EMS81644
EMS81644
EMS81644
Author Manuscript
Nature. Author manuscript; available in PMC 2019 October 03.
Published in final edited form as:
Nature. 2019 May 07; 568(7751): 193–197. doi:10.1038/s41586-019-1064-z.
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Abstract
Genetic robustness, or the ability of an organism to maintain fitness in the presence of mutations,
can be achieved via protein feedback loops. Recent evidence suggests that organisms may also
respond to mutations by upregulating related gene(s) independently of protein feedback loops, a
phenomenon called transcriptional adaptation. However, the prevalence of transcriptional
adaptation and its underlying molecular mechanisms are unknown. Here, by analyzing several
models of transcriptional adaptation in zebrafish and mouse, we show a requirement for mRNA
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degradation. Alleles that fail to transcribe the mutated gene do not display transcriptional
adaptation and exhibit more severe phenotypes than alleles displaying mutant mRNA decay.
Transcriptome analysis reveals the upregulation of a substantial proportion of the genes that
exhibit sequence similarity with the mutated gene’s mRNA, suggesting a sequence dependent
mechanism. Besides implications for our understanding of disease-causing mutations, these
findings will help design mutant alleles with minimal transcriptional adaptation-derived
compensation.
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Correspondence and requests for materials should be addressed to D.Y.R.S (didier.stainier@mpi-bn.mpg.de).
aPresent address: Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany.
bPresent address: University of New Haven, New Haven, USA.
cPresent address: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
dPresent address: The Francis Crick Institute, London, UK.
Data availability
ATAC-seq and RNA-seq data were deposited to Gene Expression Omnibus under accession codes GSE107075 and GSE114212.
Author contributions M.A.E.B. designed and performed most of the experiments, analyzed data and wrote the manuscript; Z.K. and
A.R. designed and performed mESC experiments, some imaging and edited the manuscript; C.K. performed bioinformatics analyses;
S.G. performed ATAC- and RNA-seq; N.F. and K.K. performed some qPCR experiments; G.L.M.B. performed some imaging; C.T.
generated the upf1 mutant under the supervision of A.J.G; S.L., R.F., and C.G. provided unpublished mutants; D.Y.R.S. helped design
the experiments and analyze data, supervised the work, and wrote the manuscript; all authors commented on the manuscript.
Author information Reprints and permissions information is available at www.nature.com/reprints.
The authors declare no competing interests.
El-Brolosy et al. Page 2
Recent advances in reverse genetic tools have greatly enhanced our ability to analyze gene
function in a wide range of organisms. These studies have reinforced prior observations that
many engineered mutants do not exhibit an obvious phenotype, reviving interest in the
concept of genetic robustness. Several mechanisms have been proposed to explain genetic
robustness, including functional redundancy1, rewiring of genetic networks2, and the
acquisition of adaptive mutations in the case of rapidly proliferating organisms such as
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To determine whether the increased mRNA levels were due to increased transcription of the
adapting gene or increased mRNA stability, we measured pre-mRNA levels of hbegfb and
emilin3a in hbegfa and egfl7 mutants and found that they were also upregulated (Extended
Data Fig. 3a). Similar findings were obtained for Fermt1 and Rel pre-mRNA levels in
Fermt2 and Rela K.O. cells (Extended Data Fig. 3b). Together, these data indicate an
increase in transcription of the adapting genes. In addition, Fermt2 K.O. MKFs display
increased chromatin accessibility at the Fermt1 transcription start site (TSS) as observed by
ATAC-seq (Extended Data Fig. 3c).
adaptation response, we investigated two other possibilities, the DNA lesion and the mutant
mRNA. In the context of the zebrafish studies, we identified several mutant alleles which do
not display transcriptional adaptation (Extended Data Fig. 3d-f), indicating that a DNA
lesion by itself is not sufficient to trigger transcriptional adaptation, or that specific DNA
lesions are required. Notably, while analyzing various mutant alleles, we found that unlike
hbegfaΔ7 and vclaΔ13, two other PTC bearing alleles, hbegfasa18135 and vclasa14599, do not
display transcriptional adaptation (Fig. 1c, Extended Data Fig. 1). To investigate the reason
for this difference, we examined mutant mRNA levels and observed limited or no decrease
in the hbegfasa18135 and vclasa14599 alleles (Fig. 1d). This correlation between mutant
mRNA decrease and transcriptional adaptation was observed in all alleles examined (Fig. 1e,
f, Extended Data Fig. 3-f). To determine whether the decreased mRNA levels were due to
decreased transcription or reduced stability, we analyzed pre-mRNA levels of hbegfa, egfl7
and alcama in hbegfaΔ7, egfl7 and alcama mutant zebrafish, and found that unlike the
mRNA levels, the pre-mRNA levels remained unchanged, or were slightly upregulated,
compared to wt (Extended Data Fig. 4a). Similar findings were observed in Fermt2 and Rela
K.O. cells (Extended Data Fig. 4b). Moreover, metabolic labeling of newly synthesized
transcripts revealed similar or increased levels of Fermt2, Rela and Actg1 mutant transcripts
compared to wt (Extended Data Fig. 4c), while transcription inhibition assays revealed
shorter half-lives (Extended Data Fig. 4d-f). Together, these data indicate that mutants which
exhibit transcriptional adaptation have reduced mutant mRNA levels due to mRNA decay.
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injection of uncapped RNA synthesized from the non-coding strand of hif1ab or vegfaa did
not lead to an increase in epas1a or vegfab mRNA levels (Extended Data Fig. 5k),
suggesting a possible role for the RNA sequence itself in transcriptional adaptation.
If mutant mRNA degradation is required for transcriptional adaptation, alleles that fail to
transcribe the mutated gene should not display this response. To this end, we used the
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increase in zebrafish actb1 mRNA levels (Extended Data Fig. 8e, Supplementary Data), in
line with the sequence similarity analyses mentioned above.
containing sequences similar to epas1a led to an increase in epas1a mRNA levels (Fig. 4b,
Extended Data Fig. 8f, Supplementary Data). We also generated synthetic transcripts
containing hif1ab sequences similar to the promoter, exons, introns, or 3’UTR of epas1a.
Injection of uncapped versions of these transcripts revealed that those exhibiting sequence
similarity to exons or introns induced transcriptional adaptation while those exhibiting
sequence similarity to the 3’UTR did not; and the transcripts exhibiting sequence similarity
to the promoter induced only a mild response (Extended Data Fig. 8g). These data are
consistent with the transcriptome analyses of Fermt2, Actg1 and Actb K.O. cells (Extended
Data Fig. 8a-c): genes exhibiting sequence similarity to the mutated gene’s mRNA in their
3’UTR were not up-regulated while those exhibiting sequence similarity to promoters
exhibited mostly a mild up-regulation. Altogether, these data suggest that, at least in some
cases, sequence similarity plays a role in transcriptional adaptation.
While investigating this model, we noted a study reporting that transfection of short
fragments of Cdk9 or Sox9 mRNA could lead to an increase in expression of these genes18.
Mechanistically, these RNA fragments were found to downregulate native antisense
transcripts which normally function as negative regulators of Cdk9 and Sox9 expression.
Notably, we found that transfection of uncapped Cdk9 or Sox9 RNA also led to upregulation
of these genes (Extended Data Fig. 10a). Furthermore, knockdown of a BDNF antisense
transcript was reported to cause the upregulation of the sense transcript, a response that
Discussion
Despite its potential importance5, transcriptional adaptation to mutations and its underlying
molecular mechanisms remain poorly understood. Here we show that the mRNA
surveillance machinery is important not only to prevent the translation of defective
transcripts but also to buffer against mutations by triggering the transcriptional upregulation
of related genes including the mutated gene itself (See Supplementary Discussion).
For a number of human genetic diseases, missense or in frame indels, which are less likely
to lead to mutant mRNA degradation, are reportedly more common in affected individuals
than potentially RNA-destabilizing nonsense mutations or out-of-frame indels20–25.
Interestingly, a study on Marfan syndrome patients reported that when compared to
individuals with FBN1 missense mutations, the mildest form of the disease was observed in
an individual displaying very low mutant FBN1 transcript levels due to an out-of-frame indel
leading to a PTC in the FBN1 coding sequence26. Similar observations were reported for
mutations in the HBB gene27. While the current dogma is that pathogenic missense
mutations tend to be more common in affected individuals as they might lead to
constitutively active or dominant negative proteins, we propose that nonsense mutations
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might be less common as they might lead to mRNA decay-triggered upregulation of related
genes and therefore not cause significant symptoms. Detailed transcriptomic analyses of
relevant individuals will help test this hypothesis. Moreover, recent studies28,29 of healthy
individuals have reported homozygous loss-of-function mutations in several genes
(including EGFL7 and RELA, which were studied here), and it will be interesting to
investigate whether degradation of the mutant transcripts is associated with a transcriptional
adaptation response that protects them. Such analyses may help us understand why some
mutations cause disease while others do not. They may also help identify new modifier
genes whose expression levels could be further modulated for therapeutic purposes.
Methods
Statistics and reproducibility
No statistical methods were used to predetermine sample size. The experiments were not
randomized. The investigators were not blinded to allocation during experiments and
outcome assessment. All experiments were performed at least twice unless otherwise noted.
P< 0.05 was accepted as statistically significant.
Zebrafish husbandry
All zebrafish (Danio rerio, strain: Tüb/AB) husbandry was performed under standard
conditions in accordance with institutional (Max Planck Gesellschaft) and national ethical
and animal welfare guidelines approved by the ethics committee for animal experiments at
the Regierungspräsidium Darmstadt, Germany (permit number: B2/1017). All experiments
were performed on zebrafish embryos or larvae between 6 hours post fertilization (hpf) and
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6 days post fertilization (dpf). We used the following previously published mutant and
transgenic lines: hif1abbns90 30, vegfaabns1 31, egfl7s980 4, egfl7s981 4, hbegfasa18135 and
vclasa14599 (Sanger institute zebrafish mutation project; http://www.sanger.ac.uk/resources/
zebrafish/zmp/), Tg(fli1a:EGFP)y1 32, TgBAC(etsrp:eGFP)ci1 33.
Cell culture
Wt and Fermt2 knockout MKFs were a generous gift from R. Fässler (MPI for biochemistry,
Martinsried, Germany). Wt and Rela knockout MEFs were a generous gift from A.
Hoffmann (UCLA, USA). mESCs from the C57BL/6 mouse strain were a generous gift
from J. Kim (MPI for heart and lung research, Bad Nauheim, Germany). None of the cell
lines were authenticated by the authors. All cell lines tested negative for mycoplasma
contamination.
(Thermo) supplemented with 10% bovine calf serum (HyClone), 100 U/ml penicillin, 100
μg/ml streptomycin. All cells were grown at 37°C, 95% humidity with 5% CO2 and
experiments were performed on cells passaged less than 20 times.
Genotyping
Heterozygous fish were incrossed; DNA and RNA were extracted from at least 24 individual
embryos or larvae using TRIzol (Life Technologies) followed by phenol-chloroform
extraction. In brief, single embryos or larvae were lysed and homogenized in TRIzol using
the NextAdvance Bullet Blender® Homogenizer (Scientific instrument services, inc.).
Chloroform was then added and phase separation was obtained following vortexing and
centrifugation. The top aqueous phase (containing RNA) was isolated and stored at -80°C
and the bottom organic phase (containing DNA) was subjected to ethanol purification to
precipitate the DNA. Purified DNA was then dissolved in water and genotyping was
performed using high resolution melt analysis (HRMA) or PCR. RNA from genotyped +/+
and -/- zebrafish was then pooled for further purification and cDNA synthesis. For each
experiment, this process was performed on embryos from at least 3 different crosses/parents.
HRMA was used to genotype all mutant zebrafish and mouse cell lines with the exception of
the RNA-less alleles which were genotyped by PCR. Primer sequences used for genotyping
are listed in Supplementary Table 3.
using TRIzol and at least 500 ng RNA was used for reverse transcription using the Maxima
First Strand cDNA synthesis kit (Thermo). All reactions were performed in at least technical
duplicates and the results represent biological triplicates. qPCR was performed at the
embryonic or larval stage when the wild-type version of the mutated gene exhibits its
highest expression level. For hbegfa mutants treated with NMDi, data were analyzed at 6 dpf
and not at 72 hpf as the drug seemed to be effective only when used for 3 days between 72
hpf and 6 dpf; treatment of earlier stage embryos was not possible due to toxicity. Primers
were designed using Primer3 (http://biotools.umassmed.edu/bioapps/primer3_www.cgi).
Primers to detect pre-mRNA were designed to bind around intron-exon boundaries. Allele-
specific primers were designed such that they amplify the wt but not the mutant allele. To
determine the injected capped and uncapped RNA levels, a universal reverse primer
corresponding to the adaptor sequence at the 3’ end of the injected RNAs was used along
with forward primers designed in close proximity; in this way we were able to distinguish
between endogenous and injected RNAs. Several primer pairs along the gene’s cDNA were
used to assess transcription in the candidate promoter-less alleles. Only mutant alleles that
exhibited less than 10% of wt mRNA levels with all the above mentioned primer pairs were
used in the study. For the upf1 double mutant data, the figures show expression levels of the
adapting gene in the double mutants relative to its expression levels in the upf1 single
mutants. Expression levels of the mutated gene in the upf1 double mutants are relative to
those in hbegfa, vegfaa, or vcla single mutants. Equal numbers of cells were used for the
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mouse cell line qPCR experiments. rpl13, gapdh and Actb, 18srRNA, Gapdh were used to
normalize zebrafish and mouse experiments, respectively. rpl13 was chosen as a reference
gene for zebrafish experiments as its expression levels were not changed between wild-type
and egfl74, hbegfa, vcla, vegfaa and alcama mutant embryos (unpublished microarray data).
Primer sequences used for the qPCR experiments are listed in Supplementary Table 4. Fold
changes were calculated using the ΔΔCt method. All Ct and dCt values are listed in the
source data files. Ct values for the reference genes ranged between 11 and 18 except for
18srRNA which ranged between 6 and 8.
To transcribe uncapped RNAs, cDNA was used to amplify zebrafish hif1ab and vegfaa and
mouse Actb, while genomic DNA was used to amplify mouse Cdk9 and Sox9 (single exon
for Cdk9 and the entire genomic locus (exons + introns) for Sox9) since their expression
levels were too low to amplify from cDNA, all using a reverse primer that contains the
adapter sequence 5’ GCCAAGCTATTTAGGTGACACTATAG 3’, subsequently used for
qPCR detection of the injected transcripts as described above. To generate uncapped
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xrFRAG transcripts, the xrFRAG sequence8 was cloned upstream of the hif1ab and vegfaa
coding sequences in pCS2+ which was then linearized using Not1 for in vitro transcription.
In vitro transcription of uncapped transcripts was performed using T7 RNA polymerase
(Promega) and the RNA was purified using an RNA Clean and Concentrator kit. The RNAs
were injected into one-cell stage zebrafish embryos (50 pg) or transfected into cells (1 μg) in
12 well plates.
gRNA design
gRNAs were designed using the CRISPR design tool CHOPCHOP (http://
chopchop.cbu.uib.no/)34. To generate RNA-less alleles, double gRNAs were designed to
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flank the region to be deleted. gRNAs used to delete promoter regions were designed at least
500 bp upstream and downstream from the transcription start site of the respective gene.
Sequences of the gRNAs used in this study and their genomic binding sites are listed in
Supplementary Table 5.
Actg1NSD (Actg1 K.O.) and Actg1full locus del. mutant MEFs do not exhibit any gross
morphological defects.
Overexpression plasmids
Fermt2 and Rela cDNAs were amplified using MEF cDNA as template. PCR fragments
were ligated into the pcDNA3.1 mammalian expression vector (Thermo) between the
BamHI and XbaI sites. Plasmids were transfected into the respective mutant cells using
FuGene 6 (Promega) according to manufacturer’s protocol. Two days post-transfection, cells
were selected with 0.5 mg/ml and 2 mg/ml G418 (Sigma) for Fermt2 and Rela K.O. cells,
respectively. A week later, cells were lysed in modified RIPA buffer for western blot
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CRISPRi
3 gRNAs targeting the Fermt2 promoter and transcription start site were cloned into a
plasmid encoding a fusion protein of catalytically-dead Cas9 and Krüppel-associated box
(KRAB) repressor (Addgene, #71237) as previously described42. Fermt2 K.O. cells were
transfected with the three plasmids and 48 hours post-transfection, cells positive for eGFP
were sorted into TRIzol for RNA extraction.
RNA interference
siRNAs are listed in Supplementary Table 2. siRNA transfections were performed using the
RNAimax transfection reagent (Thermo) according to manufacturer’s protocol. 48 hours
post-transfection, cells were collected in TRIzol for RNA extraction except for knockdowns
of SMG6, ERF1 and XRN1 in which case cells were collected 24 hours post-transfection.
All siRNAs were used at 10 nM and gave a knockdown efficiency of 70 to 90% (as assessed
by mRNA levels); to knock down XRN1 in mESCs, a lower concentration of siRNAs was
used (2.5 nM; which led to a knockdown efficiency of 20%) as stronger knockdown affected
housekeeping genes as well (data not shown). A scrambled (Scr) siRNA (Sigma, #SIC00),
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which does not bind to any of the mouse transcripts, was used as a negative control.
For each experiment, the figures show expression levels of the adapting gene in K.O. cells
relative to its expression levels in wt cells treated with the same siRNA. Expression levels of
the mutant gene in the K.O. cells treated with a given siRNA are relative to those in K.O.
cells treated with Scr siRNA. All Ct and dCt values are shown in the source data files.
Pharmacological treatments
To inhibit NMD, 72 hpf wild-type and hbegfa mutant larvae were treated with 10 uM
NMDi1443 or DMSO, and 3 days later, they were collected in TRIzol for qPCR analysis.
No gross alterations were observed in the drug treated larvae. To inhibit RNA degradation
through translation blockade, wild-type and Rela K.O. cells were treated with 200 µg/ml
cycloheximide (sigma) or DMSO for 5 hours then collected in TRIzol for RNA extraction.
For each experiment, the figures show expression levels of the adapting gene in mutant
larvae, or K.O. cells, relative to its expression levels in wt larvae, or cells treated with the
same drug. Expression levels of the mutated gene in the mutant larvae, or K.O. cells treated
with a given drug, are relative to those in untreated mutant larvae, or K.O. cells treated with
DMSO. All Ct and dCt values are shown in the source data files.
Metabolic labeling was performed as previously described44,45. Briefly, cells were treated
with 200 µM 4-thiouridine (4sU, Sigma) for 1 hour followed by phenol-chloroform RNA
extraction. 80 µg of RNA was incubated with biotin-HPDP (Thermo) to specifically
biotinylate the newly transcribed 4sU labeled RNAs. Biotinylated RNAs were then pulled
down using the μMacs Streptavidin Kit (Miltenyi) and at least 100 ng of pulled RNA was
used for reverse transcription and downstream qPCR analysis. This experiment was
performed once with two biological replicates.
Cytotoxicity assay
7,000 cells were seeded per well in a 96-well plate and incubated with media containing 25
ng/ml of mouse TNFα. 24 hours later, cells were washed with PBS and incubated for 5
mESC staining
Cells were fixed with 4% PFA for 15 min at room temperature (RT), permeabilized with
0,3% Triton X-100 (in PBS) for 10 min, and then incubated with 1:1000 phalloidin-568
(Thermo) in 3% BSA for 1 hr at RT. After 3x 15 min washes with PBT (0,1% Triton X-100
in PBS), samples were counterstained for 5 min at RT with 1:5000 DAPI (Sigma) and
mounted for imaging with Dako Fluorescent mounting medium. Images were obtained on a
Zeiss LSM700 confocal microscope using a LD C-Apochromat 63x/1.15 W Corr M27
objective. Protrusion length was measured using ImageJ.
Immunostaining
100 hpf larvae from alcamapromoter-less +/- and alcama Δ8 +/- incrosses were collected and
fixed in 4% paraformaldehyde. After removing the fixative with PBS/0.1% Tween washes,
larvae were incubated with 3 µg/ml proteinase K for 1 hour then washed with PBS/1% BSA/
1%DMSO/0.5% Triton-X (PBDT) before being incubated for 2 hours with phalloidin
Alexa-568 (Invitrogen) at RT to label F-Actin. They were then washed with PBS/0.1%
Tween and mounted for imaging the heart. Images of the hearts were acquired using a Zeiss
LSM880 Axio Examiner confocal microscope with a W Plan-Apochromat 20x/1.0 objective.
The ventricle length, or long axis of the ventricle, was measured from the apex of the
ventricle to the junction of the ventricle with the bulbus arteriosus using Zen Black. In the
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Confocal microscopy
A Zeiss LSM 700 confocal microscope was used for live imaging of the trunk and brain
vasculature. Embryos were anaesthetized with a low dose of tricaine, placed in a glass-
bottomed Petri dish (MatTek) on a layer of 1.2% low melt agarose and imaged using Plan-
Apochromat 10×/0.45 and LCI Plan-Neofluar 25×/0.8 objectives.
In wt embryos, at 60 hpf, an average of 13 CtAs have connected to the basilar artery (BA)46.
To measure blood flow velocity, we performed time-lapse imaging of the trunk region of 78
hpf wild-type, maternal zygotic hbegfaΔ7 -/- and maternal zygotic hbegfafull locus del. -/-
larvae using a Zeiss Spinning disc CSU-X1 confocal microscope with a high-speed camera,
and quantified blood flow velocity in the dorsal aorta by measuring the time needed by
erythrocytes to move 200 μm at the level of the fifth and sixth somites using Zen Blue as
previously described47.
hbegfafull locus del. larvae were obtained from F2 homozygous parents generated by
outcrossing the founder.
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hbegfaΔ7 and vegfaaΔ10 larvae were obtained from an incross of F3 heterozygous parents
that were generated by consecutive outcrosses.
Chromatin immunoprecipitation
ChIP was performed using the truChIP Chromatin Shearing Reagent kit (Covaris) using 30
million cells/IP according to manufacturer’s protocol. Chromatin was sheared using
Bioruptor (Diagenode) to generate fragments between 200-400 bp. Immunoprecipitation
was then performed as previously described48. The following antibodies were used: rabbit
IgG (4μg/IP, # 026102, Thermo Fischer), WDR5 (4μg/IP, #13105, Cell signaling) and
H3K4me3 (4μg/IP, #9751, Cell signaling). Following immunoprecipitation and reverse
cross-linking, samples were purified using the NucleoSpin Gel and PCR Clean-up kit
(Macherey-Nagel) following manufacturer’s protocol for samples containing SDS.
ATAC-seq analysis
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The samples were assessed for quality using FastQC (Andrews S. 2010, FastQC: a quality
control tool for high throughput sequence data. Available online at: http://
www.bioinformatics.babraham.ac.uk/projects/fastqc). Trimmomatic version 0.3350 was
employed to trim reads after a quality drop below a mean of Q20 in a window of 5
nucleotides. Only reads above 30 nucleotides were cleared for further analyses. In order to
normalize all samples to the same sequencing depth, 27 million reads per sample were
randomly selected for further analysis. These reads were mapped versus the mm10 version
of the mouse genome with STAR 2.4.2a51 using only unique alignments to exclude reads
with unclear placing. The reads were further deduplicated using Picard 1.136 (Picard: A set
of tools (in Java) for working with next generation sequencing data in the BAM format;
http://broadinstitute.github.io/picard/) to avoid PCR artefacts leading to multiple copies of
the same original fragment. The Macs2 peak caller version 2.1.0 was employed to identify
peaks. The minimum qvalue was set to -1.5 and FDR was changed to 0.01. In order to
determine thresholds for significant peaks, the data were manually inspected in IGV
2.3.5252. Peaks overlapping ENCODE blacklisted regions (known mis-assemblies, satellite
repeats) were excluded. In order to be able to compare peaks between samples, the resulting
lists of significant peaks were overlapped and unified to represent identical regions. After
conversion of BAM files to BigWig format with deep Tools bamCoverage53, the counts per
unified peak per sample were computed with BigWigAverageOverBed (UCSC Genome
Browser Utilities, http://hgdownload.cse.ucsc.edu/downloads.html). Raw counts for unified
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peaks were submitted to DESeq2 for normalization54. Spearman correlations were produced
to identify the degree of reproducibility between samples using R. To allow a normalized
display of samples in IGV, the raw BAM files were normalized for sequencing depth
(number of mapped deduplicated reads per sample) and noise level (number of reads inside
peaks). Two factors were computed and applied to the original BAM files using bedtools
genomecov55 resulting in normalized BigWig files for IGV.
RNA-seq
RNA was isolated using the miRNeasy micro Kit (Qiagen). To avoid contamination by
genomic DNA, samples were treated by on-column DNase digestion (DNase-Free DNase
Set, Qiagen). Total RNA and library integrity were verified on LabChip Gx Touch 24
(Perkin Elmer). 1µg of total RNA was used as input for SMARTer Stranded Total RNA
Sample Prep Kit - HI Mammalian (Clontech). Sequencing was performed on a NextSeq500
instrument (Illumina) using v2 chemistry, resulting in an average of 25-30M reads per
library with 1x75bp single end setup.
RNA-seq analysis
The resulting raw reads were assessed for quality, adapter content and duplication rates with
FastQC. Reaper version 13-100 was employed to trim reads after a quality drop below a
A subsampling approach was used to calculate a ranked P value for the significance of the
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and 2) epas1a including its promoter (TSS-2000) using BLASTn. The uncapped RNA
composed solely of the hif1ab sequences similar to epas1a was 1277 nucleotides in length,
and that composed solely of the hif1ab sequences not similar to epas1a was 1929
nucleotides in length. The similarity of the coding sequences of zebrafish actb1 transcript
ENSDART00000054987 (Query) to the mouse Actb transcript ENSMUST00000100497
(Subject) was assessed with a MUSCLE alignment. All these alignments can be found in
Supplementary Data.
Extended Data
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Extended Data Figure 1. Schematic illustration of the mutant alleles generated for this study.
Partial DNA sequences of the different mutant alleles generated for this study, and images of
gels providing evidence for deletions in RNA-less alleles. Red: mutation; green: stop codon
in alleles with a PTC; arrows: genotyping primers.
qPCR analysis of Fermt1 and Rel mRNA levels in wt and Fermt2 and Rela K.O. cells
transfected with empty vectors (control) or plasmids encoding wt Fermt2 or RELA. e,
Western blot analysis of Fermt2 and ACTB levels in Fermt2 K.O. cells transfected with
empty vectors (control) or plasmids encoding wt Fermt2. f, Western blot analysis of RELA
and ACTB levels in Rela K.O. cells transfected with empty vectors (control) or plasmids
encoding wt RELA. g, qPCR analysis of Actg1 mRNA levels in wt and heterozygous Actb
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and egfl7 wt and 5’UTR mutant zebrafish. f, qPCR analysis of vcla and vclb mRNA levels
in vcla wt and last exon (exon 22) mutant zebrafish. a, b, d-f, n = 3 biologically independent
samples. wt expression set at 1 for each assay. Error bars, mean, s.d. Two-tailed student’s t-
test used to assess P values.
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Extended Data Figure 4. Reduction in mutant transcript levels is due to mRNA decay.
a, qPCR analysis of hbegfa, egfl7 and alcama mRNA and pre-mRNA levels in hbegfa, egfl7
and alcama wt and mutant zebrafish. b, qPCR analysis of Fermt2 and Rela mRNA and pre-
mRNA levels in Fermt2 and Rela wt and K.O. cells. c, qPCR analysis of 4sU labeled
Fermt-2, Rela and Actg1 mRNA and pre-mRNA levels in Fermt-2, Rela and Actg1 wt and
K.O. cells. d, Fitted exponential decay curves of Fermt2 mRNA levels in wt and Fermt2
K.O. cells. e, Fitted exponential decay curves of Rela mRNA levels in wt and Rela K.O.
cells. f, Fitted exponential decay curves of Actg1 mRNA levels in wt and Actg1 K.O. cells.
a-f, n = 3 (a, b, d-f); 2 (c) biologically independent samples. a-c, wt expression set at 1 for
each assay. Error bars, mean, s.d. Two-tailed student’s t-test used to assess P values.
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(CHX). g, qPCR analysis of Rel mRNA levels in Rela K.O. cells treated with CHX. h,
qPCR analysis of endogenous hif1ab and vegfaa mRNA levels in 6 hpf wt embryos injected
with uncapped hif1ab or vegfaa RNA. i, qPCR analysis of Actg1 mRNA levels in mESCs
transfected with uncapped Actb RNA at different times post-transfection. j, qPCR analysis
of injected hif1ab, epas1a and injected vegfaa, vegfab RNA levels in 6 hpf wt embryos
injected with uncapped hif1ab or vegfaa transcripts with or without a 5’ xrFRAG sequence.
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hr, hour. k, qPCR analysis of epas1a and vegfab mRNA levels in 6 hpf wt zebrafish embryos
injected with uncapped sense or antisense hif1ab or vegfaa RNA. b, c, Scr: Scrambled
siRNA control. a-d, f, h-k, wt or control expression set at 1 for each assay. a-k, n = 3
biologically independent samples. Error bars, mean, s.d. Two-tailed student’s t-test used to
assess P values.
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Extended Data Figure 6. Mutant mRNA decay helps confer genetic robustness
a, qPCR analysis of Fermt2 and Fermt1 mRNA levels following CRISPRi mediated
knockdown of Fermt2 transcription in Fermt2 K.O. cells. b, qPCR analysis of emilin3a,
emilin3b and emilin2a mRNA levels in wt, egfl7Δ4 mutant and egfl7full locus del. mutant 20
hpf zebrafish. c, Number of CtAs connecting to the basilar artery (BA) in 58 hpf vegfaaΔ10
and vegfaapromoter-less mutants. d, Blood flow velocity in 78 hpf wt, hbegfaΔ7 and
hbegfafull locus del. mutants. e, Quantification of the cardiac ventricle length in 100 hpf wt,
alcama Δ8 and alcamapromoter-less mutant larvae. a, b, wt or control expression set at 1 for
each assay. a-e, n = 3 (a, b); 13 and 19 (c); 25 (d); 18, 7, 22 and 15 (e) animals. Error bars,
mean, s.d. Two-tailed student’s t-test used to assess P values.
chance (lower = better), while Bit score evaluates the combination of alignment quality and
length (higher = better). Each diagram shows the negative log10 of the significance P value
(higher = better) on the Y-axis, and the respective parameter value on the X-axis. A P value
of 0.05 is marked with a black horizontal line. The E-value thresholds used in our analyses
are highlighted with a circle. Lines ending preliminarily indicate a lack of any remaining
alignments after that point. The first row of diagrams explores large variations of thresholds
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in an attempt to identify the total range, while the second row focuses on the most relevant
window for the three genes investigated. The optimal thresholds differ considerably
depending on the gene analyzed. a-d, n = 2 biologically independent samples. DESeq2 tests
for significance of coefficients in a negative binomial GLM (Generalized Linear Model)
with the Wald test. P values were not multiple testing corrected.
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Extended Data Figure 8. Expression level of genes exhibiting sequence similarity in the different
mouse cell line models.
a-c, RNA-seq analysis of genes exhibiting sequence similarity with Fermt2 (a), Actg1 (b) or
Actb (c) in K.O. cells compared to wt. L2F: Log2 Fold change. Bold: Significantly
upregulated in K.O. cells relative to wt. Red: L2F>0, blue: L2F<0. Green: P value or P
adjusted value ≤ 0.05. Purple: Genes that exhibit sequence similarity with the mutated
gene’s mRNA in their promoter region. Yellow: Genes that exhibit sequence similarity with
the mutated gene’s mRNA in their 3’UTR region. Other non-colored genes exhibit sequence
similarity with the mutated gene’s mRNA in their exons or introns. Boxed: upregulated in
K.O. but not RNA-less cells; no Fermt2 RNA-less allele was analyzed. d, qPCR analysis of
Ubapl, Fmnl2, Cdk12 and Actr1a pre-mRNA levels in Actg1 K.O. cells relative to wt. e,
qPCR analysis of actb1 mRNA levels in 6 hpf wt zebrafish injected with uncapped mouse
Actb RNA. f, Schematic representation of regions of sequence similarity between hif1ab
mRNA and epas1a locus. TSS: transcription start site. Grey shaded triangles represent the
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alignments; intensity represents alignment quality and width at the base represents length of
the similarity region. g, qPCR analysis of epas1a mRNA levels in 6 hpf wt zebrafish
embryos injected with uncapped RNA composed solely of the hif1ab sequences similar to
epas1a promoter, exons, introns, or 3’UTR. a-e, g, n = 2 (a-c); 3 (d, e, g) biologically
independent samples. a-c, DESeq2 tests for significance of coefficients in a negative
binomial GLM (Generalized Linear Model) with the Wald test. P values were not multiple
testing corrected. d, e, g, wt or control expression set at 1 for each assay. Error bars, mean,
s.d. Two-tailed student’s t-test used to assess P values.
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biologically independent samples. Error bars, mean, s.d. Two-tailed student’s t-test used to
assess P values.
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Extended Data Figure 10. Antisense transcripts as potential players in the transcriptional
adaptation response.
a, qPCR analysis of Cdk9 and Sox9 mRNA levels in cells transfected with uncapped Cdk9
or Sox9 RNA. b, qPCR analysis of BDNF and BDNF-AS mRNA levels in HEK293T cells
transfected with uncapped BDNF RNA. c, Integrated genome viewer tracks of vclb and
hbegfb loci showing the location of the annotated antisense transcripts. Two alignments of
105 and 147 bp were observed between vcla mRNA and vclb AS RNAs, and an alignment
of 39 bp was observed between hbegfa mRNA and hbegfb AS RNA. d, qPCR analysis of
vclb and hbegfb antisense RNA levels in vcla and hbegfa wt and mutant zebrafish. a, b, d,
Control expression set at 1 for each assay. n = 3 biologically independent samples. Error
bars, mean, s.d. Two-tailed student’s t-test used to assess P values.
Supplementary Material
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Acknowledgments
We thank V. Serobyan, F. Mueller (U. Birmingham, UK), Z. Jiang, A. Beisaw and F. Gunawan for discussion and
comments on the manuscript and J. Pestel for the alcama mutant. We also thank A. Atzberger for support with cell
sorting and N. Gehring and V. Böhm for providing the xrFRAG plasmid. M.A.E.B. was supported by a Boehringer
Ingelheim Fonds PhD fellowship. Research in the Stainier lab is supported by the Max Planck Society, EU, DFG
and Leducq Foundation.
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Figure 1. Transcriptional adaptation in zebrafish and mouse correlates with mutant mRNA
decay.
a, qPCR analysis of hbegfb, vclb, epas1a and epas1b, vegfab, emilin3a and alcamb mRNA
levels in hbegfa, vcla, hif1ab, vegfaa, egfl7 and alcama wt and mutant zebrafish. b, qPCR
analysis of Fermt1, Rel, Actg2 and Actg1 mRNA levels in Fermt-2, Rela, Actg1 and Actb
wt and K.O. cells. c, d, qPCR analysis of hbegfb and vclb (c) and hbegfa and vcla (d)
mRNA levels in the indicated hbegfa and vcla mutant alleles. e, qPCR analysis of hif1ab,
vegfaa, egfl7 and alcama mRNA levels in hif1ab, vegfaa, egfl7 and alcama wt and mutant
zebrafish. f, qPCR analysis of Fermt-2, Rela, Actg1 and Actb mRNA levels in Kindlin-2,
Rela, Actg1 and Actb wt and K.O. cells. a-f, n = 3 biologically independent samples. wt
expression set at 1. Error bars, mean, s.d. Two-tailed student’s t-test used to assess P values.
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control expression set at 1. a-d, n = 3 biologically independent samples. Error bars, mean,
s.d. Two-tailed student’s t-test used to assess P values.
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Figure 3. Alleles that fail to transcribe the mutated gene do not display transcriptional
adaptation.
a, qPCR analysis of hbegfa, hbegfb, vegfaa, vegfab, alcama and alcamb mRNA levels in
zebrafish lacking the full hbegfa locus or the vegfaa or alcama promoter compared to wt
siblings. b, qPCR analysis of Rela, Rel, Actb, Actg1, Actg1 and Actg2 mRNA levels in
MEFs and mESCs lacking the Rela promoter or the full Actg1 or Actb locus compared to wt
cells. c, Cytotoxicity assay following treatment with TNFα of wt, Rela K.O. and Rela
promoter-less MEFs. Percentages normalized relative to DMSO-treated cells. d, Confocal
micrographs of wt, Actb K.O. and Actb full locus deletion mESCs. Actin filaments are
depicted in white, nuclei in red. e, Actin filament protrusion length in wt, Actb K.O. and
Actb full locus deletion mESCs. f, Confocal micrographs of 48 hpf Tg(fli1a:eGFP) wt and
egfl7 full locus deletion mutant siblings; lateral views, anterior to the left. Higher
magnifications of dashed boxes shown in f’. Scale bars: e, 20 µm; f, 500 µm. a, b, wt
expression set at 1. a-c, e, n = 3 (a, b); 5 (c); 189, 219 and 205 (e) independent experiments.
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Error bars, mean, s.d. Two-tailed student’s t-test used to assess P values. d, f, These
experiments were repeated twice independently with similar results.
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Figure 4. Transcriptional adaptation favors genes exhibiting sequence similarity to the mutated
gene’s mRNA and is associated with permissive histone marks.
a, Percentage of significantly upregulated (Log2 Fold Change K.O. > wt and P ≤ 0.05)
protein-coding genes exhibiting sequence similarity with Fermt-2, Actg1 or Actb and those
not exhibiting sequence similarity. b, qPCR analysis of epas1a mRNA levels in 6 hpf wt
zebrafish injected with uncapped RNA composed solely of the hif1ab mRNA sequences
similar to epas1a or uncapped RNA composed solely of the hif1ab mRNA sequences not
similar to epas1a. c, d, ChIP-qPCR analysis of Wdr5 (c) and H3K4me3 (d) occupancy near
the TSS of Fermt1, Rel and Actg2 in Fermt-2, Rela and Actg1 K.O. cells, respectively,
compared to wt. Quantification of enrichment shown as fold-enrichment over IgG control. e,
Current putative simplified model of transcriptional adaptation to mutations. TC: termination
codon; DFs: decay factors; RBPs: RNA binding proteins. b, Control expression set at 1. b-d,
n = 3 (b); 2 (c, d); biologically independent samples. Error bars, mean, s.d. Two-tailed
student’s t-test used to assess P values.