Letter | Published:

Active chromatin marks drive spatial sequestration of heterochromatin in C. elegans nuclei

Abstract

The execution of developmental programs of gene expression requires an accurate partitioning of the genome into subnuclear compartments, with active euchromatin enriched centrally and silent heterochromatin at the nuclear periphery1. The existence of degenerative diseases linked to lamin A mutations suggests that perinuclear binding of chromatin contributes to cell-type integrity2,3. The methylation of lysine 9 of histone H3 (H3K9me) characterizes heterochromatin and mediates both transcriptional repression and chromatin anchoring at the inner nuclear membrane4. In Caenorhabditis elegans embryos, chromodomain protein CEC-4 bound to the inner nuclear membrane tethers heterochromatin through H3K9me3,5, whereas in differentiated tissues, a second heterochromatin-sequestering pathway is induced. Here we use an RNA interference screen in the cec-4 background and identify MRG-1 as a broadly expressed factor that is necessary for this second chromatin anchor in intestinal cells. However, MRG-1 is exclusively bound to euchromatin, suggesting that it acts indirectly. Heterochromatin detachment in double mrg-1; cec-4 mutants is rescued by depleting the histone acetyltransferase CBP-1/p300 or the transcription factor ATF-8, a member of the bZIP family (which is known to recruit CBP/p300). Overexpression of CBP-1 in cec-4 mutants is sufficient to delocalize heterochromatin in an ATF-8-dependent manner. CBP-1 and H3K27ac levels increase in heterochromatin upon mrg-1 knockdown, coincident with delocalization. This suggests that the spatial organization of chromatin in C. elegans is regulated both by the direct perinuclear attachment of silent chromatin, and by an active retention of CBP-1/p300 in euchromatin. The two pathways contribute differentially in embryos and larval tissues, with CBP-1 sequestration by MRG-1 having a major role in differentiated cells.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

All primers sequences are provided in the Methods. All other data are available from the corresponding author upon reasonable request. RNA sequencing and EMR-1-DamID data are available under the GEO accession codes GSE116037 and GSE126028, respectively.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Solovei, I., Thanisch, K. & Feodorova, Y. How to rule the nucleus: divide et impera. Curr. Opin. Cell Biol. 40, 47–59 (2016).

  2. 2.

    Perovanovic, J. et al. Laminopathies disrupt epigenomic developmental programs and cell fate. Sci. Transl. Med. 8, 335ra58 (2016).

  3. 3.

    Gonzalez-Sandoval, A. et al. Perinuclear anchoring of H3K9-methylated chromatin stabilizes induced cell fate in C. elegans embryos. Cell 163, 1333–1347 (2015).

  4. 4.

    Mattout, A., Cabianca, D. S. & Gasser, S. M. Chromatin states and nuclear organization in development-a view from the nuclear lamina. Genome Biol. 16, 174 (2015).

  5. 5.

    Towbin, B. D. et al. Step-wise methylation of histone H3K9 positions heterochromatin at the nuclear periphery. Cell 150, 934–947 (2012).

  6. 6.

    Meister, P., Towbin, B. D., Pike, B. L., Ponti, A. & Gasser, S. M. The spatial dynamics of tissue-specific promoters during C. elegans development. Genes Dev. 24, 766–782 (2010).

  7. 7.

    Meister, P., Gehlen, L. R., Varela, E., Kalck, V. & Gasser, S. M. Visualizing yeast chromosomes and nuclear architecture. Methods Enzymol. 470, 535–567 (2010).

  8. 8.

    Sulston, J. E., Schierenberg, E., White, J. G. & Thomson, J. N. The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64–119 (1983).

  9. 9.

    González-Aguilera, C. et al. Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans. Genome Biol. 15, R21 (2014).

  10. 10.

    Muñoz-Jiménez, C. et al. An efficient FLP-based toolkit for spatiotemporal control of gene expression in Caenorhabditis elegans. Genetics 206, 1763–1778 (2017).

  11. 11.

    Ikegami, K., Egelhofer, T. A., Strome, S. & Lieb, J. D. Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2. Genome Biol. 11, R120 (2010).

  12. 12.

    Doyon, Y., Selleck, W., Lane, W. S., Tan, S. & Côté, J. Structural and functional conservation of the NuA4 histone acetyltransferase complex from yeast to humans. Mol. Cell. Biol. 24, 1884–1896 (2004).

  13. 13.

    Carrozza, M. J. et al. Histone H3 methylation by Set2 directs deacetylation of coding regions by Rpd3S to suppress spurious intragenic transcription. Cell 123, 581–592 (2005).

  14. 14.

    Filion, G. J. et al. Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143, 212–224 (2010).

  15. 15.

    Zhang, P. et al. Structure of human MRG15 chromo domain and its binding to Lys36-methylated histone H3. Nucleic Acids Res. 34, 6621–6628 (2006).

  16. 16.

    Eisen, A. et al. The yeast NuA4 and Drosophila MSL complexes contain homologous subunits important for transcription regulation. J. Biol. Chem. 276, 3484–3491 (2001).

  17. 17.

    Cai, Y. et al. Identification of new subunits of the multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase complex. J. Biol. Chem. 278, 42733–42736 (2003).

  18. 18.

    Celniker, S. E. et al. Unlocking the secrets of the genome. Nature 459, 927–930 (2009).

  19. 19.

    Ahringer, J. & Gasser, S. M. Repressive chromatin in Caenorhabditis elegans: establishment, composition, and function. Genetics 208, 491–511 (2018).

  20. 20.

    Klymenko, T. & Müller, J. The histone methyltransferases Trithorax and Ash1 prevent transcriptional silencing by Polycomb group proteins. EMBO Rep. 5, 373–377 (2004).

  21. 21.

    Yuan, W. et al. H3K36 methylation antagonizes PRC2-mediated H3K27 methylation. J. Biol. Chem. 286, 7983–7989 (2011).

  22. 22.

    Gaydos, L. J., Rechtsteiner, A., Egelhofer, T. A., Carroll, C. R. & Strome, S. Antagonism between MES-4 and Polycomb repressive complex 2 promotes appropriate gene expression in C. elegans germ cells. Cell Reports 2, 1169–1177 (2012).

  23. 23.

    Hajduskova, M. et al. MRG-1/MRG15 is a barrier for germ cell to neuron reprogramming in Caenorhabditis elegans. Genetics 211, 121–139 (2019).

  24. 24.

    Vielle, A. et al. H4K20me1 contributes to downregulation of X-linked genes for C. elegans dosage compensation. PLoS Genet. 8, e1002933 (2012).

  25. 25.

    Flury, V. et al. The histone acetyltransferase Mst2 protects active chromatin from epigenetic silencing by acetylating the ubiquitin ligase Brl1. Mol. Cell 67, 294–307 (2017).

  26. 26.

    Blobel, G. A. CREB-binding protein and p300: molecular integrators of hematopoietic transcription. Blood 95, 745–755 (2000).

  27. 27.

    Narasimhan, K. et al. Mapping and analysis of Caenorhabditis elegans transcription factor sequence specificities. eLife 4, e06967 (2015).

  28. 28.

    Schaufele, F. et al. CCAAT/enhancer binding protein α assembles essential cooperating factors in common subnuclear domains. Mol. Endocrinol. 15, 1665–1676 (2001).

  29. 29.

    Kovács, K. A., Steinmann, M., Magistretti, P. J., Halfon, O. & Cardinaux, J. R. CCAAT/enhancer-binding protein family members recruit the coactivator CREB-binding protein and trigger its phosphorylation. J. Biol. Chem. 278, 36959–36965 (2003).

  30. 30.

    Reinke, A. W., Baek, J., Ashenberg, O. & Keating, A. E. Networks of bZIP protein-protein interactions diversified over a billion years of evolution. Science 340, 730–734 (2013).

  31. 31.

    Jin, Q. et al. Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation. EMBO J. 30, 249–262 (2011).

  32. 32.

    Lussi, Y. C. et al. Impaired removal of H3K4 methylation affects cell fate determination and gene transcription. Development 143, 3751–3762 (2016).

  33. 33.

    Polansky, H. & Schwab, H. Latent viruses can cause disease by disrupting the competition for the limiting factor p300/CBP. Cell. Mol. Biol. Lett. 23, 56 (2018).

  34. 34.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  35. 35.

    Frøkjaer-Jensen, C. et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40, 1375–1383 (2008).

  36. 36.

    Timmons, L., Court, D. L. & Fire, A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103–112 (2001).

  37. 37.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  38. 38.

    Gómez-Saldivar, G., Meister, P., Askjaer, P. & Dam, I. D. DamID analysis of nuclear organization in Caenorhabditis elegans. Methods Mol. Biol. 1411, 341–358 (2016).

  39. 39.

    Sharma, R., Ritler, D. & Meister, P. Tools for DNA adenine methyltransferase identification analysis of nuclear organization during C. elegans development. Genesis 54, 151–159 (2016).

  40. 40.

    Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

  41. 41.

    Rechtsteiner, A. et al. The histone H3K36 methyltransferase MES-4 acts epigenetically to transmit the memory of germline gene expression to progeny. PLoS Genet. 6, e1001091 (2010).

  42. 42.

    Strome, S. & Wood, W. B. Generation of asymmetry and segregation of germ-line granules in early C. elegans embryos. Cell 35, 15–25 (1983).

  43. 43.

    Au, K. F., Jiang, H., Lin, L., Xing, Y. & Wong, W. H. Detection of splice junctions from paired-end RNA-seq data by SpliceMap. Nucleic Acids Res. 38, 4570–4578 (2010).

  44. 44.

    Szklarczyk, D. et al. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 43, D447–D452 (2015).

  45. 45.

    Therizols, P. et al. Chromatin decondensation is sufficient to alter nuclear organization in embryonic stem cells. Science 346, 1238–1242 (2014).

Download references

Acknowledgements

We thank the Caenorhabditis Genetics Center (CGC) of the NIH Office of Research Infrastructure Programs (P40 OD010440) for strains, S. Strome for the immunofluorescence protocol and H. Kimura for the H3K36me2 antibody. We are grateful to I. Katiç and R. Sack for invaluable technical support. We thank C. Schmid and FMI facilities as well as EMBL GeneCore for advice and support. D.S.C. was supported by an EMBO Long Term Fellowship and a FP7 Marie Curie Action Intra-European Fellowship. J.P. was supported by an EMBO Long Term Fellowship. C.M.-J. and P.A. thank the Spanish Ministry of Economy and Competitiveness for support (BFU2016-79313-P and MDM-2016-0687). S.M.G. thanks the Swiss National Science Foundation and the Novartis Research Foundation for support. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 Research and Innovation programme (Epiherigans - grant agreement No 743312).

Peer review information

Nature thanks Katsuhiko Shirahige and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

D.S.C. performed most experiments, interpreted results, designed the study and wrote the manuscript; C.M.-J. performed DamID experiments and analysis; V.K. contributed to microscopy experiments; D.G. performed bioinformatic analyses; J.P. analysed modENCODE ChIP–seq data; A.S. performed colocalization analysis; P.A. analysed DamID data, interpreted results and provided funding; S.M.G. interpreted results, designed the study, wrote the manuscript and provided funding.

Competing interests

The authors declare no competing interests.

Correspondence to Susan M. Gasser.

Extended data figures and tables

  1. Extended Data Fig. 1 Posterior intestinal cells have impaired perinuclear anchoring capabilities.

    a, Quantification of gwIs4 and gwIs42 reporters distribution in zone 1, as described in Fig. 1b, in compartments of the intestine of wild-type L1 larvae, as indicated. Strains used: GW1056 and GW447. For both reporters, posterior nuclei were compared pairwise to the rest of the intestine by χ2-test. ***P < 0.001. P and n (foci scored per condition) values are listed in Supplementary Table 1. b, Schematic of gwIs42, and cartoon showing that it gains peripheral localization during differentiation6. Focal section of 1 representative wild-type L1 larvae of 15 bearing gwIs4 and EMR-1–mCherry (left), and of 1 of 11 bearing gwIs42 and LMN-1–GFP (right). Insets show intestinal single nuclei as indicated. c, Single focal planes of 1 of 20 representative L1 larvae bearing gwIs4 and EMR-1–mCherry in wild type and mrg-1; cec-4 double mutants. Insets show enlarged single intestinal nucleus of the indicated compartment. Source data

  2. Extended Data Fig. 2 MRG-1 and CEC-4 regulate the spatial distribution of endogenous chromatin in intestine.

    a, Schematic of the single-copy transgenes used to perform intestine-specific emerin (EMR-1) DamID. b, Spearman correlation analysis between biological replicates of sample used in EMR-1- and GFP-DamID experiments. c, Same as in Fig. 1i using three biological replicates (two replicates for mrg-1 single depletion), but also showing the single mrg-1 (RNAi) and cec-4 mutant samples. d, Same as in Fig. 1h using three biological replicates (two replicates for mrg-1 single depletion) but for chromosomes I, II, IV and V. e, Same as d, but for chromosome X. Strains used throughout: GW1377, GW1378, GW1379 and GW1380. Source data

  3. Extended Data Fig. 3 H3K36me2/me3 depletion mimics MRG-1 loss.

    a, Quantification of H3K36 methylation levels in met-1 (GW1183) and mes-4 (RNAi) single- and double-depleted L1 larvae by quantitative mass spectrometry. Data are shown relative to wild-type L1 (N2) on control RNAi bacteria (dotted line, 100%) and as a mean of three biological replicates (shown as dots). b, L1 larvae of the indicated genotype stained for H3K36me2 and DAPI in strains GW554, GW566 and GW637. The image is representative of nine independent larvae. c, gwIs4 reporter distribution in zone 1 in indicated compartments of the intestine of strains GW1056, GW1041, GW1088, GW1133, GW1151 and GW1090 (genotypes indicated). d, Focal section of 1 representative L1 larvae of 18 bearing gwIs4 and EMR-1–mCherry in met-1; cec-4; mes-4 mutants. Insets show single intestinal nucleus of the indicated intestine compartment. e, Reporter distribution in zone 1 in hypoderm and intestine of the indicated genotypes (GW1056 and GW1090). Whole-intestine values reflect results pooled in relation to relative cell-type abundance in the tissue. In c and e, each sample was compared pairwise to wild type by χ2 test. *P < 0.05, **P < 0.01 and ***P < 0.001; P and n (number of foci scored) values are listed in Supplementary Table 1. Source data

  4. Extended Data Fig. 4 MRG-1 mutation causes decompaction of the heterochromatic reporter in embryos.

    a, z-projection of 1 representative 100–250-cell embryo of 10, bearing gwIs4 in wild type, mrg-1, cec-4 and mrg-1; cec-4 mutants. Strains used: GW1047, GW1048, GW1039 and GW1038. b, Single focal planes of representative 100–250-cell embryos bearing gwIs4 and EMR-1–mCherry in wild type, mrg-1, cec-4 and mrg-1; cec-4 mutants. Insets show enlarged single nucleus. Strains as in a. c, Quantification of gwIs4 reporter distribution in zone 1, as described in Fig. 1b, in 100–250-cell embryos of the indicated genotype. Strains as in a. Each sample was compared pairwise to wild type or cec-4 by χ2 test. **P < 0.01 and ***P < 0.001. P and n (number of foci scored) values are shown in Supplementary Table 1. d, Box plots comparing, the indicated genotypes and the circularity of the GFP-tagged gwIs4 reporter The median is shown as a thick line and box limits are 25th and 75th percentiles Strains as in a. Probability values from two-sided Wilcoxon rank-sum tests are indicated: *P < 0.05 and **P < 0.001. n (foci analysed) = 91 (WT), 92 (cec-4), 91 (mrg-1) and 97 (mrg-1; cec-4) from left to right of the indicated genotype. Exact P values are listed in Supplementary Table 2. e, Box plots and n as in d, but comparing the aspect ratio of the GFP-tagged gwIs4 reporter. f, Box plot as in d, comparing, in intestine or hypoderm of L1 of the indicated genotype, GFP–Laci fluorescence intensity deriving from the gwIs4 reporter. Strains used: GW1047, GW1038 and GW1090. Left, n (nuclei analysed) = 86, 94, 42 and 58 (from left to right of the indicated genotype and tissue). Right, n (nuclei analysed) = 99, 99, 53 and 58 (from left to right of the indicated genotype and tissue). Whole-intestine values were pooled in proportion to their relative abundance in the tissue. Two-sided Wilcoxon rank-sum tests; ***P < 0.001. Exact P values are listed in Supplementary Table 2. g, Scatter plot comparing, gene expression in wild-type (N2) L1 larvae (x axis) to enrichment for H3K4me3 and H3K27ac in wild type (y axis) in the three different classes of upregulated genes as identified and colour-coded in Fig. 3c. h, ChIP analysis in cec-4 L1 larvae either under mrg-1 or control RNAi for the enrichment of H3K27me3 over total H3 in the indicated regions. Two-tailed Student’s t-tests; ns, not significant. P values are listed in Supplementary Table 2. Dots represent three biological replicates. Source data

  5. Extended Data Fig. 5 MRG-1 and H3K36me genomic sites correlate with H3K27ac.

    a, Zone 1 gwIs4 reporter distribution in anterior intestine of mrg-1; cec-4 mutant L1 larvae (strain GW1038) with RNAi of control or of the indicated gene. Combined results of two biological replicates are shown. b, Schematic of CBP-1 and CBP-3. c, Mean zone 1 reporter distribution in (strain GW1037) control or RNAi treated cec-4 mutants. Dots represent three biological replicates. Each sample was compared pairwise to control RNAi by χ2 test. P and n (foci scored per condition) values are listed in Supplementary Table 1. d, mRNA expression of endogenous and overexpressed cbp-1 in L1s of the indicated genotypes analysed by RT–qPCR (strains GW1343, GW1345, GW1641, GW1658 and GW1646). The mean of two biological replicates is shown together with dots showing data distribution. e, mRNA expression analysis of the cbp-1 overexpressed allele in L1s of the indicated genotypes by RT–qPCR (strains GW1641, GW1658 and GW1646). The mean of two biological replicates is shown together with dots showing data distribution. f, Scheme of the gwIs4 reporter with relevant features. g, Sequence of the baf-1 promoter present in the gwIs4 heterochromatic reporter. The two C/EBP family motifs are in red. h, STRING44 graph showing experimental evidence of protein interaction between the indicated bZIP transcription factors. i, Correlation analysis between ChIP–seq for the indicated histone mark and MRG-1. H3K27ac is shown in red. Source data

  6. Extended Data Fig. 6 Model of MRG-1 mode of function for chromatin positioning.

    H3K36me2/me3 and MRG-1 mark open euchromatin and promote the retention of the HAT CBP-1. Upon ablation of MRG-1 or H3K36me, CBP-1 is recruited to heterochromatin through C/EBP transcription factors, favouring the detachment of heterochromatin from the INM by counteracting a yet-to-be-characterized anchoring pathway that is induced as cells differentiate. This pathway acts in parallel to and is independent of H3K9me–CEC-4 interaction. Transcriptional activation is not sufficient to cause relocation of perinuclear chromatin, but when it is coupled with chromatin changes in differentiated nuclei, then subnuclear positioning and transcriptional activation do correlate (this work and refs 5,45) for a subset of genes (class 3 in Figs. 3c, 4e). Our work suggests that the sequestration of CBP/p300 in euchromatic compartments by MRG-1, or a complex that requires this factor, is essential to keep it from modifying promoters that bind bZIP transcription factors with affinity for CBP/p300 (C/EBP-like transcription factors, including pioneer factors). The recruitment of CBP/p300 to such promoters within heterochromatin is sufficient to induce a specific chromatin state (correlated with increased H3K27ac) that promotes both transcription and chromatin repositioning. Transcriptional activation per se can occur at the INM, and CEC-4 binding to H3K9me is able to prevent the internalization of the reporter both in embryos and intestinal cells. Compromising the CEC-4–H3K9me pathway reveals the function of MRG-1–CBP-1 in counteracting the differentiation-induced anchoring pathway.

Supplementary information

  1. Supplementary Information

    This file contains Supplementary Tables 1-3.

  2. Reporting Summary

Source data

  1. Source Data Fig. 1

  2. Source Data Fig. 2

  3. Source Data Fig. 3

  4. Source Data Fig. 4

  5. Source Data Extended Data Fig. 1

  6. Source Data Extended Data Fig. 2

  7. Source Data Extended Data Fig. 3

  8. Source Data Extended Data Fig. 4

  9. Source Data Extended Data Fig. 5

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1: MRG-1 functions redundantly with CEC-4–H3K9me to anchor heterochromatin in intestine.
Fig. 2: MRG-1 interaction with euchromatic regions marked by H3K36me2/me3 is required for heterochromatic reporter sequestration.
Fig. 3: Loss of MRG-1 and H3K36me2/me3 derepresses endogenous heterochromatin.
Fig. 4: A CBP-1 gain of function drives heterochromatin delocalization.
Extended Data Fig. 1: Posterior intestinal cells have impaired perinuclear anchoring capabilities.
Extended Data Fig. 2: MRG-1 and CEC-4 regulate the spatial distribution of endogenous chromatin in intestine.
Extended Data Fig. 3: H3K36me2/me3 depletion mimics MRG-1 loss.
Extended Data Fig. 4: MRG-1 mutation causes decompaction of the heterochromatic reporter in embryos.
Extended Data Fig. 5: MRG-1 and H3K36me genomic sites correlate with H3K27ac.
Extended Data Fig. 6: Model of MRG-1 mode of function for chromatin positioning.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.