HOXD基因簇内一系列CTCF位点反转揭示绝缘子功能

doi:10.16288/j.yczz.21-131

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Hereditas(Beijing) ›› 2021, Vol. 43 ›› Issue (8): 758-774.doi: 10.16288/j.yczz.21-131

• Research Article • Previous Articles Next Articles

Combinatorial CRISPR inversions of CTCF sites in HOXD cluster reveal complex insulator function

Xianglong He(), Jinhuan Li, Qiang Wu

Center for Comparative Biomedicine, Key laboratory of Systems Biomedicine (Ministry of Education), Institute of Systems Biomedicine, Shanghai Jiao Tong University, Shanghai 200240, China

Received:2021-04-11 Revised:2021-05-12 Online:2021-08-20 Published:2021-06-30
Supported by:
Supported by the National Nature Science Foundation of China Nos(31800636);Supported by the National Nature Science Foundation of China Nos(31630039);Supported by the National Nature Science Foundation of China Nos(91940303);Science and Technology Commission of Shanghai Municipality program No(19JC1412500)

Abstract

Abstract:

CTCF (CCCTC-binding factor) is a zinc-finger protein which plays a vital role in the three-dimensional (3D) genome architecture. A pair of forward-reverse convergent CTCF binding sites (CBS elements) mediates long-distance DNA interactions to form chromatin loops with the assistance of the cohesin complex, while CBS elements at the chromatin domain boundaries show reverse-forward divergent patterns and function as insulators to discriminate against DNA interaction between chromatin domains. However, there are still many unresolved problems regarding CTCF-mediated insulation function. In order to study the connections between chromatin loops and the insulation function of CBS elements, we combinatorically inverted CBS elements at the HOXD locus by using CRISPR/Cas9 DNA-fragment editing methods in the HEK293T cell line and obtained five different kinds of single-cell CRISPR clones. By performing quantitative high-resolution chromosome conformation capture copy (QHR-4C) experiments, we found that boundary CBS inversions abolish original chromatin loops and establish new loops from the opposite direction, thus shifting the insulator boundary to the new divergent CTCF sites. Furthermore, tandem CBS elements block cohesin permeated from the opposite orientation to function as insulators. RNA-seq experiments showed that alterations of local three-dimensional genome architecture would further influence gene expression of the HOXD cluster. In conclusion, a pair of divergent CBS elements function as insulators by forming chromatin loops within chromatin domains to block cohesin sliding.

Key words: CTCF, chromatin loop, HOXD, insulator, CBS inversion

Xianglong He, Jinhuan Li, Qiang Wu. Combinatorial CRISPR inversions of CTCF sites in HOXD cluster reveal complex insulator function[J]. Hereditas(Beijing), 2021, 43(8): 758-774.

Figures/Tables 7

Table 1

Primers used in this study"

类型	引物名称	序列(5′→3′)
sgRNA	a-sgRNA1F	accgCGAAGAGTGCGGGAGAACGG
	a-sgRNA1R	aaacCCGTTCTCCCGCACTCTTCG
	b-sgRNA1F	accgGGCGCATCAGGAATGTAAG
	b-sgRNA1R	aaacCTTACATTCCTGATGCGCC
	b-sgRNA2F	accgTTCCAGAGATTATGAGCCAG
	b-sgRNA2R	aaacCTGGCTCATAATCTCTGGAA
	c-sgRNA2F	accgAGTAAAACTGGGTGTTGCC
	c-sgRNA2R	aaacGGCAACACCCAGTTTTACT
	e-sgRNA1F	accgAACTGTGCTCAAACGCTCTC
	e-sgRNA1R	aaacGAGAGCGTTTGAGCACAGTT
	e-sgRNA2F	accgGAGGCGCAAACAGCTGTTGT
	e-sgRNA2R	aaacACAACAGCTGTTTGCGCCTC
PCR	a-1F	TTCCAGCACCTCGGCTTTGTC
	a-1R	CCCACTTTCCACCTCTGTCCTG
	b-1F	GTCCGCCCGTGAGCTTCTGAA
	b-1R	GCGTTGGCAGGGCTGGACTC
	b-1F1	TTCCCTGACCCTCCAAGCAC
	b-1R1	CTCACAGCAGCCGAAACCG
	b-2F	GACACCTTGGTTCAGGCTCG
	b-2R	CAGAGGCTGCATCAAACCAC
	c-1F	TGATGCAGCCTCTGTGACCG
	c-1R	AGTTTTCCCGTGGCGTCTGA
	c-2F	GGACAACCCCTCACCTTGAA
	c-2R	TATGGTGCCCAAAGTCCCAC
	e-1F	TTCCCTGTCCCAGCTTGATTTC
	e-1R	TCAACAGTGAAGGGCGGTGC
	e-2F	CAAGCCACTCTCCCGCCACTA
	e-2R	TCGCTCTCGTCCTCTCTTGGG
bio-primer	upstream-a-bioprimer	5′biotin-GGACGAGGGACATAGAGAGTTC
	downstream-e-bioprimer	5′biotin- AGGAGCCCAGGCATAGAGAC
	CS38-bioprimer	5′biotin-CCGAATTAAATCCCCGTGAC
	Island5-bioprimer	5′biotin-AAACACAAATGCATCAACCTG
P5 primer	upstream-a-P5	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTGTCTAGAGATTCAAGTATTTCCCA
P5 primer	downstream-e-P5	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTACGCGATACTCAAGGGAATACAAGCC
	CS38-P5	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGGTTACAGTGTCATTTTCCCTG
	Island5-P5	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTATGCATCTCATGAAGCTGGCATCT
P7 index	P7-index-1	CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-2	CAAGCAGAAGACGGCATACGAGATTCTCCGGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-3	CAAGCAGAAGACGGCATACGAGATAATGAGCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-4	CAAGCAGAAGACGGCATACGAGATGGAATCTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-5	CAAGCAGAAGACGGCATACGAGATTTCTGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-6	CAAGCAGAAGACGGCATACGAGATACGAATTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-7	CAAGCAGAAGACGGCATACGAGATAGCTTCAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-8	CAAGCAGAAGACGGCATACGAGATGCGCATTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-9	CAAGCAGAAGACGGCATACGAGATCATAGCCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-10	CAAGCAGAAGACGGCATACGAGATTTCGCGGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-11	CAAGCAGAAGACGGCATACGAGATGCGCGAGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-12	CAAGCAGAAGACGGCATACGAGATCTATCGCTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-13	CAAGCAGAAGACGGCATACGAGATAGAGTACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-14	CAAGCAGAAGACGGCATACGAGATGCTCCGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-15	CAAGCAGAAGACGGCATACGAGATCATGAGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-16	CAAGCAGAAGACGGCATACGAGATTGAATCGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-17	CAAGCAGAAGACGGCATACGAGATGTCTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-18	CAAGCAGAAGACGGCATACGAGATCTGAATGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-19	CAAGCAGAAGACGGCATACGAGATCGCTTCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-20	CAAGCAGAAGACGGCATACGAGATTCGCATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-21	CAAGCAGAAGACGGCATACGAGATAATAGCAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-22	CAAGCAGAAGACGGCATACGAGATGTCGCGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-23	CAAGCAGAAGACGGCATACGAGATACGCGATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
P7 index	P7-index-24	CAAGCAGAAGACGGCATACGAGATTGATCGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-25	CAAGCAGAAGACGGCATACGAGATCCGCATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-26	CAAGCAGAAGACGGCATACGAGATCCACAATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-27	CAAGCAGAAGACGGCATACGAGATGATGTTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-28	CAAGCAGAAGACGGCATACGAGATGAATACGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-29	CAAGCAGAAGACGGCATACGAGATTAGATACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-30	CAAGCAGAAGACGGCATACGAGATTATGGTCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
adapter	adapter-upper	GACGTGTGCTCTTCCGATCTGNNNNNN-3`NH2 C6
adapter	adapter-lower	5′P-CAGATCGGAAGAGCACACGTC-3`NH2 C6

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 6

Fig. 7

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Combinatorial CRISPR inversions of CTCF sites in HOXD cluster reveal complex insulator function

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