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YY1-controlled regulatory connectivity and transcription are influenced by the cell cycle

Nature Genetics (2024)Cite this article

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Abstract

Few transcription factors have been examined for their direct roles in physically connecting enhancers and promoters. Here acute degradation of Yin Yang 1 (YY1) in erythroid cells revealed its requirement for the maintenance of numerous enhancer–promoter loops, but not compartments or domains. Despite its reported ability to interact with cohesin, the formation of YY1-dependent enhancer–promoter loops does not involve stalling of cohesin-mediated loop extrusion. Integrating mitosis-to-G1-phase dynamics, we observed partial retention of YY1 on mitotic chromatin, predominantly at gene promoters, followed by rapid rebinding during mitotic exit, coinciding with enhancer–promoter loop establishment. YY1 degradation during the mitosis-to-G1-phase interval revealed a set of enhancer–promoter loops that require YY1 for establishment during G1-phase entry but not for maintenance in interphase, suggesting that cell cycle stage influences YY1’s architectural function. Thus, as revealed here for YY1, chromatin architectural functions of transcription factors can vary in their interplay with CTCF and cohesin as well as by cell cycle stage.

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Fig. 1: YY1 binding is necessary for the maintenance of CRE loops in asynchronous cells.
Fig. 2: YY1-anchored loops can form independently of CTCF and cohesin.
Fig. 3: Promoter-proximal YY1 binding drives YY1-dependent transcription in asynchronous cells.
Fig. 4: YY1 is partially retained on mitotic chromatin and rapidly recruited to CRE loop anchors during G1 entry.
Fig. 5: YY1 recruitment dynamics and requirements for CRE loop establishment during the mitosis-to-G1 transition.
Fig. 6: Transcriptional kinetics in G1 entry after depletion of YY1.
Fig. 7: Cell-cycle-dependent YY1 requirements for CRE loop formation.
Fig. 8: Model for CRE loop establishment and maintenance following mitosis.

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Data availability

The raw and processed Micro-C, ChIP–seq, RNA-seq and TT-seq data generated from this study are deposited in the Gene Expression Omnibus (GEO) database under accession GSE247254. External CTCF–AID Hi-C data from a previous study24 are available at GSE168251. External SMC3–AID Hi-C data from a previous study84 are available at GSE228402. External mitosis-to-G1 Hi-C and ChIP–seq data from a previous study67 are available at GSE129997. Source data are provided with this paper.

Code availability

The code used in this study is available at https://github.com/jclqrs/Lam_2024_Code and https://zenodo.org/doi/10.5281/zenodo.11992254.

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Acknowledgements

The authors would like to thank E. Nora, E. Joyce and members of the Blobel Laboratory for helpful discussions. The authors would also like to thank the Children’s Hospital of Philadelphia Flow Cytometry Core for technical assistance with cell sorting. This work was supported by grants T32GM007170, T32HG000046 and F30DK132824 (to J.C.L.), T32GM008216 and the Blavatnik Family Fellowship Award (to N.G.A.), R24DK106766 (to R.C.H. and G.A.B.), National Science Foundation of China (grant 32100422 to H.Z.) and R01DK054937, R01DK058044 and U01DK127405 (to G.A.B.).

Author information

Authors and Affiliations

  1. Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Jessica C. Lam, Nicholas G. Aboreden, Susannah C. Midla, Siqing Wang, Anran Huang, Kate A. Henderson & Gerd A. Blobel

  2. Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Jessica C. Lam, Nicholas G. Aboreden & Gerd A. Blobel

  3. Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA, USA

    Cheryl A. Keller, Belinda Giardine & Ross C. Hardison

  4. Genomics Research Incubator, Pennsylvania State University, University Park, PA, USA

    Cheryl A. Keller

  5. Institute of Molecular Physiology, Shenzhen Bay Laboratory, Shenzhen, China

    Haoyue Zhang

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  1. Jessica C. Lam
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Contributions

J.C.L. and G.A.B. conceived the study. J.C.L., N.G.A. and S.C.M. performed the Micro-C experiments. J.C.L. and S.C.M. performed cell synchronization and ChIP experiments. S.W. performed the TT-seq experiments and sequencing alignment. J.C.L. and A.H. generated the YY1–AID cell line. K.A.H. performed RT–qPCR validation. C.A.K., B.G. and R.C.H. prepared ChIP–seq and RNA-seq libraries and performed sequencing and preprocessed sequencing data. J.C.L. analyzed all datasets and interpreted the results. N.G.A. and H.Z. helped with the interpretation of results. J.C.L. and G.A.B. wrote the manuscript with input from all authors.

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Correspondence to Gerd A. Blobel.

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The authors declare no competing interests.

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Nature Genetics thanks Guillaume Andrey, Bas van Steensel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Compartments and domains are maintained in the absence of YY1.

a, YY1-AID cell counts for −auxin and +auxin conditions. Results are shown as mean ± s.d. (n = 3 biological replicates, two-sided independent t-test). b, Western blot for YY1 in parental cells and YY1-AID cells after an auxin time-course. c, Histogram of YY1 ChIP–seq signal at YY1 peaks for −auxin and +auxin conditions. d, ChIP–seq tracks for YY1 (Active Motif antibody), YY1 (Bethyl antibody) and H3K27ac from −auxin and +auxin conditions. e, Heatmaps of YY1 ChIP–seq signal (Bethyl antibody) and mean log2-fold change (+auxin/−auxin) centered on all YY1 peaks (n = 2 biological replicates). f, Micro-C contact versus distance curves for each biological replicate. g, Heatmap of Pearson correlations of compartment eigenvector 1 values (EV1) between biological replicates. h, Micro-C contact maps from −auxin (top) and +auxin (bottom) conditions along with tracks of compartment EV1, with positive values corresponding to A compartment and negative values corresponding to B compartment. i, Histogram of EV1 values from −auxin and +auxin contact maps (n = 25,257 bins). j, Aggregate domain plot for all domains called in the −auxin contact map, centered on upstream and downstream boundaries. k, Histogram of log2 insulation score values from −auxin and +auxin contact maps for all boundaries called across both conditions. l, Histogram of log2 insulation score values from −auxin and +auxin contact maps for the subset of boundaries that have YY1 binding within ±50 kb. m, Micro-C contact maps from −auxin (top) and +auxin (bottom) conditions, annotated with example TAD calls. Corresponding tracks show log2 insulation scores (IS).

Source data

Extended Data Fig. 2 Chromatin loop changes after YY1 depletion in asynchronous cells.

a, Venn diagram of loop calls from −auxin and +auxin contact maps. b, Bar plot of loop change counts, stratified by the condition in which the loop was called. c, Box plot of loop strengths in the −auxin contact map, stratified by loop change (two-sided Mann–Whitney U test). d, Box plot of loop strengths in the +auxin contact map, stratified by loop change (two-sided Mann–Whitney U test). e, Counts of uncategorized loops across categories of looping change and YY1 occupancy. f, Pileup plots of all H3K27ac-H3K27ac and YY1–YY1 loops detected in the −auxin condition, weakened YY1–YY1 loops and strengthened YY1–YY1 loops (1 kb resolution, ±30 kb window). g, Pileup plots of weakened YY1–YY1 loops for individual biological replicates. Loops are centered on YY1 ChIP–seq peaks (n = 555 loops, 1 kb resolution, ±30 kb window). h, Enrichment of different factor occupancy patterns in strengthened CRE loops (*padj < 0.05, two-sided Fisher’s exact test, Benjamini–Hochberg multiple testing correction).

Extended Data Fig. 3 CTCF, cohesin and LDB1 peaks remain stable upon YY1 depletion.

a, Heatmaps showing CTCF ChIP–seq signal at all CTCF peaks before and after YY1 depletion in asynchronous cells. b, Heatmaps showing RAD21 ChIP–seq signal at all RAD21 peaks before and after asynchronous YY1 depletion. c, Heatmaps showing LDB1 ChIP–seq signal at all LDB1 peaks before and after YY1 depletion in asynchronous cells. d, Heatmaps showing H3K27ac ChIP–seq signal at all H3K27ac peaks before and after YY1 depletion in asynchronous cells. e, Heatmaps showing CTCF, RAD21 and YY1 ChIP–seq signal at their respective peaks before and after CTCF depletion in asynchronous cells. f, Box plot of loop strength fold changes of YY1-independent structural loops after YY1, CTCF or SMC3 depletion. g, Box plot of loop strengths of YY1-independent CRE loops after YY1, CTCF or SMC3 degradation. h, Pileup plots of YY1-independent CRE loops, based on observed/expected signal from 10k resolution YY1-AID, CTCF-AID and SMC3-AID contact maps. i, Box plot of loop strength fold changes of YY1-independent CRE loops after YY1, CTCF or SMC3 depletion. j, Box plot of loop strength fold changes of YY1-dependent CRE loops after YY1, CTCF or SMC3 depletion.

Extended Data Fig. 4 Examples of YY1-dependent loops after cohesin depletion.

a, Contact maps showing an example of a YY1-dependent loop (blue arrows) that persists after SMC3 depletion. Upper heatmap shows interactions before/after YY1 depletion, and lower heatmap shows interactions before/after SMC3 depletion. Tracks show YY1, CTCF, RAD21 and H3K27ac ChIP–seq in untreated YY1-AID cells. b, A different example of a YY1-dependent loop that persists after SMC3 depletion. c, Histogram plot displaying CRE loop length versus the log2-fold change in loop strength after cohesin depletion in asynchronous cells. d, Box plot displaying CRE loop lengths, stratified for cohesin-dependence and YY1 dependence (two-sided Mann–Whitney U test).

Extended Data Fig. 5 Maintenance of transcription requires continuous presence of YY1.

a, Principal component analysis (PCA) of individual biological replicates of Pol II ChIP–seq. b, Representative tracks of RPM-normalized YY1 ChIP–seq from untreated YY1-AID cells and Pol II ChIP–seq before/after YY1 depletion from biological replicates of asynchronous cells. c, Histogram plot showing transcription change for genes versus YY1 peak strength at gene promoters (Spearman correlation coefficient = −0.41). d, Histogram of the multiplicity of active genes associated with CRE loops. e, Histogram of the multiplicity of CRE loops associated with active genes. f, Box plot showing transcription changes for different looping configurations at genes that do not have YY1 binding at the promoter (two-sided Mann–Whitney U test; from top to bottom: p = 1e−7, p = 1e−4, p = 0.28). g, Scatter plot showing loop strength change versus transcription change in asynchronous YY1-AID at loops that have YY1 at the distal enhancer but not at the promoter (Spearman correlation coefficient = 0.003). h, Box plot showing log2-fold change in traveling ratio for genes after YY1 depletion in asynchronous cells (two-sided Mann–Whitney U test).

Extended Data Fig. 6 YY1 chromatin binding dynamics during the mitosis-to-G1 transition.

a, Representative plots of the gating strategy for purification of mitosis-to-G1 populations. b, Heatmap showing the Pearson correlation in YY1 occupancy between biological replicates of YY1 ChIP–seq for mitosis-to-G1 stages. c, YY1 ChIP–qPCR in asynchronous (async) cells and synchronized/sorted prometaphase cells. Primers, labeled with the nearest or overlapping gene, include YY1 binding sites that display mitotic retention as well as sites that have no mitotic retention on ChIP–seq (n = 3 biological replicates). d, Prometaphase YY1 ChIP–seq track plotted alongside in silico generated tracks simulating prometaphase background and various levels of interphase contamination. e, Box plot showing YY1 binding at retained peaks genome-wide for different levels of simulated interphase contamination (two-sided Mann–Whitney U test).

Extended Data Fig. 7 Compartmentalization and TAD establishment following YY1 depletion.

a, YY1 ChIP–qPCR of synchronized, sorted prometaphase cells after mitosis-to-G1 depletion of YY1 (n = 3 biological replicates). b, Bar plot of the percent of cells in G1 after 2-hour release from nocodazole arrest after mitosis-to-G1 depletion of YY1, as assessed by DAPI signal from flow cytometry (two-sided Mann–Whitney U test). c, Micro-C contact probability curves for all biological replicates. d, Representative Micro-C contact maps with tracks of compartmentalization, with positive eigenvector 1 values (EV1) corresponding to A compartment and negative values corresponding to B compartment. e, Heatmap of Pearson correlations of subcompartment eigenvector 1 (EV1) values between biological replicates. f, Histogram plot of EV1 values after mitosis-to-G1 depletion. g, Histogram of log2 insulation score values for all boundaries detected in the −auxin_m contact map. h, Representative Micro-C contact map annotated with example TAD calls. Tracks show log2 insulation score values. i, Aggregate domain plot for all TADs called in control contact map, centered on upstream and downstream boundaries. j, Box plot showing loop strengths of all mid G1-detected YY1−YY1 loops weakened by mitosis-to-G1 depletion of YY1 (two-sided Mann–Whitney U test). k, Pileup plots corresponding to loops included in j.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4 and Supplementary Methods.

Reporting Summary

Supplementary Tables

Supplementary Table 1: Oligo sequences. Supplementary Table 2: TAD list for asynchronous cells. Supplementary Table 3: TAD list for mid G1 cells. Supplementary Table 4: Loop calls for asynchronous cells. Supplementary Table 5: Loop calls for mid G1 cells. Supplementary Table 6. Pol II ChIP-seq DESeq2 results for asynchronous cells. Supplementary Table 7: Pol II ChIP-seq DESeq2 results for G1 stages. Supplementary Table 8: RNA-seq DESeq2 results. Supplementary Table 9: TT-seq DESeq2 results. Supplementary Table 10: Micro-C sequencing statistics.

Source data

Source Data Extended Data Fig. 1

Unprocessed western blot for Extended Data Fig. 1b.

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Lam, J.C., Aboreden, N.G., Midla, S.C. et al. YY1-controlled regulatory connectivity and transcription are influenced by the cell cycle. Nat Genet (2024). https://doi.org/10.1038/s41588-024-01871-y

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