Abstract
Biochemical crosstalk between two or more histone modifications is often observed in epigenetic enzyme regulation, but its functional significance in cells has been difficult to discern. Previous enzymatic studies revealed that Lys14 acetylation of histone H3 can inhibit Lys4 demethylation by lysine-specific demethylase 1 (LSD1). In the present study, we engineered a mutant form of LSD1, Y391K, which renders the nucleosome demethylase activity of LSD1 insensitive to Lys14 acetylation. K562 cells with the Y391K LSD1 CRISPR knockin show decreased expression of a set of genes associated with cellular adhesion and myeloid leukocyte activation. Chromatin profiling revealed that the cis-regulatory regions of these silenced genes display a higher level of H3 Lys14 acetylation, and edited K562 cells show diminished H3 mono-methyl Lys4 near these silenced genes, consistent with a role for enhanced LSD1 demethylase activity. These findings illuminate the functional consequences of disconnecting histone modification crosstalk for a key epigenetic enzyme.
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Data availability
Structure factors and atomic coordinates have been deposited in the Protein Data Bank with IDs 8Q1G, 8Q1H and 8Q1J. RNA-seq data of the parental and edited K562 cells have been deposited with Gene Expression Omnibus (GEO) accession code GSE243427. CUT&RUN data of the parental and edited K562 cells have been deposited with GEO accession code GSE243231. CUT&RUN data analyzed using deepTools are deposited at Harvard Dataverse (https://doi.org/10.7910/DVN/AUDINC). Source data are provided with this paper.
Code availability
The codes used for processing CUT&RUN data can be found in the Supplementary Note section of the Supplementary Information. Processing scripts for CUT&RUN analysis using deepTools are available at Harvard Dataverse (https://doi.org/10.7910/DVN/AUDINC).
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Acknowledgements
We acknowledge financial supprt from the National Institutes of Health (GM62437 and GM149229 to P.A.C., GM126944 to M.I.K., 1DP2GM137494 to B.B.L. and AG068179 to S.B.); the National Science Foundation (2127882 to P.A.C.), the Leukemia & Lymphoma Society (to P.A.C.); MUR (FISR2019_00374 MeDyCa to A.M.); the American Heart Association (Postdoctoral Fellowship Award 826614 to K.L.); the American Cancer Society (PF20-105-01-DMC to S.D.W.); and the Charles A. King Trust Postdoctoral Research Fellowship (to S.D.W.). We would like to thank the Cole laboratory members for helpful advice and stimulating discussions. We also thank MedGenome for RNA-seq experiments and analysis. We thank C. Bahl (AI Proteins, Inc.) and Meiler laboratory members (Vanderbilt University) for helpful advice in designing LSD1 mutants. We appreciate R. Chivukula for technical advice on CRISPR–Cas9 and Alani laboratory members (Boston University) and the Harvard Chan Bioinformatics Core for helpful advice on bioinformatics and cellular data interpretation.
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Contributions
All listed authors performed experiments and analyzed data. K.L., E.N., S.E.D-C., K.N. and B.I. performed enzymology. K.L., E.N., S.E.D.-C., S.D.W. and Z.A.W. prepared nucleosomes containing semi-synthetic histones. M.B., J.C. and A.M. conducted crystallographic analysis. M.B., J.C. and A.M. performed SNAIL peptide-binding measurements and analysis. K.L. and H.J. conducted fluorophore-labeled CoREST complex preparation and MST binding affinity measurements. K.L., A.L.W. and B.L. conducted CRISPR–Cas9 knockin experiments. K.L. and Z.D. conducted CUT&RUN profiling experiments. K.L., Z.D., M.I.K., S.B. and P.A.C. performed genomic analysis. The manuscript was prepared by K.L. and P.A.C., with input from all authors.
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Competing interests
P.A.C. is a co-founder of Acylin Therapeutics, which is involved in developing epigenetic agents, and has been a consultant for the pharmaceutical companies AbbVie and Constellation. He also is a co-inventor on US patent 11,565,994 B2 that concerns LSD1 and CoREST complex inhibitors. B.B.L. has received research funding from Eisai and AstraZeneca and is a shareholder and member of the scientific advisory board of Light Horse Therapeutics. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Measurement of Nucleosome Demethylase Activity via Western Blotting for LSD1-CoREST1 (LC) and LSD1-CoREST1-HDAC1 (LHC).
(a) Demethylase activity of LC on H3K4me2 nucleosomes. LC at a concentration of 180 nM was incubated with 100 nM of 185 bp H3K4me2 nucleosomes, and changes in H3K4me2 levels were tracked over a 60-minute time frame. (b) Evaluation of demethylase activity for LHC. LHC, present at a concentration of 365 nM, was incubated with 100 nM of 185 bp H3K4me2 nucleosomes, and changes in H3K4me2 levels were tracked over a 60-minute time frame. In both (a) and (b), the anti-H3K4me2 signal at each time point was normalized by anti-H3. Lanes containing only nucleosomes (designated by *) were excluded from the rate calculations. (c) and (d) illustrate the relative intensities obtained from (b) and (a), subjected to fitting into an exponential decay equation, featuring constraints of Y0 at 1 and plateau at 0. In (d), the H3K4me2 level appears to plateau after 30 min. (e) V/[E] (min−1) values from (c) and (d) were extrapolated (mean ± SEM).
Extended Data Fig. 2 Potential Disruption of LC Complex’s Nucleosome Binding by HDAC1 Interaction.
(a) Illustration of the nucleosome-bound, demethylase-active configuration of LC as observed in the crystal structure (PDB: 6VYP). The SANT2 domain of CoREST1 (light blue) interfaces with the globular regions of H3 (yellow), H4 (dark gray), and DNA (purple), facilitating engagement with the nucleosome. (b) AlphaFold2-generated model of LHC fitted into the EM density map (EMD-10629). HDAC1, bound to the ELM2-SANT domain of CoREST1, remains proximal to the tower domain of LSD1 and the SANT2 domain of CoREST1. This interaction can potentially hinder CoREST1-nucleosome interaction, thereby preventing LHC from adopting the demethylase-active conformation. (c) Despite comprised nucleosome-binding, the LSD1 of LHC remains catalytically active. LC (200 nM) or LHC (100 nM) have similar demethylase activities toward H3K4me2 peptides (aa 1-21; 150 μM), even when CoREST1 is N-terminally tagged with fluorescein (~70% active compared with their untagged counterparts). HRP-coupled peptide demethylase activity assay was employed for evaluation. A total of six data points from the duplicates of continuous assays were used to obtain the V/[E] (min−1) values, presented as mean ± SEM with error bars.
Extended Data Fig. 3 Analysis of H3K4me2 and H3K4me2K14ac Nucleosome Demethylase Activity for WT and Y391K LC.
(a) Y391K LC demethylase activity on H3K4me2 nucleosomes. Y391K LC at a concentration of 180 nM was subjected to a 60 minute incubation with 100 nM of 185 bp H3K4me2 nucleosomes, and changes in H3K4me2 levels were monitored. (b) Y391K LC demethylase activity targeting H3K4me2K14ac nucleosomes. Similar to (a), 180 nM Y391K LC was incubated with 100 nM of 185 bp H3K4me2K14ac nucleosomes, and changes in H3K4me2 levels were tracked over 60 minutes. (c) WT LC demethylase activity targeting H3K4me2K14ac nucleosomes. 180 nM WT LC was incubated with 100 nM of 185 bp H3K4me2K14ac nucleosomes, and changes in H3K4me2 levels were tracked over 60 minutes. Panels (d-f) illustrate the relative intensities obtained from (a-c), subjected to fitting into an exponential decay equation that includes constraints of Y0 at 1 and plateau at 0. In (f), the H3K4me2 level remains almost constant after T30min, possibly due to product inhibition. (g) Western blots with anti-H3K4me2 and anti-H3K14ac antibodies. These images display the signals obtained for H3K4me2 and H3K4me2K14ac nucleosomes used in the demethylase assays, respectively, in one replicate (n = 1). (h) Microscale thermophoresis was used to measure the binding affinities of N-terminally fluorescein-labeled WT (green) and Y391K LC (pink) to the H3K14ac nucleosome (n = 2). Both complexes tightly engage the nucleosome with comparable binding affinities. (i) V/[E] (min−1) values from (d-f) were extrapolated (mean ± SEM).
Extended Data Fig. 4 Analysis of H3K4me1 and H3K4me1K14ac Nucleosome Demethylase Activity for WT and Y391K LC.
(a) WT LC demethylase activity on H3K4me1 nucleosomes. WT LC at a concentration of 180 nM was subjected to a 60 minute incubation with 100 nM of 185 bp H3K4me1 nucleosomes, and changes in H3K4me1 levels were monitored. (b) WT LC demethylase activity targeting H3K4me1K14ac nucleosomes. Assays conditions were identical to (a). (c) Y391K LC demethylase activity on H3K4me1 nucleosomes. Assays conditions were identical to (a). (d) Y391K LC demethylase activity targeting H3K4me1K14ac nucleosomes. Assay conditions were identical to (a). Panels (e-h) illustrate the relative intensities obtained from (a-c), subjected to fitting into an exponential decay equation that includes constraints of Y0 at 1 and plateau at 0. In (e) and (f), the H3K4me1 level remains almost constant after T30min, possibly due to product inhibition. (i) Western blots with anti-H3K4me1 and anti-H3K14ac antibodies. These images display the signals obtained for H3K4me1 and H3K4me2K14ac nucleosomes used in the demethylase assays, respectively, in one replicate (n = 1). (j) Bar plot showing the demethylase activities of WT LC and Y391K LC towards H3K4me1 and H3K4me1/K14ac nucleosomes (Two-way ANOVA; mean ± SEM; n = 4; p values are indicated above each comparison group). (k) V/[E] (min−1) values from (e-h) were extrapolated (mean ± SEM).
Extended Data Fig. 5 Structural comparison of WT LC and Y391K LC in complex with H3K4M and H3K4M/K14ac peptides.
The active site of LC with the peptides is magnified (top), highlighting key residues and CoREST1′s conformation in proximity. (a) WT LC H3K4M vs. H3K4M/K14ac: In the H3K4M/K14ac structure, K9* of H3K4M/K14ac (brown) forms a compensatory salt bridge with E559LSD1 due to K14 acetylation. (b) WT LC H3K4M vs. Y391K LC H3K4M: CoREST1, bound to Y391K LSD1 (black), adopts a distinct conformation compared to CoREST1 bound to WT LSD1 (blue), shifting downward towards H3K4M. This shift is caused by the charge repulsion from K391LSD1. (c) Y391K LC H3K4M vs. Y391K H3K4M/K14ac: In both structures, K9 of H3K4M (orange) and K9** of H3K4M/K14ac (light purple) are situated nearby H564LSD1 Q358LSD1, without forming a compensatory salt bridge with E559LSD1. CoREST1 conformation remains downward, as described in (b). (d) Y391K LC H3K4M/K14ac vs. WT LC H3K4M/K14ac: In the WT LC structure, K9* of H3K4M/K14ac makes a compensatory salt bridge with E559LSD1, whereas in the Y391K LC structure, K9** of H3K4M/K14ac remains unchanged, residing nearby H564LSD1 and Q358LSD1. Note: * and ** represent different lysine residues in the H3K4M and H3K4M/K14ac peptides, respectively.
Extended Data Fig. 6 Analysis of Nucleosome Deacetylase Activity for Fluorescein-labeled WT LHC and Y391K LHC.
(a) Assessment of WT LHC deacetylase activity against H3K9ac nucleosomes. WT LHC concentrations of 90 nM (top) and 120 nM (bottom) were subjected to a 120-minute incubation with 100 nM of 185 bp H3K9ac nucleosomes, and variations in H3K9ac levels were monitored. (b) Evaluation of Y391K LHC deacetylase activity targeting H3K9ac-marked nucleosomes. Similar to (a), 90 nM (top) and 120 nM (bottom) Y391K LHC was incubated with 100 nM of 185 bp H3K9ac nucleosomes, and changes in H3K9ac levels were tracked over 120 minutes. Panels (c) and (d) illustrate the relative intensities obtained from (a) and (b), respectively, subjected to fitting into an exponential decay equation that includes constraints of Y0 at 1 and plateau at 0. (e) V/[E] (min−1) values from (a) and (b) were extrapolated (mean ± SEM). Anti-H3 blot at each time point from every other replicate was shown as a representative loading control.
Extended Data Fig. 7 CUT&RUN Chromatin Profiling Analysis in Parental K562 Cells: Comparison within Gene Bodies of Downregulated and Unaffected Control Genes.
(a) Metagene plot (mean ± SEM) of LSD1-GFP signal (experimental, n = 2) within gene bodies of downregulated genes (black) and unaffected control genes (red). (b) Metagene plot (mean ± SEM) from CUT&RUN analysis, showcasing H3K4me2 signal (experimental, n = 2), within gene bodies of 498 downregulated genes (black) and unaffected control genes (red). (c) Metagene plots (mean ± SEM) for IgG signal (experimental, n = 2), serving as a control, within gene bodies of 498 downregulated genes (black) and unaffected control genes (red). (d) Metagene plot (mean ± SEM) for H3K79me2 signal from Encode (K562 cell, ENCFF334HSS) within gene bodies of 498 downregulated genes (black) and unaffected control genes (red). (e) Metagene plot (mean ± SEM) for H3K27ac signal from Encode (K562 cells, ENCFF465GBD), encompassing gene bodies of the indicated gene sets. (f) Metagene plots (mean ± SEM) for H3K27me3 signal from Encode (K562 cells, ENCFF665RDD), observed within gene bodies of 498 downregulated genes (black) and unaffected control genes (red). (g) Metagene plots (mean ± SEM) for SUZ12 signal from Encode (K562 cells, ENCFF974IOO), observed within gene bodies of 498 downregulated genes (black) and unaffected control genes (red). (h) Metagene plots (mean ± SEM) for EZH2 signal from Encode (K562 cells, ENCFF974IOO), observed within gene bodies of 498 downregulated genes (black) and unaffected control genes (red). This provides context for the specificity of the observed signals. All analyses include assessment within the gene bodies as well as 20 kb regions upstream and downstream of the downregulated and unaffected control genes. All Encode data originate from K562 cells.
Extended Data Fig. 8 Comparative CUT&RUN Analysis between parental and edited K562 cells.
(a) Metagene plot (mean) for H3K4me2 signal at the LSD1 peaks (± 5 kb) across the 498 downregulated genes (top) and 498 unaffected control genes (bottom). The parental K562 cells are represented in black, while the edited K562 cells are depicted in pink. Two replicates (left and right) display variations in the 498 unaffected control regions. (b) Metagene plot (mean) for H3K14ac signal at the LSD1 peaks (± 5 kb) across the 498 downregulated genes (top) and 498 unaffected control genes (bottom). Similar to (a), the parental K562 cells are shown in black, and the edited K562 cells are shown in pink. Subtle variations in the 498 unaffected control regions are seen across two biological replicates, alongside reduced read counts in the downregulated genes. (c) Metagene plot (mean ± SEM) of IgG control signal within the scaled gene bodies ± 20 kb of the 498 downregulated genes (top left) and 498 unaffected control genes (bottom left), for both parental (black) and edited K562 cells (pink). Metagene plot (mean) of IgG control signal at the LSD1-bound regions within the 498 downregulated genes (top right) and 498 unaffected control genes (bottom right), for both for both parental (black) and edited K562 cells (pink) are shown. (d) Genomic distribution of LSD1 in parental and edited K562 cells. The left Venn diagram illustrates that approximately 53% of LSD1 peaks from parental K562 cells directly overlap with LSD1 peaks from edited K562 cells. About 43% of these overlapping peaks were located in the promoter region of all LSD1-bound genes. In contrast, only around 20% of the non-overlapping peaks were found in the promoter regions of LSD1-bound genes, suggesting a redistribution of LSD1 in numerous non-promoter regions. SEACR relaxed mode was employed to identify these LSD1 peaks, which were subsequently compared with LSD1 peaks from the Encode database (K562 cells, ENCFF054XCG). Both parental and edited K562 cells’ LSD1 peaks exhibit approximately 40% direct overlap with the LSD1 peaks from the Encode database.
Extended Data Fig. 9 Metagene plot (mean ± SEM) for H3K4me1, H3K4me2, H3K14ac, and LSD1 from parental (black) and edited (red) K562 cells at various genomic locations.
Including (a) H3K14ac global peaks, (b) H3K4me1 global peaks, (c) H3K4me2 global peaks, (d) H3K27ac global peaks (adopted from Encode ENCFF544LXB), (e) Intersected peaks of H3K4me1 and H3K14ac, (f) Intersected peaks of H3K4me2 and H3K14ac, (g) Intersected peaks of H3K27ac and H3K14ac, and (h) Intersected peaks of LSD1 and H3K14ac (only showing H3K4me1 and H3K4me2).
Extended Data Fig. 10 Differential Regulation in H3K4me1 and H3K4me2 Levels in Non-Promoter Regions of the 498 Downregulated Genes.
(a) LSD1 peaks in both promoter and non-promoter regions (top) were served as reference points for metagene plot analysis (bottom) (mean ± SEM). H3K4me1 and H3K4me2 levels at promoter and non-promoter regions were evaluated (parental – black and edited - pink). The reduction of H3K4me1 and H3K4me2 at LSD1-bound non-promoter regions is more pronounced in the 498 downregulated genes. (b–e) Genomic snapshots illustrate CUT&RUN signals for H3K4me1, H3K4me2, and H3K14ac for four representative genes (ZBTB16, DAB2, TRERF1, and PTPRS). Notably, H3K4me1 and H3K4me2 signals within gene bodies (brown box) show a reduction compared to the promoter region (blue box).
Supplementary information
Supplementary information (download PDF )
Supplementary Figs. 1–8, Supplementary Tables 1-2, codes used to process the CUT&RUN data, data points used for non-linear regression used for Supplementary Fig. 4 and the raw gel images used in Supplementary Figs. 6 and 7.
Source data
Source Data Fig. 1 (download PDF )
Unprocessed and processed (with the protein ladder) western blot images for Fig 1b.
Source Data Fig. 1 (download XLSX )
Statistical Source Data. Nucleosome demethylase activity for LC and LHC (Fig. 1c); nucleosome binding affinity for LC and LHC using MST (Fig. 1e).
Source Data Fig. 2 (download XLSX )
Statistical Source Data. Fig. 2b: GST LSD1 and LC mutants' demethylation rates toward peptide substrates (H3K4me2 and H3K4me2K14ac peptides).
Source Data Fig. 3 (download XLSX )
Statistical Source Data. Fig. 3b: nucleosome demethylation rate for WT LC and Y391K toward H3K4me2 and H3K4me2K14ac nucleosome substrates.
Source Data Fig. 3 (download PDF )
Unprocessed and processed (with the protein ladder) western blot images for Fig. 3a.
Source Data Fig. 4 (download XLSX )
Statistical Source Data. Fig. 4b: Growth rate for parental and Y391K edited #1 and #2 K562 cells. Fig. 4d: IC50 calculation for parental and Y391K edited #1 K562 cells' viability treated with GSK-2879552. Fig. 4e: IC50 calculation for parental and Y391K edited #1 K562 cells' viability treated with vorinostat. Fig. 4f: IC50 calculation for parental and Y391K edited #1 K562 cells' viability treated with imatinib. Fig. 4g: IC50 calculation for parental and Y391K edited #1 K562 cells' viability treated with corin.
Source Data Fig. 4 (download PDF )
Unprocessed and processed (with the protein ladder) western blot images for Fig. 4c.
Source Data Fig. 5 (download XLSX )
Statistical Source Data. Fig. 5a: Genomic distribution of LSD1-GFP associated with the 498 downregulated genes. WT LSD1 (parental) K562 cells. Y391K LSD1 (edited) K562 cells. Fig. 5a: 498 downregulated genes (ENSG). Fig. 5a: 498 unaffected control genes (ENSG). Fig. 5b: genomic coordinates for 498 downregulated genes. Fig. 5b: genomic coordinates for 498 unaffected control genes.
Source Data Extended Data Fig./Table 1 (download PDF )
Unprocessed and processed (with the protein ladder) western blot images for Extended Data Fig. 1a,b.
Source Data Extended Data Fig./Table 2 (download XLSX )
Demethylase activity of fluorescein-labeled and unlabeled LC and LHC toward peptide substrates (H3K4me2).
Source Data Extended Data Fig./Table 3 (download PDF )
Unprocessed and processed (with the protein ladder) western blot images for Extended Data Fig. 3a–c,g.
Source Data Extended Data Fig./Table 3 (download XLSX )
Statistical Source Data. Extended Data Fig. 3h: MST binding affinity data for fluorescein-labeled WT and Y391K LC toward H3K14ac nucleosome.
Source Data Extended Data Fig./Table 4 (download PDF )
Unprocessed and processed (with the protein ladder) western blot images for Extended Data Fig. 4a–d,i.
Source Data Extended Data Fig./Table 4 (download XLSX )
Statistical Source Data. Extended Data Fig. 4j: nucleosome demethylation rate for WT LC and Y391K toward H3K4me1 and H3K4me2K14ac nucleosome substrates.
Source Data Extended Data Fig./Table 6 (download PDF )
Unprocessed and processed (with the protein ladder) western blot images for Extended Data Fig. 6a,b.
Source Data Extended Data Fig./Table 8 (download XLSX )
Statistical Source Data. Extended Data Fig. 8d: annotation of LSD1 distribution—common, WT (parental) and Y391K (edited).
Source Data Extended Data Fig./Table 10 (download XLSX )
Statistical Source Data. Extended Data Fig. 10a: genomic coordinates of LSD1 intersected with the 498 genes. Extended Data Fig. 10a: genomic coordinates of LSD1 intersected with the 498 genes at the promoter region. Extended Data Fig. 10a: genomic coordinates of LSD1 intersected with the 498 genes at the non-promoter region.
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Lee, K., Barone, M., Waterbury, A.L. et al. Uncoupling histone modification crosstalk by engineering lysine demethylase LSD1. Nat Chem Biol 21, 227–237 (2025). https://doi.org/10.1038/s41589-024-01671-9
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DOI: https://doi.org/10.1038/s41589-024-01671-9
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