Three dimensional promoter interactions

during a circadian cycle

Ando-Kuri M1, Morf Jorg2, Wingett Steven2, Crains Jonathan2, Fraser Peter2, Furlan-Magaril Mayra1,2.
1 Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico.
Abstract 2 Nuclear Dynamics Programme, Babraham Institute, Cambridge, CB22 3AT UK

To have a better understanding of how chromosome

conformation influences transcriptional regulation it is useful Promoter interacting regions are enriched in Phase correlation between circadian promoter-
to analyze 3D chromatin organization in transcriptionally enhancer features including eRNAs eRNAs interacting pairs
dynamic situations in a postmitotic tissue such as a circadian
*** 22-24
cycle in mouse liver. We applied Capture-HiC (CHiC) to study R2=0.06165394
9-21
chromosome conformation between all gene promoters and Observed
their associated genomic elements providing a map of Expected 6-18
significant interactions at 4 time points during the course of a 3-15

Phases
circadian cycle. ***

Interactions
0-12
We found that genomic regions interacting with ***
promoters of circadian genes not only show enhancer-specific 7-9
eRNAs
histone modifications but often exhibit oscillatory enhancer *** 4-6 Circadian
RNA expression. Interestingly, the peak expression for most *** promoters
1-3
eRNAs is timed around dawn while gene expression of
contacted circadian genes show an even phase distribution Pairs

around the clock. Furthermore, in general we detect no Fig. 5. Phase distribution of promoter (purple) and eRNA (orange)
eRNAs H3K27ac H3K4me1 H3K4me3 CTCF
temporal variation in interaction frequency between circadian interacting pairs. Each column contains one pair.
Fig. 2. Enrichment of histone modifications, CTCF and eRNAs at
promoters and other genomic regions. This suggests a model promoter interacting regions.
in which topological scaffolds install regulatory contacts for
circadian gene expression but other factors than spatial
chromatin organization and eRNAs determine circadian Circadian enhancers Core clock gene Interaction profiles from circadian gene
phase of transcription. preferentially contact promoters form less promoters remain mostly stable over time
circadian gene interactions than non
Experimental Strategy: Promoter promoters core clock genes. Npas2
a b
Capture-HiC *** Observed
Expected
Non core-clock genes
Core clock genes
eRNAs
ZT0
Circadian promoters

CHiC is a novel method to enrich interactions of interest from

a HiC library,1,2 (Fig. 1). Promoter-CHiC was performed in Interactions
*
triplicate at ZT0, 6, 12 and 18 during a circadian cycle in
mouse adult liver. Circadian gene expression was confirmed ZT6
by RNAseq on the same samples (not shown).

Hi-C library
Oscillatory eRNAs CHiC interactions
Promoter B
Fig. 3. a) The expected number interactions was estimated by ZT12
Promoter generating a distribution for random oscillatory eRNAs b) Average
number of interactions from 25 core clock genes.

Sequence capture Streptavidin pulldown

hybridization
Circadian promoters interact with oscillatory ZT18
B
B

Promoter B enhancers with peak expression around dawn

B RNA probe

Promoter a
B
Associated circadian

PCR Fig. 6. The Npas2 gene promoter interaction landscape. Npas2 promoter
ZT0 contacts a circadian eRNA enhancer and the interaction is stable over
C-HiC Library
promoters

time.
Bait OtherEnd

ZT18 ZT6
Conclusions
ZT12 ● Enhancer features and eRNAs are enriched at the significant
Fig. 1. Capture-HiC protocol. RNA biotinylated baits are used to pull down interactions detected by promoter CHiC.
all ligation products containing a promoter on either side of the interaction. b eRNAs phases
● Core clock gene promoters make significantly less contacts
than other oscillatory genes which suggests their
Results transcriptional regulation might not require distal elements.
● Circadian gene promoters in all transcriptional phases
Significant interactions were detected using CHiCAGO preferentially interact with oscillating eRNAs expressed in
pipeline (Capture HiC Analysis of Genomic ZT18-ZT3.
Organization)3. CHiC retrieved ~150,000 interactions per ● Overall, the interaction profiles from circadian gene promoters
timepoint with ~10 interactions on average per gene are maintained over time providing a spacial framework that
allows efficient circadian gene expression control
promoter.
We found an enrichment of enhancer features over the
Diurnal promoters Nocturnal promoters
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Fig. 4. a) Number of circadian promoters interacting with oscillating high-resolution capture Hi-C. Nature genetics, 47(6), 598-606.

occupancy of circadian transcription factors (P < 0.001 t-test, enhancers b) Phase distribution of eRNAs elements interacting with
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