Nuclear receptor activation shapes spatial genome organization essential for gene expression control: lessons learned from the vitamin D receptor
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Published online 23 March 2022 Nucleic Acids Research, 2022, Vol. 50, No. 7 3745–3763
https://doi.org/10.1093/nar/gkac178
Nuclear receptor activation shapes spatial genome
organization essential for gene expression control:
lessons learned from the vitamin D receptor
Timothy Warwick1,3 , Marcel H. Schulz 2,3
, Ralf Gilsbach1,3 , Ralf P. Brandes1,3 and
Sabine Seuter 1,3,*
1
Institute for Cardiovascular Physiology, Goethe University, Frankfurt/Main, Germany, 2 Institute for Cardiovascular
Regeneration, Goethe University, Frankfurt/Main, Germany and 3 German Center for Cardiovascular Research
(DZHK), Partner site Rhein-Main 60590, Frankfurt am Main, Germany
Downloaded from https://academic.oup.com/nar/article/50/7/3745/6553119 by guest on 10 June 2022
Received August 09, 2021; Revised February 23, 2022; Editorial Decision March 04, 2022; Accepted March 07, 2022
ABSTRACT INTRODUCTION
Spatial genome organization is tightly controlled In genome architecture, chromosome territories followed by
by several regulatory mechanisms and is essen- active A and inactive B compartments represent the high-
tial for gene expression control. Nuclear receptors est hierarchy of organization (1,2). The next scale of 3D
are ligand-activated transcription factors that modu- chromatin domains contains topologically associating do-
late physiological and pathophysiological processes mains (TADs, ∼1 Mb), which are characterized by a high
degree of self-interaction (1,3,4). Structures identified in the
and are primary pharmacological targets. DNA bind- sub-TAD range have been termed insulated neighborhoods,
ing of the important loop-forming insulator protein contact domains or loop-domains (5,6).
CCCTC-binding factor (CTCF) was modulated by The zinc finger transcription factor and insulator protein
1␣,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ). We per- CTCF binds at TAD boundaries and has been proposed to
formed CTCF HiChIP assays to produce the first form loops together with cohesin by loop extrusion (1,3,7–
genome-wide dataset of CTCF long-range interac- 14). Insulators can block the spread of active or repressive
tions in 1,25(OH)2 D3 -treated cells, and to determine chromatin (15,16), or protect a gene from ectopic activation
whether dynamic changes of spatial chromatin inter- if located between the gene and an enhancer (17). Also, the
actions are essential for fine-tuning of nuclear recep- interaction of two insulators with each other by chromatin
tor signaling. We detected changes in 3D chromatin looping can bring an enhancer in proximity to a promoter
organization upon vitamin D receptor (VDR) activa- or, contrastingly, tether it to a locus away from a promoter
(18,19).
tion at 3.1% of all observed CTCF interactions. VDR
The impact of TADs in enhancer function and gene reg-
binding was enriched at both differential loop an- ulation is diverse (20). CTCF has been proposed to main-
chors and within differential loops. Differential loops tain a nucleosome-free, accessible region at promoters (21).
were observed in several putative functional roles The observed effects of TAD boundary deletion on gene ex-
including TAD border formation, promoter-enhancer pression ranged from no functional impact to severe effects
looping, and establishment of VDR-responsive insu- (16,22–34). Consequences of chromatin rearrangements in-
lated neighborhoods. Vitamin D target genes were clude TAD fusions, enhancer hijacking, ectopic enhancer-
enriched in differential loops and at their anchors. promoter loops, and insertions of new CTCF boundaries,
Secondary vitamin D effects related to dynamic all of which may lead to changes in gene expression (22–
chromatin domain changes were linked to location 24,32–40).
of downstream transcription factors in differential Intra-TAD interactions are frequent and it has been sug-
gested that a TAD facilitates contacts between enhancers
loops. CRISPR interference and loop anchor dele-
and promoters inside the TAD. The restriction of absolute
tion experiments confirmed the functional relevance 3D distance may help to obtain robust and precise tran-
of nuclear receptor ligand-induced adjustments of scription, partly by establishing a high local concentration
the chromatin 3D structure for gene expression reg- of transcription factors (20).
ulation. The CTCF cistrome and the CTCF-mediated long-range
interactions are highly conserved across human, mam-
* To whom correspondence should be addressed. Tel: +49 69 6301 6996, Email: seuter@vrc.uni-frankfurt.de
C The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License
(http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work
is properly cited. For commercial re-use, please contact journals.permissions@oup.com
3746 Nucleic Acids Research, 2022, Vol. 50, No. 7
malian and vertebrate cell types, as shown by chromatin im- anchors. In addition, transcription factors encoded by pri-
munoprecipitation sequencing (ChIP-seq), ’Chromatin In- mary vitamin D target genes were enriched in the differ-
teraction Analysis by Paired-end tag Sequencing’ (ChIA- ential loops. Clustered regularly interspaced short palin-
PET) and Hi-C experiments in different cellular systems dromic repeats interference (CRISPRi) experiments vali-
in their basal state (3,41–50). However, gene expression dated a VDR co-bound CTCF loop anchor as functional
changes during cellular differentiation and senescence have enhancer of the prostaglandin E synthase 2 (PTGES2)
been linked to rearrangements in loops and compartments gene. Deletion of anchors of differential loops resulted in
(51–53). In addition, a subset of heat shock-upregulated modulated vitamin D regulation of some genes inside and
genes displayed a modestly increased frequency of chro- neighboring the loops. Thus, the physiological stimulus with
matin interactions (54). Depletion of CTCF or cohesin in the nuclear receptor ligand 1,25(OH)2 D3 induced function-
cell culture in a steady state dramatically perturbed TAD ally important changes in the 3D chromatin architecture in
formation leading to differential expression of hundreds living cells.
of genes (11–14,55–59). The resulting expression changes
were moderate, though. Thus, it was concluded that chro- MATERIALS AND METHODS
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matin topology is not essential for enhancer-promoter inter-
actions. A more complex role of 3D chromatin architecture Cell culture
on transcriptional regulation can be assumed that needs to The human acute monocytic leukemia cell line THP-
be elucidated in non-steady state models as development, 1 is a well responding and physiologically meaning-
differentiation or cell signaling (60). ful model system for the investigation of 1,25(OH)2 D3 -
The nuclear receptor superfamily is a large group of triggered physiological processes, such as innate im-
ligand-activated transcription factors, reviewed in (61–64). munity and cellular growth. The cells were grown in
They have a wide variety of essential functions in processes RPMI 1640 medium, supplemented with 10% fetal calf
such as cell signaling, survival, or proliferation. serum (FCS), 2 mM L-glutamine, 0.1 mg/ml streptomycin
The VDR is a classical endocrine receptor and belongs to and 100 U/ml penicillin, and were kept at 37◦ C in a humid-
the retinoid X receptor (RXR) heterodimer-forming class II ified 95% air/5% CO2 incubator. Prior to experiments, cells
nuclear receptors. It is activated by its high affinity ligand were grown overnight in phenol red-free medium supple-
1,25(OH)2 D3 , the most active metabolite of vitamin D3 . mented with charcoal-stripped FCS and then treated with
VDR preferentially binds as a heterodimer with RXR to vehicle (0.1% ethanol (EtOH)) or 100 nM 1,25(OH)2 D3
DR3-type response elements, which are formed by a direct (Sigma-Aldrich) for 24 h.
repeat of two hexameric sequence motifs spaced by three Lenti-X 293T cells (Takara Bio) were grown in DMEM
nucleotides (65,66). A prerequisite of VDR DNA binding medium supplemented with 10% FCS, 0.1 mg/ml strepto-
is the location of the binding site in accessible chromatin, mycin and 100 U/ml penicillin and were kept at 37◦ C in a
which can be achieved by the action of pioneer factors like humidified 95% air/5% CO2 incubator.
purine-rich box 1 (PU.1) and CCAAT/enhancer binding
protein alpha (CEBPA) (67,68). Ligand-bound VDR inter-
feres with chromatin modifiers (69–71), leading to an in- CTCF HiChIP
crease of active histone marks and open chromatin (72,73). HiChIP assays of three biological repeats were performed as
There are some reports on interference of nuclear recep- described by Mumbach et al. (80), with some modifications.
tors with 3D chromatin structure, often only investigating After treatment of 30 × 106 THP-1 cells, nuclear proteins
individual genomic loci. Examples include links between were cross-linked to genomic DNA by adding formaldehyde
thyroid hormone receptor (TR) stimulation and CTCF directly to the medium to a final concentration of 1% and
enhancer blocking (74,75), chromatin remodelling at the incubating at room temperature for 10 min on a rocking
HOXA locus upon stimulation of retinoic acid receptor platform. Cross-linking was stopped by adding glycine to a
(RAR) ␣,  and ␥ (76), and the interplay between CTCF final concentration of 0.125 M and incubating at room tem-
and ER loop formation in breast cancer cells (77). In the perature for 10 min on a rocking platform. The cells were
latter case, mechanisms including 3D epigenome remod- collected by centrifugation and washed twice with ice cold
elling have been proposed to influence endocrine resistance phosphate-buffered saline (PBS). Cells were lysed in 1 ml
in ER+ breast cancer (78). Hi-C Lysis Buffer (10 mM Tris–HCl pH 8.0, 10 mM NaCl,
As 1,25(OH)2 D3 modulated the binding of CTCF in 0.2% NP-40, protease inhibitors) with rotation at 4◦ C for
roughly 2,100 genomic regions in monocytic THP-1 cells 30 min and split into two aliquots. Nuclei were washed with
(73,79), we hypothesized that it thereby may modulate the Hi-C Lysis Buffer and incubated with 100 l 0.5% SDS at
CTCF-defined chromatin structure. So far, no genome- 62◦ C for 10 min. SDS was quenched with 285 l H2 O and
wide studies investigated chromatin interactions upon 50 l 10% Triton X-100 rotating at 37◦ C for 15 min. Re-
1,25(OH)2 D3 treatment of cells or VDR-related interac- striction digestion with 15 l MboI (New England Biolabs)
tions. Thus, in this study we performed CTCF HiChIP as- in NEB Buffer 2.1 was performed rotating at 37◦ C for 2 h.
says to identify 1,25(OH)2 D3 -induced rearrangements in The pellet was washed twice with and resuspended in NEB
chromatin topology. We found that the frequency of >3,000 Buffer 2.1. Overhangs were filled in and marked with bi-
CTCF interactions was changed by the VDR ligand. VDR otin by adding 37.5 l 0.4 mM Biotin-14-dATP (Thermo
binding sites and vitamin D target genes were enriched in- Fisher Scientific), 1.5 l 10 mM dCTP, 1.5 l 10 mM dGTP,
side these differential loops and often located at the loop 1.5 l 10 mM dTTP and 5 l 10 U/l DNA Polymerase I,Nucleic Acids Research, 2022, Vol. 50, No. 7 3747
Large (Klenow) Fragment (Thermo Fisher Scientific) and gest genome.py, and otherwise default parameters. Loops
rotating at 37◦ C for 1 h. Then, proximity ligation was per- were called per replicate using hichipper (v0.7.0) (82) with
formed by 4 h rotation after addition of 150 l 10× NEB the EACH,ALL parameter set for CTCF peak identifica-
T4 DNA ligase buffer with 10 mM ATP (Thermo Fisher tion, and otherwise default parameters. Loop counts per
Scientific), 125 l 10% Triton X-100, 3 l 50 mg/ml BSA, sample were subsequently used as input to diffloop (v1.20.0)
4 l 1000 CEU/l T4 DNA Ligase (Thermo Fisher Sci- (83) for identification of differential CTCF-mediated chro-
entific), and 666 l H2 O. The supernatant was removed matin interactions. Only loops with a mango (included in
by centrifugation and nuclei were resuspended in 500 l hichipper) adjusted P value 5 was observed in one
combined and sonicated in a Bioruptor Plus (Diagenode) replicate of a treatment group, but not observed at all in
for 5 min. Then, debris was removed. Chromatin Immuno- an accompanying replicate. Differential analysis was carried
precipitation was performed by adding 400 l chromatin out using the quickAssocVoom function, thereby using the
aliquots and 1600 l IP Dilution Buffer (20 mM Tris–HCl limma-voom empirical Bayes analysis pipeline used in dif-
Downloaded from https://academic.oup.com/nar/article/50/7/3745/6553119 by guest on 10 June 2022
pH 7.5, 2 mM EDTA, 1% Triton X-100, 150 mM NaCl, ferential analysis previously in the limma and voom R pack-
protease inhibitors) to each 90 l Dynabeads Protein G ages. Differential interactions were those with an adjusted
(Invitrogen), pre-coupled with 4 l CTCF antibody (Mil- P value 0.9, each with the same number of loops as
washed with Tween Wash Buffer (5 mM Tris–HCl pH 7.5, the differential set. Distances and intersects between fea-
0.5 mM EDTA, 0.05% Tween-20, 1 M NaCl) and 1× Tag- tures were computed using bedtools intersect and bedtools
mentation Buffer. Tagmentation with Tn5 transposase (Il- closest. Virtual 4C was conducted using the GENOVA R
lumina) in a 50 l reaction using the appropriate amount package (v1.0.0) (87), in conjunction with background in-
of Tn5 transposase for every sample as detailed by Mum- teraction frequency data calculated using the HiCExplorer
bach et al. (80) and incubating at 55◦ C for 10 min. The re- (v3.7.2) chicViewpointBackgroundModel tool (88), using all
action was stopped by addition of EDTA and beads were loop anchor regions as reference points to build the model.
washed with Tween Wash Buffer and 10 mM Tris pH 7.5. Li- Intersections were considered to be ≥1 bp overlaps be-
brary preparation and size selection were performed as de- tween features, with the exception of TAD borders, which
scribed in the Mumbach protocol (80). Library quality was were extended by 25 kb upstream and downstream us-
controlled by High Sensitivity DNA Assay on an Agilent ing bedtools slop. Statistically significant distances were as-
Bioanalyzer. The sequencing was performed at Novogene cribed between differential and shuffled loop sets by use of
(HK) on an Illumina Hiseq-PE150. The sequencing depths Fisher’s Exact Test for count data, or by a Kolmogorov–
obtained per sample were between 300 and 400 million Smirnov test for cumulative distributions. When compar-
reads. ing the differential loop set against the stable loop sets,
the proportion of stable loop sets which exceeded the dif-
ferential set in each particular metric was taken as the
P value. To calculate this, the enrichment of the feature
CTCF HiChIP data analysis in question was computed for each set of stable loops
Paired-end tags arising from CTCF HiChIP were pre- alongside the differential loop set. Each stable enrichment
processed, aligned to the hg19 genome, and used to was then compared to the differential enrichment, and the
construct iteratively corrected contact maps using HiC- proportion of stable loop sets where the enrichment ex-
Pro (v2.11.1) (81) in combination with MboI restriction ceeded the differential enrichment was used as the resulting
fragments generated using the HiC-Pro utility script di- P value.3748 Nucleic Acids Research, 2022, Vol. 50, No. 7
The quality control metrics are displayed in Supplemen- Hi-C data analysis
tary Figure S1A.
For the analysis, the duplicates were merged to provide
greater sequencing depths. Hi-C sequencing reads were
Hi-C aligned to the hg19 genome using Bowtie2 with the –local
and –reorder parameters set. The subsequently generated
Hi-C assays were performed in duplicate biological repeats
alignment files were then used to build Hi-C matrices with
using the Arima-HiC kit following the manufacturer’s user
the HiCExplorer (v3.7.2) tool hicBuildMatrix. The built
guide, with some modifications. Briefly, 2.5 × 106 THP-1
matrices were then normalized to the smallest of the sam-
cells were cross-linked with 2% formaldehyde for 10 min,
ples using hicNormalise, and then corrected using itera-
stopped with glycine, washed twice with PBS and snap-
tive correction with hicCorrectMatrix, with the parameter
frozen. The thawed cells were lysed in 20 l lysis buffer
–correctionMethod ICE and thresholds taken from diag-
(15 min 4◦ C), then incubated with 24 l Conditioning so-
nostic plots created using hicCorrectMatrix diagnostic plot.
lution (10 min 62◦ C) and 20 l Stop Solution 2 (15 min
Corrected matrices were then used in the construction of
37◦ C). Digestion with 12 l Enzymes A1/A2 master mix
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relevant viewpoints using chicViewpointBackgroundModel
was performed by sequential incubations of 45 min 37◦ C,
and chicViewpoint. The background model was computed
20 min 65◦ C and 10 min 25◦ C. Then, the biotin fill-in was
across all identified differential loop anchors in the CTCF
performed with 16 l Enzyme B master mix (45 min RT),
HiChIP analysis.
followed by proximity ligation with 82 l Enzyme C mas-
Differential TADs were detected using the R package
ter mix (15 min RT). Proteins were digested with 35.5 l
TADCompare (1.2.0) (89). This used eigenvector gaps to
Enzyme D master mix plus 20 l Buffer E in the sequen-
compare boundaries and subsequently identify dynamic
tial incubations of 30 min 55◦ C, 90 min 68◦ C and 10 min
and stable boundaries between conditions. Input matrices
25◦ C. The DNA was purified using 100 l AMPureXP
of 50 kb resolution were imported into R and then the TAD-
beads (Beckman Coulter). After passing the QC1 quality
Compare command was used for differential TAD detec-
control, 5 g of each sample was diluted to 100 l with Elu-
tion.
tion Buffer supplemented by 1% SDS. Fragmentation to an
The quality control metrics are displayed in Supplemen-
average fragment size of ≈400 bp was done by 15 cycles son-
tary Figure S1B and C.
ication in a Bioruptor Plus (Diagenode) with 30 s ON, 30 s
OFF at high intensity. Size selection was performed with
60 l and 40 l SPRIselect beads (Beckman Coulter). Bi- Transcription factor binding prediction
otin enrichment was performed with 0.9–2 g of the size se-
lected DNA using 50 l Enrichment Beads for 1 g DNA (15 min RT). Following washes ity at areas of open chromatin in CTCF loop anchor re-
with Wash Buffer and Elution Buffer end repair and adapter gions (90). Open chromatin regions were detected with
ligation were performed: incubation with 20 l Enzyme W2 formaldehyde-assisted isolation of regulatory elements se-
master mix (10 min 37◦ C), washes, incubation with 50 l quencing (FAIRE-seq) in THP-1 cells after 24 h of 100 nM
Enzyme G3/G4 master mix (20 min 20◦ C), washes, ligation 1,25(OH)2 D3 treatment (73). Transcription factor binding
of a uniquely indexed P7 TruSeq LT Adapter (Swift Bio- sites were predicted using position specific energy matri-
sciences Set A Indexing Kit) in 25 l Enzyme Y1 master mix ces derived from JASPAR 2020 (91), HOmo sapiens COm-
(15 min 25◦ C), washes, ligation of non-indexed P5 TruSeq prehensive MOdel COllection (HOCOMOCO) (92) and
Universal adapter (Swift Biosciences Indexing Kit) in 50 l Kellis ENCODE motif databases (93), which are part of
Enzyme B4/B5/B6 master mix, washes, resuspension. The the TEPIC 2 repository. Predicted binding of a transcrip-
sufficient cycle number was determined with an initial PCR tion factor at a loop anchor region was assumed where
using 2 l of the resuspended beads. The remainder of the the predicted transcription factor binding affinity at any
beads was subjected to library amplification. Samples, for FAIRE-seq peak in the loop anchor region was signifi-
which 100 l Enrichment Beads had been used, were dis- cantly strong. Significance was computed using a back-
tributed into six 50 l PCR reactions, while those with ground set of regions with similar GC-content and length
50 l Enrichment Beads were run in three 50 l reactions. distribution as the original FAIRE-seq peak set as de-
PCRs were performed using the KAPA 2X HiFi HotStart scribed in (94). Each TF affinity value that met a TEPIC-
Ready Mix (KAPA Biosystems), KAPA Library Amplifi- computed threshold (P < 0.05) was considered signifi-
cation Primer Mix and EvaGreen (BioRad). The replicate cant. Thus, the number of loop anchors where each tran-
PCR reactions were pooled, separated from the beads and scription factor was predicted to bind could be quanti-
purified with AMPureXP beads (Beckman Coulter). Con- fied and compared between differential and shuffled loop
centrations were measured using the Qubit dsDNA HS As- anchors.
say Kit (Thermofisher Scientific) and quality-controled by
High Sensitivity DNA Assay on an Agilent Bioanalyzer or
CRISPR/Cas9-mediated loop anchor deletions
by D1000 ScreenTape on a TapeStation (Agilent). Sequenc-
ing was performed by Dr. Stefan Günther at Max-Planck- Specific gRNAs targeting regions upstream and down-
Institute for Heart and Lung Research, Bad Nauheim, Ger- stream of the selected CTCF loop anchor sites were de-
many, on an Illumina NextSeq 500-PE100. The sequencing signed using the Benchling life sciences R&D cloud (https:
depths obtained per sample were between 76.2 and 151.3 //www.benchling.com). A combination of two gRNAs was
million reads. used to obtain a deletion. Sense and antisense oligonu-Nucleic Acids Research, 2022, Vol. 50, No. 7 3749
cleotides containing the 19 bp gRNA sequence plus the CRISPR interference
required overhangs matching the BsmBI-cut vector were
Specific gRNAs targeting CTCF loop anchor regions, VDR
purchased (Sigma-Aldrich) and annealed (Supplementary
enhancer or transcription start site (TSS) have been de-
Table S2). Then, BsmBI (Esp3I) (Thermo Fisher) and T4
signed using the Benchling life sciences R&D cloud (https://
DNA ligase (Thermo Fisher) were used to clone the an-
www.benchling.com) or the GPP sgRNA Designer (https://
nealed gRNAs into the plasmid containing expression cas-
portals.broadinstitute.org/gpp/public). Sense and antisense
settes for hSpCas9 and the chimeric guide RNA with the
oligonucleotides containing the 19 bp gRNA sequence
Golden Gate assembly protocol (95). The vector used was
plus the required overhangs matching the BsmBI-cut vec-
either LentiCRISPRv2 (Addgene #52961 (96)) conveying
tor were purchased (Sigma-Aldrich) and annealed (Sup-
puromycin resistance, or a modification thereof including
plementary Table S4). Then, BsmBI (Esp3I) (Thermo
hygromycin resistance. Thus, a combination of two gRNAs
Fisher) and T4 DNA ligase (Thermo Fisher) were used
cloned in the two different plasmids allowed for double se-
to clone the annealed gRNAs into the plasmid pLV hU6-
lection after the transduction (Supplementary Table S3).
sgRNA hUbC-dCas9-KRAB-T2a-GFP (Addgene #71237,
Correct insertions of the gRNAs into the plasmid were ver-
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gift from Charles Gersbach (97)) containing expression
ified by Sanger sequencing.
cassettes for the catalytically inactive dCas9 fused to the
Cell-free, lentiviral supernatants were produced by
transcriptional repression domain Kru¨ppel-associated box
polyethylenimine (PEI)-based transient co-transfection
(KRAB) and the chimeric guide RNA with the Golden
of Lenti-X 293T cells. Briefly, the pLentiGuide-Puro-
Gate assembly protocol (95). Correct insertions of the gR-
gRNA vector, the lentiviral gag/pol packaging plasmid
NAs into the plasmid were verified by Sanger sequencing.
psPAX (Addgene #12260; gift from Didier Trono;
Lentivirus preparation and transductions were per-
http://n2t.net/addgene:12260; RRID:Addgene 12260) and
formed as described above for the CRISPR deletions, with
the envelope plasmid encoding the glycoprotein of vesicu-
the difference that 0.4 × 106 cells have been used. Four
lar stomatitis virus (VSV-G) (pMD2.G, Addgene #12259;
days after the second spinoculation, the cells were seeded in
gift from Didier Trono; http://n2t.net/addgene:12259;
medium containing charcoal-stripped FCS. The next day,
RRID:Addgene 12259) were transfected at a molar ra-
cells were treated with 1,25(OH)2 D3 or ethanol prior to
tio of 3:1:1 by standard PEI transfection. 24 and 48 h
RNA extraction.
post-transfection, two consecutive viral supernatants were
harvested, cleared through 0.45-m pore-size PVDF
membrane filter (Millipore), combined and stored at RNA isolation, cDNA synthesis and qPCR
−80◦ C.
The transduction with the gRNA-expressing plasmids Total RNA was extracted using the RNA Mini Kit (Bio-
was performed in two consecutive spinoculations. Briefly, Sell) according to the manufacturer’s instructions. cDNA
0.2 × 106 cells were pelleted and resuspended in 1 ml of the synthesis was performed using Superscript III reverse tran-
lentiviral particles. Polybrene (8 g/ml) was added and the scriptase (Invitrogen) and oligo(dT)23 primers according to
cells were centrifuged for 90 min with 1000 × g at room the manufacturer’s instructions. PCR reactions were per-
temperature. The supernatant was discarded, the cells re- formed with the AriaMx Real-Time PCR System (Agi-
suspended in 1 ml full growth medium and incubated for lent Technologies) using 250 nM of reverse and forward
48–72 h. Then, the second spinoculation was carried out primers, cDNA and the iTaq Universal SYBRGreen Su-
with 800 l of lentivirus. After another 48–72 h incubation, permix (Bio-Rad). Primer-specific temperatures and se-
puromycin (2 g/ml) and hygromycin (400 g/ml) were quences are listed in Supplementary Table S1. Relative
added to select the transduced cells for 10–14 days. mRNA expression levels were determined using the formula
The successful deletions were confirmed by agarose gel 2–(Ct) , where Ct is Ct(target gene) – mean Ct(reference genes) .
electrophoresis of the amplicons of PCRs with genomic The genes beta-2-microglobulin (B2M), glycerinaldehyde-
DNA as template (data not shown). In cases, where no 3-phosphate-dehydrogenase (GAPDH) and hypoxanthine
complete deletion was obtained in the whole cell pop- phosphoribosyltransferase 1 (HPRT1) were used as refer-
ulation, clonal expansion was performed. Positive sin- ences as described previously (98).
gle cell clones were then selected for subsequent experi- All RT-qPCR experiments have been performed in at
ments. RT-qPCR was performed to assess possible changes least triplicate biological replicates. One-sample Student’s
of the transcriptional response to 1,25(OH)2 D3 after the t-tests were performed to determine the significance of
deletion. changes of mRNA inductions by 1,25(OH)2 D3 in reference
The deletion of the right anchor of the second differen- to solvent-treated cells and to determine the significance of
tial loop in the ADAM like decysin 1 (ADAMDEC1) lo- % expression changes compared to NTC control cells. The
cus was very efficient for two different gRNA combinations significance of all other effects was determined by two-tailed
(L66 and L67), thus the experiments were performed with Student’s t-tests (* P < 0.05; ** P < 0.01; *** P < 0.001).
both whole cell populations without clonal expansion. For
all other loop anchor deletions single cell clones were cre-
Previously published RNA and chromatin data
ated.
Prior to each experiment for RNA extraction, the VDR ChIP-seq data in THP-1 cells is publicly available
CRISPR cells were re-selected with puromycin (2 g/ml) at NCBI Gene expression Omnibus (GEO) under the ac-
and hygromycin (400 g/ml) for 3 days. cession GSE89431, originally published by Neme et al.3750 Nucleic Acids Research, 2022, Vol. 50, No. 7
(99). CTCF ChIP-seq data in THP-1 cells is publicly avail- Trends underlying observed differential CTCF HiChIP
able under the accession GSE69962 (73,79). RNA-seq time interactions could also be observed in Hi-C data from THP-
course data in THP-1 cells with 2.5 h, 4 h and 24 h of 1 cells treated with 1,25(OH)2 D3. Viewpoint analyses of dif-
100 nM 1,25(OH)2 D3 treatment is accessible at the acces- ferential CTCF HiChIP anchors revealed that even at a
sion GSE69303 (73,99). FAIRE-seq data in THP-1 cells shallow sequencing depth, a number of differential CTCF
after 24 h of 100 nM 1,25(OH)2 D3 treatment is available HiChIP interactions were also observable when not only fo-
from the same source under the accessions GSE69297 and cusing on interactions mediated by CTCF (Supplementary
GSE69303, originally published by Seuter et al. (73). ETS1 figures S4A–C).
ChIP-seq data from 1,25(OH)2 D3 -treated THP-1 cells can Taken together, 3.1% of all CTCF-mediated chromatin
be obtained from GEO under GSE157209 (100). Digestion- loops in monocytic THP-1 cells are sensitive to VDR ac-
ligation-only Hi-C (DLO-Hi-C) is available under the acces- tivation and the overrepresented upregulated loops are en-
sion GSE89663 (101). riched for promoter-enhancer interactions.
The Integrative Genomics Viewer (IGV) browser was
used to visualize the genome-wide datasets (102).
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1,25(OH)2 D3 treatment changes chromatin conformation
close to TAD boundaries
RESULTS
As CTCF binding is important for the formation of TADs,
1,25(OH)2 D3 treatment modulates functional CTCF-defined
we sought to examine whether VDR activation leads to
chromatin structure in monocytic cells
changes in chromatin domain organization, such as the fre-
In this study, we have aimed to decipher dynamic changes quency of individual TADs in a cell. We intersected dif-
in CTCF-mediated long-range chromatin interactions teth- ferential CTCF loops with published TADs called from
ered by CTCF in THP-1 cells treated for 24 h with 100 nM DLO Hi-C data in THP-1 cells (101). We found that the an-
1,25(OH)2 D3 using CTCF HiChIP assays (Figure 1A). A chors of differential CTCF loops were significantly enriched
replicate-based analysis identified in total 2,408,953 loops for TAD borders when compared to shuffled loops, but
across 260,099 anchors present across any replicate. 160,291 not when compared to stable loops (Supplementary Figure
interactions were present in every control sample and S5A, P < 0.001, Fisher’s Exact Test). The locus of the vi-
190,889 loops in every 1,25(OH)2 D3 -treated sample (Sup- tamin D target gene zinc finger protein 618 (ZNF618) is a
plementary Figures S2A and B). Of those, 109,764 were de- representative example, where a differential CTCF interac-
tected in both conditions (Figure 1B). tion coincides with a TAD (Supplementary Figure S5B).
Differential chromatin topology analysis identified To further develop this finding, we used differential TAD
3,433 loops, whose frequency was significantly changed border detection to identify whether any differential CTCF
(Padj < 0.05, diffloop) by the stimulation. Of these dif- HiChIP loops matched modulated TADs observed in our
ferential loops, 2,480 were more frequently observed in own Hi-C data from vehicle- and 1,25(OH)2 D3 -treated
1,25(OH)2 D3 -treated cells, and 953 were less frequent in THP-1 cells. Of the 8,215 TAD borders identified by TAD-
treated cells (Figure 1C). The number of 1,25(OH)2 D3 - Compare, 1,700 were classified as differential with differ-
sensitive loops was similar to the number of vitamin D ent consequences on the TADs they defined (745 complex,
target genes that has been published in THP-1 cells (73,99), 493 split, 429 merged, 33 changed in strength) (Supplemen-
slightly higher than the number of 1,25(OH)2 D3 -modified tary Figure S6A). Representative examples of differential
CTCF ChIP-seq peaks (73,79), but much lower than the TADs, which reflected putative differential CTCF HiChIP
number of differential VDR ChIP-seq peaks (99) (Supple- loops, were those in the loci of the vitamin D target genes
mentary Figure S2C). CTCF ChIP-seq peaks were also androgen induced 1 (AIG1), adenosine deaminase tRNA
reproduced in CTCF HiChIP, with ∼84% of ChIP-seq specific 2 (ADAT2) and cytochrome P450 family 24 sub-
peaks also being called in HiChIP data (Supplementary family A member 1 (CYP24A1), where differential loop
Figures S2D and E), although the crossover between anchors clearly intersected with TAD boundaries enriched
differential CTCF ChIP-seq peaks and differential loop after 1,25(OH)2 D3 treatment (Supplementary Figures S6B
anchors was not extensive (Supplementary Figure S2F). We and C).
found that the differential loops were enriched for putative This indicates that VDR-mediated changes of chromatin
promoter-promoter, promoter-enhancer and enhancer- interactions may affect CTCF-marked TAD boundaries.
enhancer interactions (Figure 1D, Supplementary figures
S3A and B). Notably, a higher ratio of 1,25(OH)2 D3 -
VDR binding sites are enriched at 1,25(OH)2 D3 -responsive
upregulated loops (30.5%) was classified as putative
CTCF loop regions
promoter-enhancer loops compared to downregulated
loops (23.6%) (P < 0.0001, Fisher’s exact test) (Figure 1D, To elucidate the interplay between VDR and CTCF-defined
Supplementary figures S3C and D). chromatin structure, we integrated previously published
Virtual 4C viewpoint analyses visually confirmed for a VDR ChIP-seq data (99) with differential loops. Of spe-
number of loci the differential chromatin interactions iden- cial interest was the identification of differential loops where
tified by the replicate-based analysis. A representative dif- VDR binding in proximity to the loop anchor regions
ferential loop encapsulates the differentially expressed gene could affect differential loop formation or VDR promoter-
(DEG) potassium channel tetramerization domain contain- enhancer interactions. VDR ChIP-seq peaks were enriched
ing 12 (KCTD12), along with several VDR ChIP-seq peaks in proximity of differential CTCF loop anchors when
(Figure 1E). compared to shuffled and stable loop sets (Figure 2A):Nucleic Acids Research, 2022, Vol. 50, No. 7 3751
CTCF HiChIP
A
1,25D
24 h CTCF control
CTCF
CTCF
CTCF
CTCF
CTCF
THP-1 CTCF
CTCF
C
CTCF
CTCF
compare contact frequencies
1,25D
F
CTCF
CTCF
CTCF
CTCF
CTCF
CTCF
CTCF
CTCF
1,25D HCHO crosslinks
VDR
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VDR Biotin
relate to VDR-bound loci
VDR
VDR
CTCF CTCF protein
V
F
CTCF
CTCF
CTCF
CTCF
CTCF
CTCF
CTCF
CTCF
B C D
Proportion of differential loops
EtOH 1,25D 1.00
(160,291) (190,889)
Differential loops
2000 0.75
Other
27
0.50 Promoter-enhancer
,5
50
109,764 1000 Enhancer-enhancer
25
0.25
,1
81
0 0.00
Down Up Down Up
Loop frequency change Loop frequency change
Chr13: 76.000 kb 76.500 kb 77.000 kb 77.500 kb
E 0.4
Treatment
0.3
Background
CTCF
EtOH
HiChIP
0.2 1,25D
Viewpoint
signal
0.1
0.0
ChIP-seq ChIP-seq
EtOH
CTCF
1,25D
EtOH
VDR
1,25D
Genes
KCTD12
HiChIP
CTCF
Diff.
loops
Figure 1. Treatment of THP-1 cells with 1,25(OH)2 D3 provoked changes in CTCF-directed chromatin conformation, identified by differential chromatin
topology analysis of HiChIP data. (A) Experimental setup of this study. THP-1 cells were treated for 24 h with 100 nM 1,25(OH)2 D3 or control (EtOH).
Triplicate CTCF HiChIP experiments were performed and the interactions were called using hichipper. The resulting loop data was integrated with previous
VDR ChIP-seq data (99). (B) Intersect between consistent (i.e. present in all three replicates) CTCF loops in control and 1,25(OH)2 D3 -treated cells. (C)
Numbers of upregulated and downregulated differential (Padj < 0.05, diffloop) loops in 1,25(OH)2 D3 treatment versus control (vehicle) HiChIP samples.
(D) Annotation of upregulated and downregulated differential CTCF loops as enhancer-enhancer, promoter-enhancer, or other loops (see Supplementary
Figures S3A–D for all types). Enhancers were taken from EpiRegioDB, and promoters were considered as a 5 kb (2.5 kb upstream/downstream) window
around the TSSs of all hg19 genes. (E) Virtual 4C viewpoint analysis of a differential loop anchor region in both control and 1,25(OH)2 D3 -treated cells
(n = 3, summed interaction frequencies), shown alongside CTCF ChIP-seq (violet tracks, (73,79)) and VDR ChIP-seq (red tracks, (99)) data. There are
two strong 1,25(OH)2 D3 -induced VDR peaks (shaded in grey) in the locus, while the strong VDR site at the TSS of the vitamin D target gene KCTD12
(24 h 1,25(OH)2 D3 : log2 FC = 3.44; Padj < 0.001) is persistently occupied with and without 1,25(OH)2 D3 stimulation.3752 Nucleic Acids Research, 2022, Vol. 50, No. 7
A Shuffled Differential Stable B C
VDR peaks per kb of loop
0.008 *** 0.008
VDR peaks per kb of loop
***
***
1.5e-05
0.006 0.006
1.0e-05
Density
0.004 0.004
5.0e-06 0.002 0.002
0.000 0.000
0.0e+00 Down Up
led ble Diff
0 100 200 300 400 500+
h uff Sta Loop regulation
Closest VDR peak to loop anchor [kb] S
Loop type
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D E
% of all VDR peaks
Shuffled Differential 60%
Differential loops
1000 K-S test: p < 8.3e -184
Non-differential
40%
Downregulated
500 Upregulated
20%
0 0%
0 5 10 15 No Yes
VDR peaks in differential loop Peak in differential loop
F chr 20: 52,200 kb 52,300 kb 52,400 kb 52,500 kb 52,600 kb 52,700 kb 52,800 kb
[0-2.00]
VDR EtOH
[0-2.00]
ChIP-seq 1,25D
ZNF271 LOC105372672 SUMO1P1 BCAS1 MIR4756 CYP24A1 PFDN4
RNA-seq FC 0.67x
CTCF [0-2.00]
EtOH
ChIP-seq 1,25D [0-2.00]
[0-600]
EtOH
[0-600]
1,25D
CTCF
HiChIP Diff.
loops
log2 FC = 3.0
Figure 2. VDR binding loci were enriched inside differential CTCF loops and preferentially located close to differential CTCF loop anchors. (A) Densities
of the minimum linear distances between differential, stable, and shuffled differential loop anchors and VDR ChIP-seq peaks (99). The dashed lines
represent the median distances per loop set. (B) Occurrence rate of previously identified VDR ChIP-seq peaks (99) present in THP-1 cells after 24 h
of 100 nM 1,25(OH)2 D3 treatment per kb of differential, stable and shuffled loops. The difference between the regions was computed using Fisher’s
exact test for shuffled loops, and the proportion of stable loop sets, which exceeded the differential value for stable loops. The standard deviations are
too small to be visible. (C) Number of VDR ChIP-seq peaks per kb of upregulated and downregulated loops. (D) Numbers of shuffled and differential
CTCF loop regions containing different numbers of VDR ChIP-seq peaks (99). Difference between the two distributions was determined using a two-
sided Kolmogorov–Smirnov (K–S) test. There were 2,231 differential CTCF loops intersecting with more than two VDR peaks versus 1,131 shuffled
loop regions. (E) Percentage of all 9,763 VDR ChIP-seq peaks (99) intersecting with either upregulated differential CTCF loops (44.9%), downregulated
differential CTCF loops (20.7%), or not intersecting with any differential CTCF loop (34.4%). (F) An upregulated differential CTCF loop (violet arc) in
the locus of the strong vitamin D target gene CYP24A1 contains several VDR sites (red peak tracks) (99), one of which is located within 25 kb distance
of one of the loop anchors. Gene structures are shown in blue. The violet peak track displays CTCF ChIP-seq data (73,79) and CTCF HiChIP peaks. The
expression change (FC, linear fold change) 24 h after stimulation with 1,25(OH)2 D3 as determined by RNA-seq (73,99) is given for significant DEGs. The
scale for the VDR peak tracks has been chosen such as so allow weaker peaks to be visible, which is why the summit of strong peaks is cut.Nucleic Acids Research, 2022, Vol. 50, No. 7 3753
differential CTCF loops, stable CTCF loops, and shuf- above observations (Figure 3H). The highly 1,25(OH)2 D3 -
fled loops had median loop anchor-VDR distances of responsive gene CD14 and several VDR sites were in close
26.7, 38.7 and 88.4 kb, respectively. proximity to the loop anchors.
To also characterize those differential loops, which These data show that primary vitamin D target genes are
were not putative promoter-enhancer interactions, we enriched in differential loops and tend to be close to their
then analyzed whether VDR peaks were located inside anchors. A high proportion of the 1,25(OH)2 D3 -sensitive
1,25(OH)2 D3 -modulated loops. The number of VDR peaks CTCF loops contain both VDR site(s) and DEG(s).
per kb of loop was significantly greater in differential loops
(0.0064) when compared to stable (0.0053) and shuffled
1,25(OH)2 D3 -sensitive CTCF loops are enriched in VDR or
(0.0035) loop sets (P < 0.05, Fisher’s exact test for shuf-
downstream transcription factor binding at anchor regions
fled, proportion exceeded for stable) (Figure 2B). The en-
richment of VDR peaks was higher in upregulated than in Our previous analyses suggested a prominent role of VDR
downregulated loops (Figure 2C). There were also signifi- binding in the vicinity of loop anchor regions in differen-
cantly more instances of multiple VDR peaks intersecting tial CTCF-looping. However, VDR binding may occur at
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with differential loop regions, compared to shuffled loop other regulatory genes that work downstream of VDR to
regions (Figure 2D). Of the 9,763 VDR peaks used in the regulate gene expression (100). To gauge whether differen-
analysis, 4,386 (44.9%) lay within upregulated differential tial CTCF loops can also be a consequence of association of
CTCF loops and 2,020 (20.7%) within downregulated loops the previously identified 47 downstream transcription fac-
(Figure 2E). tors (100), we used VDR and ETS1 ChIP-seq and FAIRE-
The locus of the vitamin D target gene CYP24A1 il- seq data (73) in combination with transcription factor bind-
lustrates a differential loop, containing a DEG and sev- ing prediction (90).
eral strong VDR binding sites. CYP24A1 is located closely More than half (55.3%) of the differential CTCF
(∼25 kb distance) to one of the loop anchors (Figure 2F). loops had a VDR ChIP-seq peak or a predicted
In summary, VDR binding sites are preferentially located VDR/VDR:RXRA binding motif in a FAIRE-seq
in CTCF loops that are modulated by 1,25(OH)2 D3 treat- peak within 25 kb of at least one of their loop anchors.
ment. This enrichment was even stronger when considering only
upregulated loops (59.0%) (Figure 4A). VDR was shown
to bind at 48.3% (1,658) of all 3,433 differential loops.
VDR-dependent gene expression is linked to dynamic CTCF-
At anchors of an additional 240 differential loops VDR
loops
binding was only predicted by motif occurrence, while
To elucidate whether the observed VDR-dependent at the remaining 1,534 loops there was no obvious VDR
changes in CTCF-mediated loops correlate with changes binding event at their anchor regions. VDR-downstream
in gene expression, we incorporated previously published transcription factors, encoded by primary vitamin D
RNA-seq data (73,99). The number of DEGs observed target genes, were predicted to bind at 76.7% (1,176)
per kb of loop was significantly (P < 0.05, Fisher’s exact of these (100). Indeed, of all differential CTCF loops,
test for shuffled, proportion exceeded for stable) greater only 10.5% (359) did not have a VDR peak, predicted
in differential loops (0.0024) when compared to stable VDR/VDR:RXRA, or downstream VDR target tran-
(0.0018) and shuffled (0.0013) loop sets (Figure 3A). The scription factor motif at their loop anchor regions (Figure
majority of the 3,372 differentially expressed genes after 4B). Among the downstream transcription factors with
1,25(OH)2 D3 stimulation (2,272, 67.4%) resided inside the highest enrichment in predicted affinity at differential
differential CTCF loops (Figure 3B). Significantly more loop anchors were E2F transcription factor 4 (E2F4) and
differential CTCF loops contained multiple DEG loci zinc finger and BTB domain containing 7A (ZBTB7A)
when compared to shuffled loops (Figure 3C). Along the (Figure 4C). As expected, predicted CTCF binding cor-
same lines, the co-occurrence of DEGs and VDR peaks relation with loop frequency change was highest, while
was enriched in differential CTCF loops (Figure 3D). VDR:RXR ranked seventh in the list of transcription
DEGs and VDR loci co-occurred in 73.3% and 58.8% of factors. Some downstream transcription factors tended to
upregulated and downregulated differential CTCF loops, appear less in differential loop anchors (Supplementary
respectively (Figure 3E). Interestingly, predicted primary Figure S7). Using previously published ChIP-seq data for
VDR target genes (100) were enriched inside differential the transcription factor ETS1 (100), we could validate the
CTCF loops. Of DEGs residing within differential CTCF predicted enrichment of ETS1 at anchors of differential
loops, 64.5% were predicted primary VDR targets. This loops (Figure 4D).
was significantly different (P < 4.915e−15, Fisher’s exact The possible scenarios of 1,25(OH)2 D3 -modulated
test) compared to only 50.4% direct VDR targets of the CTCF interactions that can be deduced from our analyses
DEGs located outside of differential loop regions (Figure include VDR enhancer-promoter interactions. VDR may
3F). associate to one of the CTCF loop anchors, while the TSS
Similar to VDR binding sites, DEGs were located close to of its target gene is located at the other loop anchor (Figure
differential loop anchors, indicating that the interaction of 4E). In contrast, VDR may also interact with the TSS of
VDR at CTCF-loop anchors is important for the regulation the primary DEG independently from CTCF, while the
of associated target genes (Figure 3G). frequency of the CTCF-formed insulated neighborhood or
A differential loop containing eight vitamin D target TAD is influenced by the colocation of VDR (Figure 4F)
genes and several VDR-bound sites is an example for the or a downstream transcription factor (Figure 4G) to one of3754 Nucleic Acids Research, 2022, Vol. 50, No. 7
A B Non-differential C D E
Downregulated 2000 ***
Proportion of differential loops
Shuffled Differential 0.08
Proportion of loops
*** Upregulated ***
0.003 1.00
with VDR & DEG
K-S test: p < 3.2e-13 None
DEGs per kb of loop
Differential loops
*** 1500 0.06 VDR
0.75
0.002 60%
0.04 DEG
1000
% of all DEGs
0.50
VDR
40% 0.02
0.001 & DEG
500 0.25
0.00
20%
led le f
0.000
uff ab Dif 0.00
0 Sh St Down Up
led ble ff
uff Sta Di 0% 0 2 4 6 8 10 Loop type Loop frequency change
Sh No Yes DEGs in differential loop
Loop type DEG in differential loop
chr 5: 139,400 kb 139,500 kb 139,600 kb 139,700 kb 139,800 kb 139,900 kb 140,000 kb
H
EtOH
F Secondary
G VDR
ChIP-seq 1,25D
Shuffled Differential Stable NRG2 MALINC1 PURA CYSTM1 PFDN1 HBEGF SLC4A9 ANKHD1 EIF4EBP3 SRA1 CD14 NDUFA2
Primary
LOC101929719 ANKHD1-EIF4EBP3 APBB3 TMCO6 DND1
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1.5e-05
SLC35A4 WDR55
1.00
Proportion of DEGs
RNA-seq FC 3.97x 1.53x 6.37x 1.25x 2.51x 83.86x 1.86x
2.60x 2.10x 1.96x
0.75 1.0e-05 1.78x 1.44x
Density
CTCF EtOH
0.50 ChIP-seq 1,25D
5.0e-06
EtOH
0.25 1,25D
CTCF
0.00 0.0e+00
HiChIP
Yes No 0 100 200 300 400 500+ Diff.
DEG in diff loop Closest DEG to loop anchor [kb] loops
log2 FC = -2.45
Figure 3. 1,25(OH)2 D3 -responsive genes were enriched inside differential CTCF loops and close to differential CTCF loop anchor regions. (A) Occurrence
rate of 1,25(OH)2 D3 -responsive gene loci in differential, stable, and shuffled differential loops. The difference between the loop types was computed using
a Fisher’s exact test for shuffled loops, and the proportion of stable loop sets which exceeded the differential value for stable loops. (B) Percentages of genes
previously identified as being differentially expressed after 24 h 1,25(OH)2 D3 treatment in THP-1 cells where the gene loci intersected with upregulated
differential CTCF HiChIP loops, downregulated differential CTCF HiChIP loops, or which did not intersect with any differential loop. (C) Numbers of
differential CTCF HiChIP loops and shuffled differential loops intersecting with different numbers of 1,25(OH)2 D3 -responsive genes (73,99). Difference
between the distributions was evaluated using a two-sided Kolmogorov–Smirnov (K–S) test. Overall, 1,605 differential CTCF loops contained more than
one DEG, in contrast to 741 shuffled loops. (D) Proportions of differential, stable, and shuffled loops, which intersected with both a 1,25(OH)2 D3 -responsive
gene (73,99) and a VDR ChIP-seq peak (99) present in THP-1 cells after 1,25(OH)2 D3 treatment. The difference between the loop sets was computed
using Fisher’s exact test for shuffled loops, and the proportion of stable loop sets, which exceeded the differential value for stable loops. (E) Proportions
of upregulated and downregulated differential CTCF HiChIP loops which intersected with either 1,25(OH)2 D3 -responsive genes (73,99), VDR ChIP-seq
peaks (99), both features, or neither feature. (F) Relative proportions of 1,25(OH)2 D3 -responsive genes (73,99) that intersected with differential CTCF
HiChIP loop regions, which were previously classified as either being directly regulated by VDR (primary vitamin D target gene) or as being regulated by
transcription factors downstream of VDR (secondary target) (100). The enrichment of primary vitamin D target genes in differential loops was significant
(P < 4.915e−15, Fisher’s exact test). (G) Densities of minimum linear distances between differential, stable, and shuffled CTCF HiChIP loop anchors and
1,25(OH)2 D3 -responsive gene loci (73,99): median distances were 150 kb (upregulated loops), 211 kb (downregulated loops) and 524 kb (shuffled loops).
(H) Representative example of a 1,25(OH)2 D3 -sensitive CTCF loop (violet arc) containing eight vitamin D target genes, among them CD14 and HBEGF,
together with several VDR ChIP-seq peaks (99). Four DEGs and four VDR sites (red peak tracks) are in proximity (within 25 kb) of either loop anchor.
Gene structures are shown in blue. The violet peak track displays CTCF ChIP-seq data (73,79) and CTCF HiChIP peaks (lighter violet). The expression
change (FC, linear fold change) 24 h after stimulation with 1,25(OH)2 D3 as determined by RNA-seq (73,99) is given for significant DEGs. The scale for
the VDR peak tracks was chosen to enable visualization of weaker peaks, thus the summit of strong peaks is cut.
the CTCF loop anchors. Correspondingly, the binding of rectly VDR-regulated transcription factors in the respective
a VDR-regulated transcription factor at one CTCF loop loops.
anchor may facilitate its contact with the TSS of its target
gene located at the other CTCF loop anchor (Figure 4H).
VDR promoter-enhancer loops are facilitated by CTCF-
A downstream transcription factor residing in a differential
CTCF interactions
CTCF loop with VDR at its anchors may contact the TSS
of a secondary vitamin D target gene independently from In order to determine functional consequences of
CTCF (Figure 4I). Finally, VDR may also be present in the VDR/CTCF promoter-enhancer loops, we performed
loop without directly regulating the secondary vitamin D CRISPRi experiments in THP-1 cells. The locus of the
target gene (Figure 4J). Differential CTCF HiChIP loops vitamin D target gene jagged canonical Notch ligand 1
were assigned to one or more of the aforementioned (JAG1) contains three CTCF loops which were not called
categories, with the majority falling under putative VDR differential, but were consistently present only after the
transcriptional neighborhoods bounded by CTCF with 1,25(OH)2 D3 treatment (Figure 5A). One of them rep-
or without VDR or a downstream transcription factor resented a putative VDR enhancer-promoter interaction
binding at one of the loop anchors (Figures 4E–J and as VDR co-located to one loop anchor while the JAG1
Supplementary Table S5). TSS was located at the other anchor. Inside the loop
In summary, the majority of all differential CTCF there were some additional VDR sites, one of them being
loops can be linked to the presence of VDR or di- much stronger than the one at the loop anchor. Thus, thisNucleic Acids Research, 2022, Vol. 50, No. 7 3755
1,176
A Downregulated Upregulated B 1200 1,067
Differential loops
loops loops
1600 900
Differential loops
1200 B 600
359 327
300 235 219
800
29 21
400 0
Secondary TF motif (2789)
VDR ChIP-seq (1658)
0 VDR motif (596)
Yes No Yes No
Unknown (359)
VDR (peak or motif) at loop anchor
TF binding at loop anchor
C D
% change in diff. loop anchors
Loops with ETS1 at anchor
with motif (diff. vs. shuffled)
1500 ***
**
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10 1000
5 500
0
0
d
le
iff
fle
ab
D
IR L
E2 7A
VD 4
E 2C
EN R
A B
N PA
TB RA
EL PB
K A
M G
SI ES
uf
C F4
TB 13
N 5
ZN 15
B R
EL 41
A 6
E 4
TB 1
JD T6
M 2
IR 4
N P2
A R2
C F4
F
K 2
XR S 1
C ITF
IP
ES F1
LF
St
F
C VD
TF BP
PP CL
K
ZB LK
FI
EOR1I
X
R
FE
R O TS
TC
LF
X
P
LX
F
EB
Sh
R
R
::
E
M
Loop type
Transcription factor
E F G R
R
VD
RX
R
R
VD
R
RX
R
VD
R
RX TF
VDR
R
VD
RX
CTCF
enhancer- C
CTCF
CT
CTCF
CTCF
promoter CTCF
CTCF
interaction TSS (prim. DEG)
TSS (prim. DEG)
TSS (prim. DEG)
(95) (467) (1,911)
H I J
R
RX
R
R
TF
VD
VD
downstream TF TF
RX
R TF
enhancer- CTCF CT
CTCF CTCF
promoter CTCF CTCF CTCF
interaction TSS (sec. DEG) TSS (sec. DEG)
TSS (sec. DEG)
(124) (827)
(12)
1,25D-liganded
RXR
VDR
differential CTCF CTCF VDR RXR
loop
potential CTCF- TF
1,25D-regulated
independent interaction transcription factor
Figure 4. Most differential CTCF HiChIP loop anchor regions intersected with either VDR binding sites, or binding motifs of transcription factors
downstream of VDR. (A) The number of the upregulated (in total 2,480) and downregulated (in total 980) differential CTCF HiChIP loops where either a
VDR ChIP-seq peak (99) or a VDR/VDR:RXRA binding motif was or was not identified in at least one loop anchor region. (B) An upset plot characterizes
the sets of differential CTCF HiChIP loops where a VDR ChIP-seq peak (1,658), VDR/VDR:RXRA binding motif (596), or downstream transcription
factor binding motif (2,789) was present in either of the loop anchor regions. In total, VDR peaks or its binding motif were identified at an anchor of
1,898 differential loops. At 1,534 1,25(OH)2 D3 -sensitive loops no VDR binding was shown (ChIP-seq (99)) or predicted (motif), but at 1,176 of these
association of a 1,25(OH)2 D3 -regulated transcription factor was predicted. Loops where none of the features were present were designated as unknown
(359). (C) Percentage change in the numbers of differential CTCF loop anchors predicted to be bound by CTCF, VDR, VDR:RXRA or transcription
factors previously identified to be primary targets of VDR, relative to shuffled loops. (D) The number of differential, stable, and random (shuffled) CTCF
HiChIP loops where an ETS1 ChIP-seq peak (100) was detected in at least one loop anchor region. Difference was evaluated using a Fisher’s exact test. (E–
J) Models depicting different potential scenarios involving VDR/RXR or downstream transcription factors bound at differential loops in the regulation of
primary and secondary vitamin D target genes. The frequency of each model among the differential CTCF loops is indicated below the respective model.
locus represents a more complex variation of the model in the strong VDR enhancer significantly reduced mainly the
Figure 4E (Supplementary Figure S8A). It was primarily 1,25(OH)2 D3 -stimulated JAG1 expression by 28.9% (L98)
chosen to validate our CRISPRi system by targeting both up to 63.3% (L99) (Figure 5F, Supplementary Figure S8B).
the TSS and a strong enhancer of a VDR target gene. The induction of JAG1 mRNA expression by 1,25(OH)2 D3
Thus, we first designed gRNAs targeting either the TSS was also decreased when targeting the strong VDR site
of the JAG1 gene or the strong VDR site in ≈ 170 kb (Supplementary Figure S8C). The distal loop anchor region
distance of the TSS (Figure 5C and D). Targeting the contained both a VDR and a CTCF site (Figure 5B). These
dCas9-KRAB fusion protein to the JAG1 TSS significantly results confirmed that our CRISPRi system works fine. In
reduced the basal and stimulated JAG1 mRNA levels by contrast to the inhibitions observed at the JAG1 TSS and
39.8% and 44.7%, respectively (Figure 5F). CRISPRi for strong VDR enhancer, CRISPRi targeting both the VDR3756 Nucleic Acids Research, 2022, Vol. 50, No. 7
chr 20: 10,000 kb 10,100 kb 10,200 kb 10,300 kb 10,400 kb 10,500 kb 10,600 kb
A
[0-2.00]
VDR EtOH
[0-2.00]
ChIP-seq 1,25D
PARAL1
ARA SNAP25-AS1 SNAP25 MKKS SLX4IP JAG1
G1
RNA-seq FC 1.65x 2.84x
.84x
[0-0.81]
CTCF EtOH
[0-0.81]
ChIP-seq 1,25D
EtOH
CTCF
HiChIP
Downloaded from https://academic.oup.com/nar/article/50/7/3745/6553119 by guest on 10 June 2022
1,25D
B C D
chr 20: 9,965 kb 9,969 kb 9,973 kb 9,977 kb 10,480 kb 10,484 kb 10,488 kb 10,492 kb 10,648 kb 10,652 kb 10,656 kb 10,660 kb
[0-1.50] [0-16] [0-1.50]
VDR EtOH
[0-1.50] [0-16] [0-1.50]
ChIP-seq 1,25D
PARAL1 SLX4IP JAG1
RNA-seq FC 2.84x
[0-0.50] [0-1.20] [0-1.20]
CTCF EtOH
[0-0.50] [0-1.20] [0-1.20]
ChIP-seq 1,25D
[0-240] [0-240] [0-240]
EtOH
[0-240] [0-240] [0-240]
CTCF 1,25D
HiChIP
1,25D
L96-99
L81
L86
L89
E F *
** *
Relative expression
0.01 * *
***
Relative expression
**
0.0075 ** 24h EtOH 0.006
0.005 24h 1,25(OH)2D3 0.004
0.0025
0.002
0
NTC L86 L89 0
NTC L81 L99
CTCF/VDR TSS VDR
anchor enhancer
Figure 5. CRISPRi identified a strong VDR enhancer in the JAG1 locus regulating JAG1, while the functionality of a distal VDR/CTCF site that interacted
with the CTCF anchor at the JAG1 TSS remained uncertain. (A) A CTCF HiChIP loop (violet arcs) in the JAG1 locus, which was present consistently
only after stimulation with 1,25(OH)2 D3 , represents a potential VDR enhancer-promoter interaction as VDR co-located to one of the loop anchors, while
the TSS of the vitamin D target JAG1 was located at the other anchor. Both loop anchors as well as a strong VDR ChIP-seq peak (red peak tracks) (99)
inside of the loop are marked by a grey shade. Gene structures are shown in blue. The violet peak tracks display CTCF ChIP-seq data (73,79), as well as the
CTCF HiChIP peaks (lighter violet). The expression change (FC, linear fold change) 24 h after stimulation with 1,25(OH)2 D3 as determined by RNA-seq
(73,99) is given for significant DEGs. The scale for the VDR peak tracks has been chosen such as so allow weaker peaks to be visible, which is why the
summit of strong peaks is cut. (B-D) Zoomed-in representations of the regions to which the CRISPRi experiments were targeted. (E) Lentiviral CRISPRi
using two different gRNAs targeting the distal CTCF/VDR site (L86 and L89) or a non-targeting control (NTC) followed by stimulation was performed
with THP-1 cells. After the transduction, the cells were treated for 24 h with 1,25(OH)2 D3 or EtOH, followed by RT-qPCR experiments for JAG1. (F)
Lentiviral CRISPRi using one gRNA targeting the JAG1 TSS (L81), a gRNA targeting the strong VDR site (L99), or NTC. Transduction, stimulation
and RT-qPCR for JAG1 were performed as described for (E). Columns in (E) and (F) represent the means of three independent experiments and the
bars indicate standard deviations. Two-tailed Student’s t-tests were performed to determine the significance of the mRNA induction by 1,25(OH)2 D3 in
reference to solvent-treated cells and of expression changes compared to the NTC control cells (* P < 0.05; ** P < 0.01; *** P < 0.001).You can also read
