RFX5调控原钙粘蛋白α基因簇的表达

doi:10.16288/j.yczz.20-184

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Hereditas(Beijing) ›› 2020, Vol. 42 ›› Issue (8): 760-774.doi: 10.16288/j.yczz.20-184

• Research Article • Previous Articles Next Articles

RFX5 regulates gene expression of the Pcdhα cluster

Na Wang, Zhilian Jia, 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:2020-06-19 Revised:2020-07-08 Online:2020-08-20 Published:2020-07-29
Contact: Wu Qiang E-mail:qwu123@gmail.com
Supported by:
Supported by the National Natural Science Foundation of China Nos(91640118);Supported by the National Natural Science Foundation of China Nos(31630039)

Abstract

Abstract:

Gene expression and three-dimensional (3D) genome organization are thought to be closely related. The protocadherin (Pcdh) clusters are central for neuronal self-avoidance in brain development and have been used as model genes to explore the role of 3D genome structures in gene regulation. Transcription factor RFX5 (regulatory factor x 5) is a member of the winged-helix subfamily of the helix-turn-helix superfamily proteins. The RFX5 protein contains four domains: oligomerization, DNA-binding, helical, and transactivation domains. RFX5 plays an important role in regulating expression of the major histocompatibility complex class II (MHC II) gene complex. Here we report a role of RFX5 in the regulation of clustered Pcdh genes. We first knocked out the RFX5 gene by using CRISPR/Cas9 DNA-fragment editing in HEC-1-B cell line. By RNA-seq experiments, we found that RFX5 deletion results in a significant increase of expression levels of the Pcdhα6, Pcdhα12 and Pcdhαc2 genes. By ChIP-nexus experiments, we found that RFX5 deletion leads to the increased binding of CTCF and RAD21 in the Pcdhα cluster. Finally, through quantitative high-resolution chromosome conformation capture copy (QHR-4C) experiments, we found that RFX5 deletion results in a significant increase of long-distance chromatin interactions between the HS5-1 enhancer and its target promoters in the Pcdhα cluster. In conclusion, RFX5 regulates gene expression of the Pcdhα cluster through higher-order chromatin structure.

Key words: RFX5, Pcdh, CTCF, cohesin, CRISPR/Cas9

Na Wang, Zhilian Jia, Qiang Wu. RFX5 regulates gene expression of the Pcdhα cluster[J]. Hereditas(Beijing), 2020, 42(8): 760-774.

Figures/Tables 6

Table 1

Primer sequences used in this study"

类型	引物名称	序列(5′→3′)
PCR	F1	GGGAAGTCGTGGCGAGATTA
	F2	GTGCCCTGAAAGTGGCTACA
	R1	CTGGGTGACTCAGCTGTCTG
	R2	GCTGGAGGTCACACACAAGA
	R3	AGAGTAGCCTGGTGTTAGCG
sgRNA	sgRNA1F	ACCGACCCTTCTTCAGAGGCTCCG
	sgRNA1R	AAACCGGAGCCTCTGAAGAAGGGT
	sgRNA2F	ACCGAAGCAACACCCCCATGATAC
	sgRNA2R	AAACGTATCATGGGGGTGTTGCTT
Index	P5-HS5-1-F2	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGTTTTGGCGGCGACAAATTCG
	P5-α12-F3	AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGTCCAATCATTCACGGAATAGGATC
	P7-index-1	CAAGCAGAAGACGGCATACGAGATCGAGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-2	CAAGCAGAAGACGGCATACGAGATTCTCCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-3	CAAGCAGAAGACGGCATACGAGATAATGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-4	CAAGCAGAAGACGGCATACGAGATGGAATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-5	CAAGCAGAAGACGGCATACGAGATAGCTTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-6	CAAGCAGAAGACGGCATACGAGATGCGCATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-7	CAAGCAGAAGACGGCATACGAGATGCGCGAGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-8	CAAGCAGAAGACGGCATACGAGATAGAGTACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-9	CAAGCAGAAGACGGCATACGAGATGCTCCGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-10	CAAGCAGAAGACGGCATACGAGATCATGAGAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-11	CAAGCAGAAGACGGCATACGAGATTGAATCGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-12	CAAGCAGAAGACGGCATACGAGATGTCGCGTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-13	CAAGCAGAAGACGGCATACGAGATCCGCATGAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-14	CAAGCAGAAGACGGCATACGAGATCCACAATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-15	CAAGCAGAAGACGGCATACGAGATTTCATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-16	CAAGCAGAAGACGGCATACGAGATTAGAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-17	CAAGCAGAAGACGGCATACGAGATTAACTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-18	CAAGCAGAAGACGGCATACGAGATTATACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-19	CAAGCAGAAGACGGCATACGAGATGAGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-20	CAAGCAGAAGACGGCATACGAGATGACATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-21	CAAGCAGAAGACGGCATACGAGATGTATTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-22	CAAGCAGAAGACGGCATACGAGATGACACAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-23	CAAGCAGAAGACGGCATACGAGATGCGATAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-24	CAAGCAGAAGACGGCATACGAGATCTCTATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
	P7-index-25	CAAGCAGAAGACGGCATACGAGATTACGCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

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