reSeArcH Article
The PDGF signalling is highly activated in smooth muscle and
endothelial cells, and is initiated through the activation of two
major receptors belonging to the PDGF receptor-α (PDGFRA)
and PDGF receptor-β (PDGFRB) family^30. During cardiomyocyte
differentiation, PDGFRA and PDGFRB are highly upregulated in
the early stages of differentiation but become downregulated after
generating functional cardiomyocytes^31 (Extended Data Fig. 9a).
In particular, expression of PDGFRB mRNA and PDGFRB pro-
tein is low in adult iPSC-CMs and normal heart tissues, but can be
increased by stress conditions^32 ,^33 , which suggests that the PDGF
signalling pathway is silenced in cardiomyocytes under phys-
iological conditions (Extended Data Fig. 9a–c). However, we
fo und that a significant increase in PDGFRB mRNA and protein
expression occurred in K117fs iPSC-CMs compared to control
iPSC-CMs (Fig. 5e–g and Extended Data Fig. 9d–f). In addition, a
kinase array showed hyperactivation of PDGFRB in K117fs iPSC-CMs
compared to isogenic control iPSC-CMs (Extended Data Fig. 9g).
Furthermore, we found that the promoter region of the PDGFRB was
more accessible in K117fs iPSC-CMs, as demonstrated by high enrich-
ment of an active histone marker (H3K4me3) and open chromatin inLMNA enrichment (log(MUT/WT)) 2WT/cor-WT
ATAC WT/MUT
WT/cor-WT
WT/MUT
WT/cor-WT
WT/MUT
WT/cor-WT
WT/MUT
WT/cor-WT
WT/MUTH3K4me3H3K27me3LMNALADs
Loss in WT/MUT
Overlap
Gain in WT/MUTLADsaGenomic coverage (%) 0 Mean of LAD length (Mb)1020302Location of LADs
Loss GainNone
Partial
Entire44 464214
3123UpDown0200400600800LAD peaksWT LADs
MUT LADs
Overlap
Loss in MUT
Gain in MUTbcde
fgATAC enrichment (log 2 (MUT/WT))h k Chr20:36,833,000–42,088,878Differentially
expressed genes (%)Down
i Up48312192No changeATAC-seq difference inMUT vs WT (%)LossOverGain(^636652)
(^373448)
Chromosome 20 (64 Mb)
0–8
0–8
0–30
0–30
0–30
0–30
–2–+2
–2–+2
100
80
60
40
20
0
100
50
0
1.2
0.8
0.4
0.0
–0.4
–0.8
–1.2
–0.6 –0.4 –0.2 0.0 0.2 0.4 0.6
P < 0.0001
Slope = –1.0753
–0.973 0.810
FPKM differencein MUT vs WT
100
80
60
40
20
0
100
50
0
WT/cor-WT
ATAC WT/MUT
WT/cor-WT
WT/MUT
WT/cor-WT
WT/MUT
WT/cor-WT
WT/MUT
WT/cor-WT
WT/MUT
H3K4me3
H3K27me3
LMNA
LADs
WT/cor-WT
RNA WT/MUT
0–30
0–30
0–150
0–150
0–80
0–80
0–100
0–100
–2–+2
–2–+2
Gain in MUT Overlap Loss in MUT
WT MUT WT MUT WT MUT WT MUT
Loss in MUT Overlap Gain in MUT Non-LADs
Gene distance (kb)
20
(^1510)
5
12.510.0
7.55.0
2.5
10
5
10
5
15
25
20
15
10
5
0
j
–1.5
TSS
1.5–1.5 1.5
TSS –1.5TSS1.5–1.5TSS1.5
–1.5
TSS
1.5–1.5 1.5
TSS
–1.5
TSS
1.5–1.5 1.5
TSS
Gene distance (kb) Gene distance (kb) Gene distance (kb)
2.0
0
0.5
1.0
1.5
Fig. 4 | Lamin A/C haploinsufficiency results in reduced lamin A/C
enrichment and increased open chromatin formation of each LAD.
a, Representative images of chromatin immunoprecipitation followed by
sequencing (ChIP–seq), ATAC-seq and RNA-seq of chromosome 20. The
sc-376248 anti-LMNA antibody was used for ChIP–seq. b–d, Number (b),
genomic coverage (c) and mean of length of LADs (d) in control, mutant,
gained, overlapping and lost LADs. e, Location of LADs in the loss or gain
category. LADs located within ± 100 kb of overlapping LADs are shown
as ‘entire’. LADs partially shared with ± 100 kb of overlapping LADs are
showed as ‘partial’. LADs located outside of ± 100 kb of overlapping LADs
are shown as ‘none’. f, Comparison of normalized ATAC enrichment of
each LAD in control and mutant iPSC-CMs. Red, percentage of LADs that
showed upregulated normalized ATAC enrichment in mutant iPSC-CMs
compared to control iPSC-CMs. Blue, percentage of LADs that showed
downregulated normalized ATAC enrichment in mutant iPSC-CMs
compared to control iPSC-CMs. g, Normalized ATAC-seq signal intensity
around the transcription start site (TSS) of genes located in each LAD
category. h, Scatter plot of normalized lamin A/C and ATAC enrichment
of each LAD (n = 588). The y axis shows the log 2 -transformed relative
normalized lamin A/C enrichment of each LAD in mutant iPSC-CMs
compared to control iPSC-CMs. Note, in the graph, log 2 (MUT/WT)
indicates log 2 (lamin A/C enrichment of each LAD in MUT/lamin A/C
enrichment of each LAD in WT). The x axis shows the log 2 -transformed
relative normalized ATAC enrichment of each LAD in mutant iPSC-CMs
compared to control iPSC-CMs (shown as log 2 (MUT/WT)). Note, in the
graph, log 2 (MUT/WT) indicates log 2 (ATAC enrichment of each LAD
in MUT/ATAC enrichment of each LAD in WT). One dot represents
one LAD. i, Comparison of normalized fragments per kb per million
aligned reads (FPKM) of each LAD in control and mutant iPSC-CMs.
Red, percentage of LADs with upregulated normalized FPKM in mutant
iPSC-CMs compared to control iPSC-CMs; blue, percentage of LADs
with downregulated normalized FPKM in mutant iPSC-CMs compared
to control iPSC-CMs; grey, no change. j, Percentage of differentially
expressed genes located in LADs. False-discovery rate (FDR)-corrected
P < 0.01; log 2 -transformed fold change in expression of >1 or <−1.
k, Representative images of ChIP–seq, ATAC-seq and RNA-seq. Blue
boxes, LADs with lower enrichment of lamin A/C and higher expression
in mutant iPSC-CMs compared to control iPSC-CMs.
338 | NAtUre | VOl 572 | 15 AUGUSt 2019