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Inducible histone K-to-M mutations are dynamic tools to probe the physiological role of site-specific histone methylation in vitro and in vivo

Nature Cell Biology volume 21, pages 1449–1461 (2019)Cite this article

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Abstract

Development and differentiation are associated with profound changes to histone modifications, yet their in vivo function remains incompletely understood. Here, we generated mouse models expressing inducible histone H3 lysine-to-methionine (K-to-M) mutants, which globally inhibit methylation at specific sites. Mice expressing H3K36M developed severe anaemia with arrested erythropoiesis, a marked haematopoietic stem cell defect, and rapid lethality. By contrast, mice expressing H3K9M survived up to a year and showed expansion of multipotent progenitors, aberrant lymphopoiesis and thrombocytosis. Additionally, some H3K9M mice succumbed to aggressive T cell leukaemia/lymphoma, while H3K36M mice exhibited differentiation defects in testis and intestine. Mechanistically, induction of either mutant reduced corresponding histone trimethylation patterns genome-wide and altered chromatin accessibility as well as gene expression landscapes. Strikingly, discontinuation of transgene expression largely restored differentiation programmes. Our work shows that individual chromatin modifications are required at several specific stages of differentiation and introduces powerful tools to interrogate their roles in vivo.

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Fig. 1: Doxycycline-inducible K-to-M histone mutants globally suppress site-specific histone methylation and impair differentiation of ES cells.
Fig. 2: In vivo expression of H3K36M leads to rapid death with severe anaemia and organ abnormalities.
Fig. 3: In vivo expression of H3K36M dramatically alters myeloid and lymphoid differentiation.
Fig. 4: Expression of H3K36M and H3K9M have opposing, cell autonomous effects on HSPC function.
Fig. 5: H3K36M and H3K9M induction in HSPCs leads to widespread changes in the chromatin landscape and altered gene expression.
Fig. 6: The physiological consequences of suppressing H3K9 and H3K36 methylation are reversible both in vivo and in vitro.
Fig. 7: Prolonged expression of H3K9M/I is associated with T cell leukaemia/lymphoma.

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Acknowledgements

The authors thank M. Handley, A. Galvin, M. Gesner and E. Surette of the MGH/HSCI Rodent Histopathology Core for technical assistance. They also thank members of the Hochedlinger Lab for helpful discussions. A.J.H. is supported by an American Cancer Society—New England Division—Ellison Foundation Postdoctoral Fellowship (PF-15-130-01-DDC). B.D.S. was supported by an EMBO long-term fellowship (no. ALTF 1143-2015) and a MGH Tosteson and FMD postdoctoral fellowship. K.H. was supported by funds from the MGH, NIH (R01 HD058013-06) and the Gerald and Darlene Jordan Chair in Regenerative Medicine. H.H. was supported by a Hyundai Hope on Wheels Scholar Grant. J.B. is grateful for support from the NIH (1F32HD078029-01A1). R.I.S. was supported by funds from the NIH (P30-DK40561).

Author information

Author notes

  1. Justin Brumbaugh

    Present address: Department of Molecular, Cellular, and Developmental Biology, University of Colorado-Boulder, Boulder, CO, USA

  2. These authors contributed equally: Justin Brumbaugh, Ik Soo Kim.

Authors and Affiliations

  1. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA

    Justin Brumbaugh, Fei Ji, Aaron J. Huebner, Bruno Di Stefano, Benjamin A. Schwarz, Amy Coffey, Jiho Choi, Ryan M. Walsh, Anthony Anselmo, Ruslan I. Sadreyev & Konrad Hochedlinger

  2. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA

    Justin Brumbaugh, Aaron J. Huebner, Bruno Di Stefano, Benjamin A. Schwarz, Amy Coffey, Jiho Choi, Ryan M. Walsh, Jeffrey W. Schindler, Hanno Hock & Konrad Hochedlinger

  3. Center for Cancer Research, Massachusetts General Hospital, Boston, MA, USA

    Justin Brumbaugh, Ik Soo Kim, Aaron J. Huebner, Bruno Di Stefano, Benjamin A. Schwarz, Amy Coffey, Jiho Choi, Ryan M. Walsh, Jeffrey W. Schindler, Bradley E. Bernstein, Hanno Hock & Konrad Hochedlinger

  4. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA

    Justin Brumbaugh, Aaron J. Huebner, Bruno Di Stefano, Benjamin A. Schwarz, Jocelyn Charlton, Amy Coffey, Jiho Choi, Ryan M. Walsh, Alexander Meissner & Konrad Hochedlinger

  5. Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA

    Justin Brumbaugh, Aaron J. Huebner, Bruno Di Stefano, Benjamin A. Schwarz, Amy Coffey, Jiho Choi, Ryan M. Walsh, Jeffrey W. Schindler, Hanno Hock & Konrad Hochedlinger

  6. Harvard Medical School, Boston, MA, USA

    Justin Brumbaugh, Fei Ji, Aaron J. Huebner, Bruno Di Stefano, Benjamin A. Schwarz, Amy Coffey, Jiho Choi, Ryan M. Walsh, Jeffrey W. Schindler, Anthony Anselmo, Ruslan I. Sadreyev, Hanno Hock & Konrad Hochedlinger

  7. Department of Pathology, Harvard Medical School, Boston, MA, USA

    Ik Soo Kim, Ruslan I. Sadreyev & Bradley E. Bernstein

  8. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Fei Ji, Anthony Anselmo & Konrad Hochedlinger

  9. Department of Genome Regulation, Max Planck Institute for Molecular Genetics, Berlin, Germany

    Jocelyn Charlton & Alexander Meissner

  10. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Ik Soo Kim, Alexander Meissner, Bradley E. Bernstein & Konrad Hochedlinger

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Contributions

J.B., H.H. and K.H. conceived the study and wrote the manuscript. J.B., B.A.S., A.J.H., J. Choi, J. Charlton, J.W.S., A.C., I.S.K., B.D.S., A.M., B.B. and R.M.W. designed and performed the experiments and analysed the data. F.J., A.A. and R.I.S. the performed bioinformatics analysis.

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Correspondence to Hanno Hock or Konrad Hochedlinger.

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Extended data

Extended Data Fig. 1 Expression of H3K9M and H3K36M suppresses methylation and blocks differentiation of pluripotent stem cells.

(a) Western blot analysis for tri-methyl H3K9 and H3K36 with and without mutant histone expression in mouse ES cells. (b) Pie charts showing the distribution of ATAC-seq peaks in EBs expressing H3, H3K9M, and H3K36M. (c) Images of teratomas derived from ES cells expressing H3, H3K9M, or H3K36M. Scale bar = 200 μm; endo = endoderm, meso = mesoderm, ecto = ectoderm. (d) qRT-PCR analysis for knockdown of H3K9 and H3K36 KMTs. One sample is shown. (e) Flow cytometry for Rex1-GFP at days 2 and 3 of embryoid body formation. (f) Western blot analysis showing H3K36me3 levels following knockdown of H3K36 KMTs. (g) Western blot analysis showing H3K9me3 levels following knockdown of H3K9 KMTs. See source data for full membrane Western blot images. Data in a,c,e,f,g are representative of 3 independent experiments.

Source data

Source data

Extended Data Fig. 2 In vivo expression of H3K9M and H3K36M disrupts tissue homeostasis.

(a) Histological analysis of testis in mice induced for 4 weeks. Scale bar = 20 μm. (b) GSEA based on RNA-seq data from testes isolated from H3K9M and H3K36M mice induced for 4 weeks (H3, n = 2; H3K9M, n = 2; H3K36M, n = 2). Enrichment is shown for transcriptional signatures58 related to spermatogenesis. Statistics were generated in accordance with the published GSEA algorithm51 (c) Gene tracks showing expression for genes characteristic of spermatogonia (left panel) or spermatocytes (right panel). (d) Histological analysis of intestine in mice induced for 4 weeks. Black and white arrowheads indicate goblet cells and paneth cells, respectively. Scale bar = 20 μm. (e) Staining for markers of Paneth cells (Lysozyme; left panel) and goblet cells (periodic acid Schiff; right panel) (f) GSEA based on RNA-seq data from intestine isolated from H3K9M and H3K36M mice induced for 4 weeks (H3, n = 2; H3K9M, n = 2; H3K36M, n = 2). Enrichment is shown for transcriptional signatures related to secretory lineages57 and Goblet cells59. Statistics were generated in accordance with the published GSEA algorithm51. (g) Gene tracks showing expression for genes characteristic of secretory lineages (left panel) or goblet cells (right panel). (h) Tibia from mice expressing mutant histones for 4 weeks. Scale bar = 5 mm. Data in a,d,e,h are representative of 3 independent experiments.

Extended Data Fig. 3 H3K9M and H3K36M induction cause abnormalities in the erythroid, megakaryocyte, granulocyte and T cell lineages.

(a) cKit expression in early erythroid progenitors. Data are representative of three independent experiments. (b) Quantification of cKit positive erythroid progenitors (right panel). Columns represent the mean and error bars represent standard deviation of the mean for biological replicates (rtTA, n = 6; H3K9M, n = 3; H3K36M, n = 3). Statistical significance was determined using a two-tailed unpaired Student’s t-test. (c) Time course analysis for hemoglobin assessed by CBC. Columns represent the mean and error bars represent standard deviation of the mean for biological replicates (rtTA, n = 5; H3K9M, n = 4; H3K36M, n = 3). Statistical significance was determined using the Holm-Sidak method. (d) An infarcted spleen from a H3K9M mouse induced for 4 weeks. Scale bar = 5 mm. Infarctions were present in 2 out of 3 mice inspected. (e) Flow cytometry analysis for granulocyte cells. Gr1+ cells are shown in green. (f) Granularity of transgenic cells assessed by side scatter. (g) Cytospin images of Gr1+ granulocytes sorted from H3K36M and rtTA mice. Scale bar = 5 μm. (h) Flow cytometry analysis for thymocytes. Frequencies for each cell type are indicated as a percentage of the parent gate. (i) Quantification of CD4/CD8 double negative thymocytes (left panel) and double positive CD4/CD8 thymocytes (right panel). Columns represent the mean and error bars represent standard deviation of the mean for biological replicates (rtTA, n = 6; H3K9M, n = 3; H3K36M, n = 3). Statistical significance was determined using a two-tailed unpaired Student’s t-test. Data in e,f,g,h are representative of 3 independent experiments.

Source data

Extended Data Fig. 4 Mutant histone expression impacts HSC function in competitive bone marrow transplantation assays.

(a) Frequency of granulocytes derived from donor bone marrow (CD45.2+) harboring control or mutant histones before and after 12 weeks of induction with doxycyline. Columns represent the mean and error bars represent standard deviation of the mean for biological replicates (n = 4). Statistical significance was determined using a two-tailed unpaired Student’s t-test. (b) Flow cytometry analysis for donor-derived, CD45.2+ LKS cells, hematopoietic progenitor cells, and stem cells at the conclusion of competitive bone marrow transplant assays. Frequencies for each cell type are indicated as a percentage of the parent gate. (c) Flow cytometry showing granularity of both wild-type competitor marrow (CD45.1) and marrow expressing histone transgenes (CD45.2) as assessed by side scatter. (d) Brightfield images of colonies in methylcellulose. Black arrowheads indicate megakaryocytes. Scale bar = 200 μm or 50 μm. (e) May Grünwald Giemsa stains of cells grown in methylcellulose. Representative images are shown for rtTA, H3K9M, and H3K36M. Scale bar = 20 μm. Black arrowheads indicate megakaryocytes, white arrowheads indicate granulocytes, and red arrowheads indicate erythroid cells. Data in b,c,d,e are representative of 3 independent experiments.

Source data

Extended Data Fig. 5 Mutant histone expression impacts HSC gene expression.

(a) GSEA analysis based on RNA-seq data for hematopoietic stem and progenitor cells sorted from H3K9M and H3K36M mice induced for 4 weeks (H3, n = 2; H3K9M, n = 2; H3K36M, n = 2). Enrichment is shown for transcriptional signatures related to Lymphoid/Myeloid progenitors (CMP, LMPP, and proB)33 and B cells50 (K9M) as well as erythroid cells33 and B cells50 (K36M). Statistics were generated in accordance with the published GSEA algorithm51. (b) Principal component analysis based on the union of differentially expressed genes between rtTA control and H3K9M or H3K36M samples. Each circle represents a biological replicate (rtTA, n = 3; H3K9M, n = 3; H3K36M, n = 3) while squares represent the mean. (c) Expression (RPKM) values for select genes relative to average expression in rtTA replicates (shown in log2 scale) for biological triplicates from LKS cells sorted at day 7 of induction.

Extended Data Fig. 6 H3K36M/H3K9M induction change the chromatin landscape in HSPCs.

(a) Distribution of H3K9me3 across genomic elements. (b) Heat-maps showing H3K9me3 methylation levels genome-wide following four weeks of H3 or H3K9M induction (averages, two replicates). (c) Representative tracks for loci with decreased H3K9me3 signal following H3K9M expression. (d) RNA-seq scatter plot based on RPKM. Red lines marks 1.5-fold difference between WT and K9M, gray line marks RPKM = 1 (cutoff for expressed repeats). Differentially expressed repeats (1.5-fold, FDR < 0.1; RNA-Seq data) are marked in orange and green dots. Red circles mark loci where H3K9me3 is differentially bound. Repeats overlapping protein-coding exons were excluded. (e) Heat-maps showing H3K36me3 methylation levels genome-wide following four weeks of H3 or H3K36M induction (averages, two replicates). (f) Distribution of H3K36me3 across genomic elements. (g) Levels of H3K36me3 in genes with increased expression, decreased expression, and all genes in H3K36M vs control HSPCs. The center bar represents the mean and the whiskers represent the standard deviation of the mean. (h) Replicate heat-maps showing H3K27me3 methylation for genes that are downregulated following H3K36M induction. (i) Correlation plot comparing H3K27me3 levels in H3K36M vs. H3 HSPCs. Levels of H3K36me3 overlaid on data points. (j) Meta-analysis showing H3K27me3 levels for a 200 kb region surrounding genes that are downregulated with H3K36M expression (top) and at random regions (bottom). (k) ATAC-seq scatter plot. Loci with reduced H3K36me3 following H3K36M expression are in blue. (l) Replicate tracks for Gfi1 in Fig. 5g. (m) Representative tracks for Prdm5, which is downregulated following H3K36M expression. (n) Correlation plots comparing gene body methylation between H3 or H3K36M HSPCs. Significant differences in green (left), hematopoietic genes in blue (right). (o) Gene tracks for Gfi1 showing DNA methylation in H3 or H3K36M HSPCs. (p) Replicate heat-maps showing H3K36me3 methylation at gene bodies for genes upregulated in H3K36M HSPCs. (q) Replicate tracks for Klf5 in Fig. 5h. (r) Representative tracks for Zfpm1, which is upregulated following H3K36M expression. N numbers in d represent repetitive elements. N numbers in g,n represent genes. All n numbers are stated in the figure.

Extended Data Fig. 7 Reversibility of K-to-M dependent phenotypes.

(a) Histological analysis of testis in mice induced for 4 weeks with an additional 4 weeks of dox withdrawal. Scale bar = 50 μm. (b) Histological analysis of intestine in mice induced for 4 weeks with an additional 4 weeks of dox withdrawal. Black and white arrowheads indicate goblet cells and paneth cells, respectively. Scale bar = 50 μm. (c) Brightfield images of colonies in methylcellulose with dox removal. Black arrowheads indicate megakaryocytes. Scale bar = 200 μm or 50 μm. (d) Western blot analysis for tri-methyl H3K9 and H3K36 from EBs induced for 3 days with dox and EBs induced for 3 days with dox followed by 3 days of dox withdrawal. (e) Western blot analysis for H3K9me3 and H3K36me3 from HSPCs in mice induced for 7 days with dox and mice induced for 7 days with dox followed by 7 days of dox withdrawal. See source data for full membrane Western blot images. Data in a-e are representative of 3 independent experiments.

Source data

Extended Data Fig. 8 K9I mice phenocopy K9M mice.

(a) Flow cytometry analysis of megakaryocyte progenitors (MkPs) cells, 4 weeks of dox. Frequencies as percentages of the parent gate. (b) Quantification of MkPs in bone marrow. Columns represent the mean +/− standard deviation (rtTA, n = 1; H3K9I, n = 3, biological replicates). (c) Platelet counts after 4 weeks of dox. Columns represent the mean +/− standard deviation (rtTA, n = 1; H3K9I, n = 3, biological replicates). (d) Flow cytometry of B cells in the bone marrow, 4 weeks of dox. Frequencies as percentages of the parent gates. (e) Quantification of mature B cells and IGM+ B cells. Columns represent the mean +/− standard deviation (rtTA, n = 1; H3K9I, n = 2, biological replicates). (f) Flow cytometry analysis of B cell progenitors in bone marrow, 4 weeks of dox. Frequencies as percentages of parent gates. (g) Quantification of ProB cells and PreB cells. Columns represent the mean +/− standard deviation (rtTA, n = 1; H3K9I, n = 2, biological replicates). (h) Flow cytometry analysis of LKS cells. Frequencies as percentages of parent gates. (i) Quantification of LKS cells in bone marrow. Columns represent the mean +/− standard deviation (rtTA, n = 1; H3K9I, n = 3, biological replicates). (j) Flow cytometry of stem cell populations gated on LKS cells. Frequencies indicated as percentages of parent gates. (k) Quantification of LMPPs, defined as Flt3high cells (top 25% of Flt3+ cells set on rtTA control). Columns represent means and error bars standard deviation (rtTA, n = 1; H3K9I, n = 3, biological replicates). (l) Flow cytometry of SLAM markers gated on LKS cells. Frequencies as percentages of parent gates. (m) Quantification of MPPs (left panel) and long-term HSCs (right panel) in bone marrow. Columns represent the mean +/− standard deviation (rtTA, n = 1; H3K9I, n = 3 biological replicates). Data in a,d,f,h,j,l are representative of 3 independent experiments.

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Brumbaugh, J., Kim, I.S., Ji, F. et al. Inducible histone K-to-M mutations are dynamic tools to probe the physiological role of site-specific histone methylation in vitro and in vivo. Nat Cell Biol 21, 1449–1461 (2019). https://doi.org/10.1038/s41556-019-0403-5

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