Dendritic cell (DC) lineages coordinate immune system activity through functional specialization. • Irf4, a transcription factor(TF), is required for CD11b+ DC lineage development from bone marrow stem cells and has been implicated in multiple inflammatory diseases, eg. asthma. • The epigenetic consequences of immune specialization in CD11b+ DCs and relation to inflammatory diseases remain largely unexplored partly due to the difficulty of using highly purified, and typically, limited populations of cells in ChIP-seq (chromatin immunoprecipitation then sequencing) assays. • A robust, multiplexed ChIP-seq protocol – using an input control, TF (CTCF) and histone modification marks (H3K9me3- methylation, H3K27ac-acetylation) - was developed using limited amounts of K562 cells, for the Ion ProtonTM system. • Peak-calling analysis was performed using using MACS2. • Significant data correlations were observed with ENCODE. • The Ion ProtonTM results are based on chromatin derived from 1 million(M) cells, making it viable for generating data from a limited number of primary cells. This is in contrast to the 10M cells recommended by ENCODE. • The developed methodology was used to compare Irf4 genomic binding sites generated from flow-sorted populations of 1, 3, 5, and 20M CD11b+ lineage murine DCs. • Comparable Irf4 ChIP-seq results were obtained from 5M versus 20M cells, indicating that as low as 5M flow-sorted cells can be used to acquire high quality(FDR: 10-19) data. • We identified genomic Irf4 binding sites proximal to genes, whose activity is consistent with CD11b+ DC lineage activity and/or known to contribute to inflammatory disease. • We examined Irf4 functional regulation of the identified gene targets via RNA-seq analysis with CD11b+ DCs and a related lineage, CD103+ DCs. Integrating expression analysis with ChIP-seq indicates a unique CD11b+ DC gene expression program concordant with Irf4 loci association in comparison to CD103+ DC (data not shown).

1. 1
Approach for limited cell ChIP-Seq on a
semiconductor-based sequencing platform
S. Ghosh1, K. Giorda1, R. Marcus2, M. Taylor1, E. Farias-Hesson1,2, D. Bluestein2, G. Meredith1, S. Leach2, B. O'Conner2
1Thermo Fisher Scientific, 180 Oyster Point Blvd., South San Francisco, CA 94080; 2National Jewish Health, Center for Genes, Environment
& Health, 1400 Jackson St., Denver, CO 80206
ABSTRACT
• Dendritic cell (DC) lineages coordinate immune system activity
through functional specialization.
• Irf4, a transcription factor(TF), is required for CD11b+ DC
lineage development from bone marrow stem cells and has
been implicated in multiple inflammatory diseases, eg. asthma.
• The epigenetic consequences of immune specialization in
CD11b+ DCs and relation to inflammatory diseases remain
largely unexplored partly due to the difficulty of using highly
purified, and typically, limited populations of cells in ChIP-seq
(chromatin immunoprecipitation then sequencing) assays.
• A robust, multiplexed ChIP-seq protocol – using an input
control, TF (CTCF) and histone modification marks (H3K9me3-
methylation, H3K27ac-acetylation) - was developed using
limited amounts of K562 cells, for the Ion ProtonTM system.
• Peak-calling analysis was performed using using MACS2.
• Significant data correlations were observed with ENCODE.
• The Ion ProtonTM results are based on chromatin derived from
1 million(M) cells, making it viable for generating data from a
limited number of primary cells. This is in contrast to the 10M
cells recommended by ENCODE.
• The developed methodology was used to compare Irf4 genomic
binding sites generated from flow-sorted populations of 1, 3, 5,
and 20M CD11b+ lineage murine DCs.
• Comparable Irf4 ChIP-seq results were obtained from 5M
versus 20M cells, indicating that as low as 5M flow-sorted cells
can be used to acquire high quality(FDR: 10-19) data.
• We identified genomic Irf4 binding sites proximal to genes,
whose activity is consistent with CD11b+ DC lineage activity
and/or known to contribute to inflammatory disease.
• We examined Irf4 functional regulation of the identified gene
targets via RNA-seq analysis with CD11b+ DCs and a related
lineage, CD103+ DCs. Integrating expression analysis with
ChIP-seq indicates a unique CD11b+ DC gene expression
program concordant with Irf4 loci association in comparison to
CD103+ DC (data not shown).
INTRODUCTION
ChIP-seq (Fig. 1) is used to enrich
and map binding sites for
transcription factors, histone
modification marks, and other
chromatin modifying complexes.
The limited cell, ChIP-seq,
multiplexed on a single P1 chip is
comprised of input control, a TF,
transcriptionally active histone
(H3K27ac) and histone silencer
(H3K27me3). The simplicity and
comprehensiveness of the
experimental design coupled with
limited cell ChIP-seq from 1M
cells that yields high quality data,
makes it an effective tool to study
salient aspects of an epigenetic
mechanism
u Approach for limited cell ChIP-Seq
Stem
cell
CD10
3+ DC
CD11
b+
DC
Irf8
Irf4
Fix Flow
sort Library
1 Million 3 Million 5 Million 20 Million
1 Million 3 Million 5 Million 20 Million
Master regulator of the antigen presentation pathway required
for DC function; known target of Irf4 [7]
Transcription factor that defines classical DC pathway
differentiation; known target of Irf4 [7]
Regulator of the antigen presentation pathway required for
DC function; known target of Irf4 [7]
Transcription factor regulator of immunity and DC function;
known target of Irf4 [7]
Transcription factor regulator of immunity and DC function;
known target of Irf4 [7]
Part of the coagulation pathway and known mediator of innate
immune defense, which includes DC
An E3 ligase with known immune regulation properties and
asthma activity
Complement receptor that regulates adaptive immunity and
DC function
Life Technologies • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
RESULTS
u A multiplexed template that would enable a
functional study via the triangulation of transcription
histone activation and silencing
Table 2. CTCF Motif analysis
CONCLUSIONS
• A proof of principle ChIP-seq design on the Ion
Proton™ system that allows for a functional analysis on
a single chip has been demonstrated.
• Robust profiling of binding sites can be done from
limited numbers of cells, i.e. 3-5M flow-sorted cells.
• The simple and comprehensive experimental design
highlighted by low input volume and quick turnaround
time – and potentially coupled with RNA-seq provides a
powerful tool for exploring regulation mechanism.
REFERENCES
1. MACS v2.0.10 https://github.com/taoliu/MACS/
2. ENCODE Consortium, (2012) Nature 489:57-74.
3. ENCODE comparisons are presented against BroadChromHMM, K562
cell-line data for Broad Histone, Hudson Alpha(HAIB) CTCF, HAIB RR
DNA methylation – genome.ucsc.edu
4. M Herold et al. (2012) Development 139:1045-57.
5. GEM(Motif analysis): Y Guo et al, (2012), PLoS Comput Biol 8(8)
6. D Martin et al, (2011), Nat Struct Mol Biol, 18(6):708-714
7. BV Lugt et al. (2014) Nat Immunol 15: 161–167
ACKNOWLEDGEMENTS
Colin Davidson, Ion Torrent
srinka.ghosh@thermofisher.com
.
CTCF - A transcription factor :
• A transcriptional repressor encoded in
human by the CTCF gene, is also an
insulator binding protein.
• ~41% of the loci[3] show significant
enrichment at insulator domains (Fig. 3).
• ~17% of the loci show enrichment for
promoter and enhancer domains, each[3].
• GEM[5] analysis (k_min=16, k_max=23)
recapitulates the MEME[6] based 17-bp
CTCF motif (Table 2).
Figure 3. [top] A schematic of
insulator locations [4];
[bottom] Example of CTCF
enrichment at insulators [2-3].
Chr1:11091-11530: depth:98,
log10.qValue:145, fold-change :47
DC lineage specialization of CD103+ and CD11b+
cells is controlled by the TFs Irf8 and Irf4, respectively.
• To monitor the epigenetic consequences of CD11b+
lineage differentiation, cells were collected 6 days
after induction and formaldehyde fixed for flow
cytometry sorting and ChIP-Seq library construction.
• Libraries were sequenced using Ion PITM v3
templating and sequencing.
• Loci detected by Irf4 ChIP-seq from CD11b+ DCs
were rank-ordered by significance, confirming the
Irf4 gene targets highlighted by Lugt et al[7], (Fig. 7).
• Additionally, 61% of significant peaks [–log10 q-value
≥20] associated with Irf4 binding, were in
common across all cell-sorted populations of 1, 3,
5, 20M CD11b+ DCs (Fig. 8).
Figure 7. [left] Genes and biological pathways identified by Irf4
ChIP-seq loci. Known Irf4 binding targets are noted.
[right]: Peaks from the immunoprecipitation of 1, 3, 5, 20M mouse
CD11b+ DCs for Zbtb46 (A) and F13a1 (B) genes.
Figure 8. Significant overlap in
ChIP-seq loci (with q-values of
≥20) (total n = 17,417) from cell
sorted populations of 1, 3, 5,
and 20M CD11b+ lineage mouse
dendritic cells.
Figure 1. ChIP-Seq
METHODS
Input
≤10ng
End
repair
Ligate
adapters
Nick
translate
& Amp
Size
select
145 300 Figure 2. ChIP-Seq Library Protocol
• Input control and immunoprecitated chromatin was quantitated
using the Qubit® HS kit.
• 10 ng (max) of DNA was used for end repair reactions.
• Samples were purified using 1.8x AMPure® XP beads prior to
adapter ligation.
• Libraries were purified with 1.5x AMPure® XP beads to remove
excess adapter dimer.
• This was followed by nick translation, amplification for 18 cycles,
size-selection using either double SPRI clean up (0.7/1.5x)
AMPure® XP or the Pippin PrepTM with internal size standards
for a range of 145-300 bp.
4-plexed
Proton
data
Barcode
balanced:
%of reads/PI
Redundancy
Rate
# Putative
Loci @
FDR
cutoff: 10-3
Confirmation against ENCODE* @
variable levels of overlap
Any At least
50% 100%
CTCF
(BC=38) 24.0 0.44 17,538 68.5%
(12,018)
67.9%
(11,888)
60.3%
(10,571)
H3K27ac
(BC=39) 22.1 0.11 52,861 73.2%
(38,716)
70.2%
(37,105)
66.4%
(35,101)
H3K27me3
(BC=45) 29.5 0.11 54,742 89.1%
(48,772)
87.9%
(48,123)
87.0%
(47,632)
Input
(BC=51) 24.3 0.12
*wgEncodeBroa
dHistoneK562H3
k27acStdPk
*wgEncodeBroa
dHistoneK562H3
k27me3StdPk
*wgEncodeHaibTfb
sK562CtcfcPcr1xPk
Rep2
Table 1. 4-plex Ion PI™ v3 ChIP-seq w/ significant confirmation rates vs ENCODE
Low input-volume samples – using the human leukemia cell-line K562, and
antibody from Abcam - were 4-plexed (Table 1) onto a single P1 chip; the
protocol ensured a barcode balance such that the background estimate
from the input matched all immunoprecipitated samples. MACS2 2.0.10[1]
was used for peak-calling.
H3K27ac – an activating acetylation mark:
• A confirmation rate of 73% is observed against ENCODE[2].
• ~55% of loci enrich for enhancers and 35% for promoters[2].
Fig 4. shows, representative association with the active
promoter (red) and strong enhancer (beige) of CHD8 – a
chromatin remodeling factor[2].
Figure 4. Confirmation of H3K27ac against ENCODE
H3K27me3 – a repressive methylation mark:
• A confirmation rate against ENCODE of ~87%[2] is observed.
• The moderate presence of DNA methylation[2] concomitant with
H3K27me3, a signature for polycomb repression is attested to by the
Broad ChromHMM data[2-3] (Fig. 5).
• ~74% enrichment is observed for the repressed segments of the
genome. Since this mark is typically associated with closed/inactive
chromatin, a no/negative correlation(R:-0.1) is observed with the
activating acetylation mark(H3K27ac) (Fig. 5).
• ~19% of the putative loci are associated with the heterochromatin.
The tri-methyl form has been noted as an important mark for
facultative heterochromatin, in the literature.
Figure 5. Confirmation of H3K27me3 against ENCODE
Figure 6. [top] Lineage specialization of dendritic cells;
[bottom] Flow cytometry sorting of differentiated DCs
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