. 2012 Jun 22;149(7):1447-60.
Epub 2012 Jun 14.
Dynamics and Memory of Heterochromatin in Living Cells
Free PMC article
Item in Clipboard
Dynamics and Memory of Heterochromatin in Living Cells
Free PMC article
Posttranslational histone modifications are important for gene regulation, yet the mode of propagation and the contribution to heritable gene expression states remains controversial. To address these questions, we developed a chromatin in vivo assay (CiA) system employing chemically induced proximity to initiate and terminate chromatin modifications in living cells. We selectively recruited HP1α to induce H3K9me3-dependent gene silencing and describe the kinetics and extent of chromatin modifications at the Oct4 locus in fibroblasts and pluripotent cells. H3K9me3 propagated symmetrically and continuously at average rates of ~0.18 nucleosomes/hr to produce domains of up to 10 kb. After removal of the HP1α stimulus, heterochromatic domains were heritably transmitted, undiminished through multiple cell generations. Our data enabled quantitative modeling of reaction kinetics, which revealed that dynamic competition between histone marking and turnover, determines the boundaries and stability of H3K9me3 domains. This framework predicts the steady-state dynamics and spatial features of the majority of euchromatic H3K9me3 domains over the genome.
Copyright © 2012 Elsevier Inc. All rights reserved.
Figure 1. Design of Chromatin
in vivo Assay at Oct4 ( CiA:Oct4) ES cell line and mouse
(A) CIP allows direct recruitment, washout, co-occupancy, and order-of-addition experiments. (B) The
CIA:Oct4 mouse contains one modified Oct4 allele harboring two arrays of DNA binding sites (12XZFHD1 and 5XGal4) in the promoter region upstream of an in-frame EGFP reporter. Distribution of histone modifications at the Oct4 locus in murine ES cells and brain tissue (Mikkelsen et al., 2007) reveals the distinct chromatin substrates for CiA modulation.
Figure 2. Kinetics of heterochromatin formation following HP1α recruitment in ES cells
A) Experimental design: rapamycin addition recruits HP1α chromoshadow fragment (csHP1α) to the
CiA:Oct4 promoter. B) Schematic representation of wild-type and CiA alleles depicts location of allele-specific and common real-time PCR primers C) ChIP analysis shows rapamycin-mediated csHP1α recruitment over time. D) ChIP analysis reveals dynamic changes of active (H3K4me3, H3K27ac) and repressive (H3K9me3, HP1 gamma) chromatin modifications at the CiA:Oct4 locus. Upper panel summarizes time course of chromatin remodeling at 0h, 24h, 48h, 72h, 96h, 120h, and 192h. Data rotated 180° as indicated to display loss of active marks. Lower panels display ChIP analysis of histone modifications (y-axis) across the CiA:Oct4 locus (x-axis) at selected time points. GFP expression was measured by flow cytometry at each time point. Schematic of the reporter allele indicates CiA:Oct4-specific primer pairs in black. E) Dnase I sensitivity across the CiA:Oct4 locus before and after csHP1α recruitment. F) ChIP analysis of OCT4 transcription factor binding at Oct4 enhancer before and after csHP1α recruitment. G) Bisulfite sequencing analysis of DNA methylation changes at the CiA:Oct4 promoter following csHP1α targeting, with percentage methylated CpGs. White lines in schematic below mark relative positions of CpG dinucleotides.
Figure 3. Maintenance of heterochromatin and heritable
Oct4 repression in ES cells
A) Experimental design: rapamycin was added for either 7 days or 4.5 weeks and then washed-out with or without Dnmt-inhibitor 5azaC. B) Flow cytometry analysis after release from csHP1α after 7 days (low promoter DNAme), or after 4.5 weeks (high promoter DNAme). C) Colony growth assay tests heritability of
CiA:Oct4 repression. GFP expression of individual colonies was quantified by microscopy (7 days, n=158; 4.5 weeks, n=199). D) ChIP analysis of histone modifications after rapamycin washout. The bottom panels depict bisulfite sequencing analysis of DNA methylation at CiA:Oct4 promoter after csHP1α washout for 4 days. White lines in schematic below mark relative positions of CpG dinucleotides.
CiA:Oct4 activation and kinetics of heterochromatin formation in MEFs
(A) CiA E14.5 p4 MEFs were infected with lentiviral constructs of either GAL4 alone or a GAL4-VP16 fusion. Puromycin was added at 48 hrs and cells analyzed by flow cytometry. (B) Experimental design: the
CiA:Oct4 allele was reactivated by GAL4-VP16 in transformed CiA MEFs. GFP-positive cells were enriched by FACS and treated with rapamycin to induce csHP1α targeting. C) ChIP analysis of rapamycin-mediated csHP1α recruitment over time. D) ChIP analysis reveals dynamic changes of active (H3K4me3, H3K27ac) and repressive (H3K9me3) histone modifications at the CiA:Oct4 locus. Upper panel summarizes time course of chromatin remodeling at 0h, 24h, 48h, 72h, 96h, 120h, and 192h. Data rotated 180° as indicated to display loss of active marks. Lower panels display loss of active and gain of repressive histone modifications (y-axis) across the CiA:Oct4 locus (x-axis) at selected time points. GFP expression was measured by flow cytometry analysis at each time point.
Figure 5. Maintenance of heterochromatin and dependence on transcription
A) Experimental design: the
CiA:Oct4 allele was reactivated in transformed CiA MEFs by abscisic acid (ABA)-mediated recruitment of VP16. GFP-positive reactivated cells were enriched by FACS. Rapamycin was added for 7 days to recruit csHP1α. GFP-negative cells were sorted by FACS. Finally, rapamycin was washed out in the presence or absence of ABA-recruited VP16. Cells were analyzed four and eight days later. B) Flow cytometry analysis after removal of csHP1α in the presence and absence of ABA-recruited VP16. C) Cartoon depicts recruitment strategy to form heterochromatin and test its maintenance. ChIP analysis of H3K9me3 along the CiA:Oct4 allele during heterochromatin formation and after csHP1 α removal with or without ABA-mediated VP16 recruitment for 4 and 8 days. H3K9me3 is maintained after rapamycin washout when not opposed by ABA-mediated transcription (P-values: * p=0.052, n/s=not significant, ** p=0.007, *** p=0.004).
Figure 6. Kinetic model of H3K9me3 dynamics
A) We consider chromatin as a one-dimensional beads-on-a-string lattice. Three processes: nucleation, propagation and turnover, yield a bounded steady-state island of marks. Nucleation occurs at the target site at rate
k +. Propagation of the mark continues at sites immediately adjacent to marked sites, at rate k +. Turnover of the mark is equally likely everywhere at rate k −. When these processes are allowed to occur at the same time, a stochastic, bounded island of H3K9me3 marks is established at steady state. Sample output of the model with H3K9me3 domains at steady-state (right panel; each horizontal line represents a single simulation). B) Simplified kinetic scheme of H3K9me3 dynamics. Without a feedback mechanism to reinforce placement of H3K9me3 marks, the domain collapses in the absence of continued nucleation (lower panel). In the presence of a feedback mechanism that stabilizes H3K9 methylation (denoted by H3K9me3*) the domain persists. C) The profile of the steady-state island varies with κ (defined in main text). Larger values of κ increase the size of the island until κ > 1.5; above this value, the island grows without bounds. D) Fits of the experimental H3K9me3 ChIP data shown in Figure 2 to the kinetic model. Data from ES cells are best described by κ=1.5, while the data from MEFs are described by κ=1.0. E) Specific values of k + and k − were obtained by fitting the simulations to a time course of integrated H3K9me3 ChIP enrichment at the locus (see Figure 2). Resulting values of k + and k − are shown next to the data for each cell type. Our estimated uncertainty in these values is 35% (shaded regions). F) Clustering of genomic H3K9me3 domains in ES cells (Bilodeau et al., 2009) by k-means, with k=3. Clustering identified two predominant groups ("small" and "large" H3K9me3 domains), and a very small number of aberrant domains. G) Small H3K9me3 domains (mean ± SD) are described well by our model with κ=1.0. H) Large H3K9me3 domains (mean ± SD) are described well by our model with κ=1.4.
Contribution of promoter DNA sequence to heterochromatin formation velocity and memory of gene repression in mouse embryo fibroblasts.
PLoS One. 2019 Jul 3;14(7):e0217699. doi: 10.1371/journal.pone.0217699. eCollection 2019.
PLoS One. 2019.
31269077 Free PMC article.
Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin.
Genome Res. 2011 Feb;21(2):147-63. doi: 10.1101/gr.110098.110. Epub 2010 Dec 22.
Genome Res. 2011.
21177972 Free PMC article.
Loss of RB compromises specific heterochromatin modifications and modulates HP1alpha dynamics.
J Cell Physiol. 2007 Apr;211(1):131-7. doi: 10.1002/jcp.20913.
J Cell Physiol. 2007.
H3K9me3-Dependent Heterochromatin: Barrier to Cell Fate Changes.
Trends Genet. 2016 Jan;32(1):29-41. doi: 10.1016/j.tig.2015.11.001. Epub 2015 Dec 8.
Trends Genet. 2016.
26675384 Free PMC article.
On the relations of phase separation and Hi-C maps to epigenetics.
R Soc Open Sci. 2020 Feb 26;7(2):191976. doi: 10.1098/rsos.191976. eCollection 2020 Feb.
R Soc Open Sci. 2020.
32257349 Free PMC article.
Analysis of Single-Cell Gene Pair Coexpression Landscapes by Stochastic Kinetic Modeling Reveals Gene-Pair Interactions in Development.
Front Genet. 2020 Jan 31;10:1387. doi: 10.3389/fgene.2019.01387. eCollection 2019.
Front Genet. 2020.
32082359 Free PMC article.
Epigenetic crosstalk in chronic infection with HIV-1.
Semin Immunopathol. 2020 Apr;42(2):187-200. doi: 10.1007/s00281-020-00783-3. Epub 2020 Feb 11.
Semin Immunopathol. 2020.
32047948 Free PMC article.
Chemical and Light Inducible Epigenome Editing.
Int J Mol Sci. 2020 Feb 3;21(3):998. doi: 10.3390/ijms21030998.
Int J Mol Sci. 2020.
32028669 Free PMC article.
Epigenetic Control of a Local Chromatin Landscape.
Int J Mol Sci. 2020 Jan 31;21(3):943. doi: 10.3390/ijms21030943.
Int J Mol Sci. 2020.
32023873 Free PMC article.
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Chromosomal Proteins, Non-Histone / metabolism
Heterochromatin / metabolism*
Octamer Transcription Factor-3 / metabolism
Chromosomal Proteins, Non-Histone
Octamer Transcription Factor-3
heterochromatin-specific nonhistone chromosomal protein HP-1
LinkOut - more resources
Full Text Sources Other Literature Sources Molecular Biology Databases Research Materials