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YY1 interacts with guanine quadruplexes to regulate DNA looping and gene expression

Abstract

The DNA guanine quadruplexes (G4) play important roles in multiple cellular processes, including DNA replication, transcription and maintenance of genome stability. Here, we showed that Yin and Yang 1 (YY1) can bind directly to G4 structures. ChIP–seq results revealed that YY1-binding sites overlap extensively with G4 structure loci in chromatin. We also observed that the dimerization of YY1 and its binding with G4 structures contribute to YY1-mediated long-range DNA looping. Displacement of YY1 from G4 structure sites disrupts substantially the YY1-mediated DNA looping. Moreover, treatment with G4-stabilizing ligands modulates the expression of not only those genes with G4 structures in their promoters, but also those associated with distal G4 structures that are brought to close proximity via YY1-mediated DNA looping. Together, we identified YY1 as a DNA G4-binding protein, and revealed that YY1-mediated long-range DNA looping requires its dimerization and occurs, in part, through its recognition of G4 structure.

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Fig. 1: Tag-free YY1 binds selectively with G-quadruplex DNA.
Fig. 2: YY1 interacts with G4 structure in cells.
Fig. 3: Disruption of the binding of YY1 with G4 structure attenuates YY1-mediated DNA looping.
Fig. 4: YY1–G4 interaction and dimerization of YY1 promote long-range DNA looping.
Fig. 5: Regulation of gene expression by YY1 promoter G4 interactions.
Fig. 6: YY1–G4 binding participates in transcription regulation of the TRMT12 gene through long-range DNA looping.

Data availability

The ChIP–seq, HiChIP–seq and RNA-seq data generated in this study have been deposited into the NCBI GEO database (for ChIP–seq and HiChIP–seq data: GEO accession number GSE128106; for RNA-seq: GEO accession number GSE142075). The ChIP–seq data for Ishikawa and SK-N-SH cells were obtained from NCBI GEO database with accession numbers of GSM1010753 and GSM1010897, respectively43. The two G4 ChIP–seq datasets were obtained from NCBI GEO database with accession numbers of GSE99205 and GSE107690 (refs. 3,7). The human hg19 reference genome was downloaded from https://hgdownload.soe.ucsc.edu/goldenPath/hg19/bigZips. Source data are provided with this paper.

Code availability

Custom codes used in this work are available from https://github.com/linliucr/UCR_code.

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Acknowledgements

This work was supported by the National Institutes of Health (R35 ES031707 to Y.W. and R35 GM119721 to J.S.). M.H. was supported in part by an NRSA T32 Institutional Research Training Grant (ES018827).

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Authors

Contributions

L.L. and Y.W. conceived the project. P.W., W.M. and M.H. performed the G-quadruplex pull-down experiments and analyzed the mass spectrometry data. L.L., M.Y.W., Z.G. and W.M. performed the plasmid construction and cell culture experiments. L.L. and Z.G. performed the in vitro binding assay. L.L. performed the ChIP–seq, HiChIP–seq, RNA-seq and relevant data analysis. W.R. and J.S. assisted with the protein expression and purification. L.L. and P.W. analyzed the data. L.L. and Y.W. wrote the manuscript, which was reviewed and commented by all co-authors.

Corresponding author

Correspondence to Yinsheng Wang.

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Extended data

Extended Data Fig. 1 Proteome-wide identification of G quadruplex-binding proteins.

Volcano plots showing the quantification results of G quadruplex-binding proteins identified from SILAC-based interaction screening. The −Log10(P value) was plotted against the log2(ratio G4/M4). YY1 is labeled in red.

Source data

Extended Data Fig. 2 Circular dichroism (CD) spectra of the three G4 sequences in the absence or presence of tag-free YY1 protein.

a-c, CD spectra of cMYC G4 (a), YY1 (b) and cMYC G4-YY1 complex (c). d-f, Comparison of CD spectra for G4 probes in the presence or absence of YY1 protein. The CD spectra for the G4 probes in the presence of YY1 protein were obtained by subtracting the CD spectrum of the YY1 protein from the composite spectra of the protein-G4 DNA complexes.

Source data

Extended Data Fig. 3 Zinc finger domain of YY1 is essential, but not sufficient for the specific binding toward G4 structure.

a, A schematic diagram depicting the domain structure of YY1 protein. b-e, Fluorescence anisotropy for monitoring the bindings of mutant (YY1C360S) and truncated (YY11-382, YY1231-414, and YY1293-414) YY1 proteins with hTEL G4 and the corresponding M4 DNA probes. The data represent the mean ± S.E.M. of results obtained from 3 independent experiments.

Source data

Extended Data Fig. 4 Statistical analysis of YY1 ChIP-Seq and BG4 ChIP-Seq data.

a, The ChIP-seq signal enrichment of YY1 ChIP-Seq and BG4 ChIP-Seq based on the overlapped peaks for the two datasets. The YY1 ChIP-Seq and BG4 ChIP-Seq average signal enrichments are plotted against the BG4 overlapped peaks. b, Analysis of peak width distribution for YY1 ChIP-seq and BG4 ChIP-seq data.

Source data

Extended Data Fig. 5 Unwinding of G4 with the overexpression of BLM helicase disrupts the YY1-mediated DNA looping.

a-b, Reads enrichment of two regions from BG4 ChIP-Seq and YY1 ChIP-Seq. The long-range DNA interactions monitored in HiChIP-PCR experiment are labeled with red arches, and the regions monitored in BG4 ChIP PCR experiments are indicated with blue triangles. c-d, BG4 ChIP and YY1 ChIP enrichments at the two sites were markedly diminished after ectopic overexpression of BLM helicase (BLM-O.E.). e, YY1-mediated DNA looping is disrupted by overexpression of BLM helicase. HiChIP-PCR quantification results of YY1-mediated DNA looping in HEK293T cells with and without the overexpression of BLM. Shown in (c-e) are mean ± S.E.M. of results obtained from 3 independent experiments. The p values were calculated by using two-tailed, unpaired Student’s t test: **, p < 0.01; ***, p < 0.001.

Source data

Extended Data Fig. 6 Analysis of YY1 dimerization and binding stoichiometry of the YY1-G4 DNA complex.

a-b, Gel filtration chromatography revealed the dimerization of YY1 and truncated YY1231-414, but not YY1293-414. c-d, The binding stoichiometry of YY1:G4 DNA was analyzed using EMSA. The quantification results showed the YY1-bound fraction of TAMRA-G4. The stoichiometry in binding of YY1 to G4 DNA matches with the theoretical curve in 1:1 binding stoichiometry. The data represent mean ± S.E.M. from 4 independent experiments.

Source data

Extended Data Fig. 7 YY1 promotes interactions between DNA elements containing its consensus sequence motifs, G4 DNA, or both.

a, A scheme depicting the in vitro proximity ligation assay for assessing the ability of YY1 to enhance DNA-DNA interactions involving G4 structures and/or YY1 consensus motifs. b, qPCR quantification results of the proximity ligation products formed between motifs, between G4 structures, and between motif and G4 structure in the presence or absence of YY1 protein. c, qPCR quantification results revealed the inability of YY1 in promoting the ligation between M4 (that is mutated sequence of G4 that can no longer fold into G4 structure) and G4, motif, or M4. d, qPCR quantification results of the proximity ligation products formed between G4 structures in the absence or presence of PDS or TMPyP4. The data represent mean ± S.E.M. of results from three independent experiments.

Source data

Extended Data Fig. 8 Dimerization of YY1 promotes long-range DNA looping.

a, SDS-PAGE for monitoring the purified recombinant truncated forms of YY1 that is incapable of dimerization, but able to discriminate G4 structure from single-stranded DNA. b, Gel filtration chromatography revealed that YY1∆231-290 (calculated monomer MW: 38.4 kDa) exists as a monomer, and GST-YY1∆231-290 is present as a dimer (calculated monomer MW: 66.3 kDa). c-d, Fluorescence anisotropy for monitoring the binding of YY1∆231-290 and GST-YY1∆231-290 protein with G4 structure and the corresponding mutated sequence (M4) derived from the MYC promoter. e, Proximity ligation assay showing that YY1 and GST-YY1∆231-290, but not YY1∆231-290, is capable of promoting ligation between G4 DNA sequences.

Source data

Extended Data Fig. 9 Regulation of gene expression by YY1-promoter G4 interactions.

a-c, Quantification of mRNA expression levels of MYC (a), SLC25A28 (b) and TMEM145 (c) genes after shRNA-mediated knockdown of YY1 and/or PDS/TMPyP4 treatment. Top of each panel shows read enrichments from BG4 ChIP-Seq and YY1 ChIP-Seq. The data represent mean ± S.E.M. of results from three independent experiments.

Source data

Extended Data Fig. 10 YY1-G4 binding participates in transcription regulation of EHD3 genes through long-range DNA looping.

a, Read enrichments obtained from BG4 ChIP-seq and YY1 ChIP-Seq experiments. b, Normalized interaction frequency between the two sites linked with a red arch in a in HEK293T cells with or without PDS/TMPyP4 treatment, as captured by YY1 HiChIP. c, Quantification results for the mRNA expression levels of EHD3 genes in HEK293T cells after knockdown of YY1 and/or with PDS/TMPyP4 treatment. The data represent mean ± S.E.M. of results from three independent experiments.

Source data

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2 and Figs. 1–13.

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Li, L., Williams, P., Ren, W. et al. YY1 interacts with guanine quadruplexes to regulate DNA looping and gene expression. Nat Chem Biol 17, 161–168 (2021). https://doi.org/10.1038/s41589-020-00695-1

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