, enriches for open chromatin
FAIRE DNA was
obtained from
protoplasts prepared
from oidia, the
monokaryotic, asexual
spore phase of the C.
cinerea lifecycle.
Defining Open Chromatin Regions in Coprinopsis cinerea Oidia by FAIRE


Virginia K. Hench and Patricia J. Pukkila
Department of Biology, University of North Carolina, Chapel Hill, NC 27599 USA
Giresi & Lieb, 2009
Obtain tissue or cell sample
Treat with formaldehyde
Wash twice
Lyse
Sonicate
Phenol/Chloroform extraction
Proteinase K/RNase, Ethanol
precipitation
Sequence
Protocol Summary

C. cinerea lifecycle.
Library prep and sequencing optimization was performed by
Shweta Deshpande, Chee-Hong Wong and Chia-Lin Wei at JGI.
Align reads and call peaks using ZINBA.
Map relative positioning of peaks and other genomic features
using Galaxy (http://galaxy.psu.edu/).

Robinson JT, Thorvaldsdttir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP. Integrative Genomics
Viewer. Nature Biotechnology 29, 2426 (2011).
Stajich JE, Wilke SK, Ahren D, Au CH, Birren BW, Borodovsky M, Burns C, Canback B, Casselton LA, Cheng CK, Deng
J, Dietrich FS, Fargo DC, Farman ML, Gathman AC, Goldberg J, Guigo R, Hoegger PJ, Hooker JB, Huggins A,
James TY, Kamada T, Kilaru S, Kodira C, Kues U, Kupfer D, Kwan HS, Lomsadze A, Li W, Lilly WW, Ma LJ,
Mackey AJ, Manning G, Martin F, Muraguchi H, Natvig DO, Palmerini H, Ramesh MA, Rehmeyer CJ, Roe BA,
Shenoy N, Stanke M, Ter-Hovhannisyan V, Tunlid A, Velagapudi R, Vision TJ, Zeng Q, Zolan ME, Pukkila PJ.
Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis
cinerea (Coprinus cinereus). Proceedings of the National Academy of Science 107: 11889-94 (2010).
FAIRE peak distribution across the C. cinerea genome
Peak calling by ZINBA revealed 7,276 FAIRE peaks covering 6.3% of the genome. The figure below shows a 59 kb
window of chromosome 2. The FAIRE peaks called by ZINBA are indicated by the royal blue track. Note that called FAIRE peaks
line up with the center of peaks in the track showing continuous FAIRE sequence alignment data. Broad version 3 gene predictions
are indicated by the green track.

Figure above shows continuous sequence alignment data (*.wig) for FAIRE & Input samples. Genome position and peak height are
indicated on the horizontal and vertical axes, respectively. The enormous peak on chromosome 6 is associated with rDNA repeats.
Input
FAIRE
1 2 3 4 5 6 7 8 9 10 11 12 13
Chromosome Number
Input
FAIRE
FAIRE peaks
called by ZINBA
Br3 gene predictions
FAIRE peaks
chiefly occur in
intergenic regions
with 78% of peak-
coding sequence
overlapping with
noncoding
sequence.
Approximately 47% of annotated
genes have a FAIRE peak in the
proximal promoter region
(defined as the region 500 bps
upstream of the predicted TSS).
Arrows drawn in green clarify the
direction of transcription.
Divergent genes
w/shared upstream
FAIRE peak
Divergent genes
w/out upstream
FAIRE peak
Gene w/proximal
upstream FAIRE
peak
Gene w/proximal
upstream FAIRE
peak
Promoter FAIRE peak status in oidia does not
predict meiotic transcriptional profile


Conclusions & Future
Directions
The mapping of FAIRE peaks in oidia
in relation to all annotated genes and
specifically in relation to genes
classified by their meiotic
transcriptional profile, suggests that
nucleosome occupancy contributes to
differential, tissue-specific gene
expression along with other factors in
C. cinerea.

Next, we will use FAIRE to analyze
meiotic chromatin because we want to
investigate potential meiotic-specific
chromatin dynamics involving both
transcription and recombination.

Burns et al previously classified
genes according to their expression
profile during meiosis. Genes could
be meiotic specific (MS),
significantly changing during
meiosis (SCDM) or both MS &
SCDM. We observed that a large
fraction of MS genes (63%) lacked
promoter FAIRE peaks in oidia
(light blue bars), including well-
characterized genes such as spo11,
dmc1 and rec8. In contrast, a
minority of SCDM genes that are
also expressed in vegetative cells
(39%) lacked promoter FAIRE
peaks in oidia.

Promoter FAIRE
peak in oidia:
Abstract

Changes in chromatin organization are
principal regulatory mechanisms controlling
multiple cellular processes including gene
expression and meiotic crossover formation.
Here we present FAIRE (formaldehye assisted
isolation of regulatory elements) data that
reveal regions of open chromatin in
Coprinopsis cinerea oidia, the asexual spore
stage of the C. cinerea life cycle. A standard
FAIRE protocol was developed and optimized
for oidia and used to enrich for nucleosome-
free stretches of chromatin. FAIRE peaks were
identified from single-end read whole genome
sequence data using ZINBA (Zero-Inflated
Negative Binomial Algorithm), which
identified 7,276 peaks covering 6.3% of the
genome. FAIRE peaks are predominantly
intergenic with 78% of FAIRE domains
overlapping noncoding sequence. The peak
widths range from 98-1390 bps, with an
average width of 310 bps. Nearly half or 47% of
annotated genes (Broad version 3) contain a
FAIRE peak in the proximal promoter region
(defined as 500 bps immediately upstream of
the gene start). Differential transcription has
been characterized throughout the
synchronous meiotic process in C. cinerea
(Burns, C. et al., PLOS Genetics, vol 6, issue 9,
2010), but the extent to which nucleosome
occupancy might contribute to gene regulation
in this multicellular fungus was not known. We
found that a minority of meiotic specific (MS;
genes expressed in meiosis and not in
vegetative tissue) genes had promoter FAIRE
peaks (of 819 genes 37% had promoter FAIRE
peaks). In contrast, 61% of genes significantly
changing during meiosis (SCDM; 2,455 genes)
had promoter FAIRE peaks. Out of 295 genes
that were MS and SCDM, 38% had promoter
FAIRE peaks in oidia. Genes with known
meiotic function including spo11, dmc1, and
rec8 were amongst the MS/SCDM genes that
did not have promoter FAIRE peaks in oidia.
In summary, the different patterns of
nucleosome occupancy in promoters of meiotic
genes indicates that complex gene regulation
mechanisms contribute to differential, tissue-
specific gene expression in C. cinerea.
Supported by the U.S. Department of Energy
Joint Genome Institute Community
Sequencing Program. The work conducted by
the U.S. DOE JGI is supported by the Office of
Science of the U.S. Department of Energy
under Contract No. DE-AC02-05CH11231.