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Iron homeostasis regulates facultative heterochromatin assembly in adaptive genome control

Nature Structural & Molecular Biology volume 25, pages 372–383 (2018)Cite this article

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Abstract

Iron metabolism is critical for sustaining life and maintaining human health. Here, we find that iron homeostasis is linked to facultative heterochromatin assembly and regulation of gene expression during adaptive genome control. We show that the fission yeast Clr4/Suv39h histone methyltransferase is part of a rheostat-like mechanism in which transcriptional upregulation of mRNAs in response to environmental change provides feedback to prevent their uncontrolled expression through heterochromatin assembly. Interestingly, proper iron homeostasis is required, as iron depletion or downregulation of iron transporters causes defects in heterochromatin assembly and unrestrained upregulation of gene expression. Remarkably, an unbiased genetic screen revealed that restoration of iron homeostasis is sufficient to re-establish facultative heterochromatin and proper gene control genome-wide. These results establish a role for iron homeostasis in facultative heterochromatin assembly and reveal a dynamic mechanism for reprogramming the genome in response to environmental changes.

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Fig. 1: Transcriptome analyses of cells grown at 18 °C.
Fig. 2: New facultative heterochromatin domains form at low temperatures.
Fig. 3: Facultative heterochromatin forms by an RNA-based mechanism at 18 °C.
Fig. 4: Mutation of fep1 or ssn6 restores growth in ccr4∆ cells grown at 18 °C.
Fig. 5: Mutation of fep1 restores gene expression in ccr4∆ cells grown at 18 °C.
Fig. 6: Mutation of fep1 restores heterochromatin assembly in ccr4∆ cells grown at 18 °C.
Fig. 7: Depletion of iron from growth medium abolishes heterochromatin assembly at a low temperature.
Fig. 8: NET-seq reveals enhanced RNAPII transcription in clr4∆ cells.

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References

  1. Berger, S. L. & Sassone-Corsi, P. Metabolic signaling to chromatin. Cold Spring Harb. Perspect. Biol. 8, a019463 (2016).

    Article  PubMed  Google Scholar 

  2. Berry, S. & Dean, C. Environmental perception and epigenetic memory: mechanistic insight through FLC. Plant J. 83, 133–148 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Feng, S., Jacobsen, S. E. & Reik, W. Epigenetic reprogramming in plant and animal development. Science 330, 622–627 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Rando, O. J. & Simmons, R. A. I’m eating for two: parental dietary effects on offspring metabolism. Cell 161, 93–105 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Grewal, S. I. & Elgin, S. C. Transcription and RNA interference in the formation of heterochromatin. Nature 447, 399–406 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 1074–1080 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Trojer, P. & Reinberg, D. Facultative heterochromatin: is there a distinctive molecular signature? Mol. Cell 28, 1–13 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Wang, J., Jia, S. T. & Jia, S. New insights into the regulation of heterochromatin. Trends Genet. 32, 284–294 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Kilchert, C., Wittmann, S. & Vasiljeva, L. The regulation and functions of the nuclear RNA exosome complex. Nat. Rev. Mol. Cell Biol. 17, 227–239 (2016).

    Article  CAS  PubMed  Google Scholar 

  10. Lykke-Andersen, S. & Jensen, T. H. Nonsense-mediated mRNA decay: an intricate machinery that shapes transcriptomes. Nat. Rev. Mol. Cell Biol. 16, 665–677 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. Rinn, J. L. & Chang, H. Y. Genome regulation by long noncoding RNAs. Annu. Rev. Biochem. 81, 145–166 (2012).

    Article  CAS  PubMed  Google Scholar 

  12. Yamamoto, M. The selective elimination of messenger RNA underlies the mitosis-meiosis switch in fission yeast. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 86, 788–797 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Nakayama, J., Rice, J. C., Strahl, B. D., Allis, C. D. & Grewal, S. I. Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292, 110–113 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Zofall, M. et al. RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin formation. Science 335, 96–100 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. Ekwall, K. Genome-wide analysis of HDAC function. Trends Genet. 21, 608–615 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Nicolas, E. et al. Distinct roles of HDAC complexes in promoter silencing, antisense suppression and DNA damage protection. Nat. Struct. Mol. Biol. 14, 372–380 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Lee, N. N. et al. Mtr4-like protein coordinates nuclear RNA processing for heterochromatin assembly and for telomere maintenance. Cell 155, 1061–1074 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sugiyama, T. & Sugioka-Sugiyama, R. Red1 promotes the elimination of meiosis-specific mRNAs in vegetatively growing fission yeast. EMBO J. 30, 1027–1039 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhou, Y. et al. The fission yeast MTREC complex targets CUTs and unspliced pre-mRNAs to the nuclear exosome. Nat. Commun. 6, 7050 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Meola, N. et al. Identification of a nuclear exosome decay pathway for processed transcripts. Mol. Cell 64, 520–533 (2016).

    Article  CAS  PubMed  Google Scholar 

  21. Miller, J. E. & Reese, J. C. Ccr4-Not complex: the control freak of eukaryotic cells. Crit. Rev. Biochem. Mol. Biol. 47, 315–333 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Brönner, C., Salvi, L., Zocco, M., Ugolini, I. & Halic, M. Accumulation of RNA on chromatin disrupts heterochromatic silencing. Genome Res. 27, 1174–1183 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Folco, H. D. et al. Untimely expression of gametogenic genes in vegetative cells causes uniparental disomy. Nature 543, 126–130 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hiriart, E. et al. Mmi1 RNA surveillance machinery directs RNAi complex RITS to specific meiotic genes in fission yeast. EMBO J. 31, 2296–2308 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Sugiyama, T. et al. Enhancer of rudimentary cooperates with conserved RNA-processing factors to promote meiotic mRNA decay and facultative heterochromatin assembly. Mol. Cell 61, 747–759 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yamanaka, S. et al. RNAi triggered by specialized machinery silences developmental genes and retrotransposons. Nature 493, 557–560 (2013).

    Article  CAS  PubMed  Google Scholar 

  27. Volpe, T. A. et al. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833–1837 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Cotobal, C. et al. Role of Ccr4-Not complex in heterochromatin formation at meiotic genes and subtelomeres in fission yeast. Epigenetics Chromatin 8, 28 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Shah, S., Wittmann, S., Kilchert, C. & Vasiljeva, L. lncRNA recruits RNAi and the exosome to dynamically regulate pho1 expression in response to phosphate levels in fission yeast. Genes Dev. 28, 231–244 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Touat-Todeschini, L. et al. Selective termination of lncRNA transcription promotes heterochromatin silencing and cell differentiation. EMBO J. 36, 2626–2641 (2017).

    CAS  PubMed  Google Scholar 

  31. Joh, R. I. et al. Survival in quiescence requires the euchromatic deployment of Clr4/SUV39H by Argonaute-associated small RNAs. Mol. Cell 64, 1088–1101 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hall, I. M. et al. Establishment and maintenance of a heterochromatin domain. Science 297, 2232–2237 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Tashiro, S., Asano, T., Kanoh, J. & Ishikawa, F. Transcription-induced chromatin association of RNA surveillance factors mediates facultative heterochromatin formation in fission yeast. Genes Cells 18, 327–339 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. Chen, D. et al. Global transcriptional responses of fission yeast to environmental stress. Mol. Biol. Cell 14, 214–229 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Chalamcharla, V. R., Folco, H. D., Dhakshnamoorthy, J. & Grewal, S. I. Conserved factor Dhp1/Rat1/Xrn2 triggers premature transcription termination and nucleates heterochromatin to promote gene silencing. Proc. Natl Acad. Sci. USA 112, 15548–15555 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Tucker, J. F. et al. A novel epigenetic silencing pathway involving the highly conserved 5′-3′ exoribonuclease Dhp1/Rat1/Xrn2 in Schizosaccharomyces pombe. PLoS Genet. 12, e1005873 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Labbé, S., Khan, M. G. & Jacques, J. F. Iron uptake and regulation in Schizosaccharomyces pombe. Curr. Opin. Microbiol. 16, 669–676 (2013).

    Article  PubMed  Google Scholar 

  38. Pelletier, B., Beaudoin, J., Mukai, Y. & Labbé, S. Fep1, an iron sensor regulating iron transporter gene expression in Schizosaccharomyces pombe. J. Biol. Chem. 277, 22950–22958 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Pelletier, B., Beaudoin, J., Philpott, C. C. & Labbé, S. Fep1 represses expression of the fission yeast Schizosaccharomyces pombe siderophore-iron transport system. Nucleic Acids Res. 31, 4332–4344 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Encinar del Dedo, J., Gabrielli, N., Carmona, M., Ayté, J. & Hidalgo, E. A cascade of iron-containing proteins governs the genetic iron starvation response to promote iron uptake and inhibit iron storage in fission yeast. PLoS Genet. 11, e1005106 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Fagerström-Billai, F. & Wright, A. P. Functional comparison of the Tup11 and Tup12 transcriptional corepressors in fission yeast. Mol. Cell. Biol. 25, 716–727 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Watson, A. D. et al. Ssn6-Tup1 interacts with class I histone deacetylases required for repression. Genes Dev. 14, 2737–2744 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Znaidi, S., Pelletier, B., Mukai, Y. & Labbé, S. The Schizosaccharomyces pombe corepressor Tup11 interacts with the iron-responsive transcription factor Fep1. J. Biol. Chem. 279, 9462–9474 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Grewal, S. I. & Jia, S. Heterochromatin revisited. Nat. Rev. Genet. 8, 35–46 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Churchman, L. S. & Weissman, J. S. Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368–373 (2011).

    Article  CAS  PubMed  Google Scholar 

  46. Chen, E. S. et al. Cell cycle control of centromeric repeat transcription and heterochromatin assembly. Nature 451, 734–737 (2008).

    Article  CAS  PubMed  Google Scholar 

  47. Cam, H. P. et al. Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome. Nat. Genet. 37, 809–819 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Wagschal, A. et al. Microprocessor, Setx, Xrn2, and Rrp6 co-operate to induce premature termination of transcription by RNAPII. Cell 150, 1147–1157 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Horn, P. J., Bastie, J. N. & Peterson, C. L. A. A Rik1-associated, cullin-dependent E3 ubiquitin ligase is essential for heterochromatin formation. Genes Dev. 19, 1705–1714 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Jia, S., Kobayashi, R. & Grewal, S. I. Ubiquitin ligase component Cul4 associates with Clr4 histone methyltransferase to assemble heterochromatin. Nat. Cell Biol. 7, 1007–1013 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. Hong, E. J., Villén, J., Gerace, E. L., Gygi, S. P. & Moazed, D. A cullin E3 ubiquitin ligase complex associates with Rik1 and the Clr4 histone H3-K9 methyltransferase and is required for RNAi-mediated heterochromatin formation. RNA Biol. 2, 106–111 (2005).

    Article  CAS  PubMed  Google Scholar 

  52. Greil, F. et al. Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev. 17, 2825–2838 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Piacentini, L., Fanti, L., Berloco, M., Perrini, B. & Pimpinelli, S. Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin. J. Cell Biol. 161, 707–714 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Vakoc, C. R., Mandat, S. A., Olenchock, B. A. & Blobel, G. A. Histone H3 lysine 9 methylation and HP1gamma are associated with transcription elongation through mammalian chromatin. Mol. Cell 19, 381–391 (2005).

    Article  CAS  PubMed  Google Scholar 

  55. Tsukada, Y. et al. Histone demethylation by a family of JmjC domain-containing proteins. Nature 439, 811–816 (2006).

    Article  CAS  PubMed  Google Scholar 

  56. Kimura, S. & Suzuki, T. Iron-sulfur proteins responsible for RNA modifications. Biochim. Biophys. Acta 1853, 1272–1283 (2015).

    Article  CAS  PubMed  Google Scholar 

  57. Roignant, J. Y. & Soller, M. m(6)A in mRNA: an ancient mechanism for fine-tuning gene expression. Trends Genet. 33, 380–390 (2017).

    Article  CAS  PubMed  Google Scholar 

  58. Andrews, N. C. Disorders of iron metabolism. N. Engl. J. Med. 341, 1986–1995 (1999).

    Article  CAS  PubMed  Google Scholar 

  59. Hentze, M. W., Muckenthaler, M. U., Galy, B. & Camaschella, C. Two to tango: regulation of Mammalian iron metabolism. Cell 142, 24–38 (2010).

    Article  CAS  PubMed  Google Scholar 

  60. Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).

    Article  CAS  PubMed  Google Scholar 

  61. Wood, V. et al. The genome sequence of Schizosaccharomyces pombe. Nature 415, 871–880 (2002).

    Article  CAS  PubMed  Google Scholar 

  62. Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Huber, W. et al. Orchestrating high-throughput genomic analysis with Bioconductor. Nat. Methods 12, 115–121 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. R Development Core Team. A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, Vienna, Austria, 2016).

    Google Scholar 

  66. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995).

    Google Scholar 

  67. Gu, Z., Eils, R. & Schlesner, M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32, 2847–2849 (2016).

    Article  CAS  PubMed  Google Scholar 

  68. Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag, New York, 2009).

    Book  Google Scholar 

  69. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Li, H. et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  71. Danecek, P. et al. The variant call format and VCFtools. Bioinformatics 27, 2156–2158 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly (Austin) 6, 80–92 (2012).

    Article  CAS  Google Scholar 

  73. Pierik, A. J., Netz, D. J. & Lill, R. Analysis of iron-sulfur protein maturation in eukaryotes. Nat. Protoc. 4, 753–766 (2009).

    Article  CAS  PubMed  Google Scholar 

  74. Churchman, L. S. & Weissman, J. S. Native elongating transcript sequencing (NET-seq). Curr. Protoc. Mol. Biol. 98, 14.4 (2012).

    Google Scholar 

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Acknowledgements

We thank E. Hidalgo (Universitat Pompeu Fabra, Spain) for yeast strains, J. Zhu, V. Bliskovsky and S. Shema for valuable technical advice, S. Holla for helpful contributions, J. Barrowman for editing the manuscript, and members of the Laboratory of Biochemistry and Molecular Biology, in particular the Grewal laboratory, for discussions. This study used the Helix Systems and Biowulf Linux cluster at the National Institutes of Health. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.

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  1. Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Pamela S. Gallagher, Madeline Larkin, Gobi Thillainadesan, Jothy Dhakshnamoorthy, Vanivilasini Balachandran, Hua Xiao, Christopher Wellman, David Wheeler & Shiv I. S. Grewal

  2. Human Genetics Unit, Indian Statistical Institute, Kolkata, India

    Raghunath Chatterjee

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S.I.S.G. and P.S.G. conceived and supervised the project. P.S.G., M.L., J.D., V.B., H.X., and C.W. performed experiments and analyzed data. R.C., G.T., and D.W. performed bioinformatics analyses of genomic datasets. S.I.S.G. and P.S.G. wrote the manuscript.

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Supplementary Figure 1 Current model showing the roles of RNA processing factors in the degradation of transcripts and facultative heterochromatin assembly.

RNA binding proteins engage a network of nuclear RNA processing factors such as MTREC and CCR4-NOT, which in turn act along with the termination factor Dhp1/Xrn2 to promote degradation of RNAs by the exosome and/or RNAi machinery. In addition to RNA degradation, these factors are believed to mediate recruitment of histone H3 lysine 9 methyltransferase Clr4 to promote the assembly of facultative heterochromatin islands.

Supplementary Figure 2 Loci showing expression changes and formation of new heterochromatin islands in wild-type cells grown at 18 °C are enriched in stress response genes.

(Left) Histogram showing the numbers of genes in broad categories with increased expression (fold change value ≥ 1 and P value < 0.05) or decreased expression (fold change value ≤ 1 and P value < 0.05) in wild-type cells grown at 18 °C relative to wild-type cells grown at 30 °C as determined by RNA-seq (n = 2 independent experiments per condition). Genes with increased expression are shown in red and genes with decreased expression are shown in blue. Statistical significance (right-hand, single-tailed P value of binomial distribution, n=6380 transcripts) of the number of genes with increased expression is listed on the right. (Right) List of categories for genes within heterochromatin islands in wild-type cells grown at 18 °C as determined by ChIP-chip. Statistical significance (P value) of the number of genes within 18 °C heterochromatin islands is listed for each category.

Supplementary Figure 3 Clr6 histone deacetylase complex regulates expression of iron transporter genes.

(a) Area proportional Venn diagrams representing the numbers of genes with increased expression (fold change value ≥ 1 and P value < 0.05) in clr6-1 mutant cells grown at 30 °C and wild-type cells grown at 18 °C (top) or clr6-1 cells grown at 30 °C and clr4Δ cells grown at 18 °C (bottom). Statistical significance (right-hand, single-tailed P value of binomial distribution, n=6380 transcripts) of the overlap between the two groups is shown above. (b) Expression levels of six representative iron transporter genes determined by RNA-seq are shown in wild-type and clr6-1 cells grown at 30 °C. (c) Pst1-HA and Clr6-HA relative enrichment in cells grown at 30 °C was determined by ChIP-chip and is shown for iron transporter genes.

Supplementary Figure 4 Clr4 represses expression of meiotic genes at low temperature.

(a) Expression levels of four representative meiotic genes determined by RNA-seq are shown in wild-type and clr4Δ cells grown at 30 °C or 18 °C. (b) Wild-type and clr4Δ cells were grown at 18 °C in YEA rich medium and optical densities (ODs) of the cultures are shown as the mean ±s.d. (n = 2 independent experiments).

Supplementary Figure 5 RNAi is dispensable for the assembly of heterochromatin islands at 18 °C.

(a) Genome-wide H3K9me distribution at 18 °C in wild-type, ago1Δ, and ccr4Δ cells was determined by ChIP-chip and is plotted on all three chromosomes of S. pombe. New 18 °C facultative heterochromatin peaks are indicated. Wild-type ChIP-chip data is also presented in Fig. 2a and ccr4Δ ChIP-chip data is also presented in Fig. 6a. (b) H3K9me enrichments at 18 °C in wild-type and ago1Δ cells were determined by ChIP-qPCR. H3K9me fold enrichments at SPAPB1A11.02, SPBC428.10, and SPBC1289.14 relative to the control leu1 gene are shown as the mean +s.d. (n = 3 independent experiments). (c) H3K9me enrichments at 18 °C in wild-type, ccr4Δ, and red1Δ cells were determined by ChIP-qPCR. H3K9me fold enrichments at SPAPB1A11.02, SPBC428.10, and SPBC1289.14 relative to the control leu1 gene are shown as the mean +s.d. (n = 3 independent experiments). Wild-type ChIP-qPCR data is also presented in Supplementary Fig. 5b.

Supplementary Figure 6 Fep1 colocalizes with Ssn6 and Clr6 at various loci, including iron-transporter gene promoters, and its loss restores iron homeostasis in ccr4Δ cells.

(a) ChIP-chip analyses of Fep1, Ssn6 and Clr6 histone deacetylase complex-I subunit Pst1. Fep1-GFP, Ssn6-Myc, and Pst1-HA relative enrichment in cells grown at 30 °C. Iron transporter loci and additional peaks are indicated. (b) Fep1-GFP, Ssn6-Myc, and Pst1-HA relative enrichments are shown for representative iron transporter genes. (c) Mutation in fep1 in ccr4Δ cells causes up-regulation of iron transporter genes that are otherwise defective in their expression at 18 °C. Expression levels of frp1, str1, and str3 relative to act1 in the indicated strains grown at 18 °C were determined by RT-qPCR and are shown as the mean +s.d. (n = 2 independent experiments). Expression levels are normalized to wild-type cells grown at 30 °C. (d) Iron-55 uptake in wild-type, ccr4Δ, and ccr4Δ fep1Δ cells grown at 18 °C. (e) Mutation in ssn6 restores facultative heterochromatin assembly in ccr4Δ cells grown at low temperature. H3K9me enrichments at 18 °C in wild-type, ccr4Δ, and ccr4Δ ssn6-1 cells were determined by ChIP-qPCR. H3K9me fold enrichments at indicated loci relative to the control leu1 gene are shown as the mean +s.d. (n = 3 independent experiments).

Supplementary Figure 7 Iron affects gene regulation and the assembly of facultative heterochromatin islands at low temperature.

(a) Heat map of fold change values relative to wild-type 30 °C cells for up-regulated transcripts in indicated cultures grown at 18 °C. Clusters are grouped according to the analysis presented in Fig. 1f. (b) H3K9me enrichments in wild-type cells untreated or treated with 250µM Dip and grown at 18 °C for 72 hours (upper) or 24 hours (lower). H3K9me fold enrichments at indicated loci were determined by ChIP-qPCR and are shown relative to the control leu1 gene. Enrichments for the 72 hour treatments are shown as the mean +s.d. (n = 3 independent experiments). (c) H3K9me enrichments at 18 °C in ccr4Δ fep1-1 cells untreated or treated with 250µM Dip were determined by ChIP-qPCR. H3K9me fold enrichments at indicated loci are shown relative to the control leu1 gene.

Supplementary Figure 8 Iron depletion causes defects in RNA processing similar to those observed in the mtl1 mutant cells.

(a) Schematic of cryptic introns detected using RNA-seq in wild-type cells untreated or treated with 250µM Dip, and mtl1-1 mutant cells grown at 30 °C. The arcs below or above the line represent intron junction reads that map to the bottom or top DNA strands. (b) Hierarchical clustering of the indicated strains based on Pearson's correlation coefficients determined using RNA-seq (log2 fold change versus wild-type cells grown at 30 °C). Pairwise comparisons were performed (n = 6380 transcripts per comparison) and Pearson's correlation coefficients were converted into a color gradient. RNA-seq data for Dip-treated wild-type and clr4Δ cells grown at 18 °C and 30 °C were compared to various RNA processing mutants such as RNAi components (ago1Δ and dcr1Δ), nuclear RNA elimination factors (mmi1Δ, erh1Δ, red1Δ and mtl1-1), and CCR4-NOT complex (ccr4Δ) cultured at 30 °C. cwf10-1 splicing factor mutant was cultured at 26 °C. Also included is Clr6 HDAC mutant (clr6-1) grown at 30 °C that shows de-repression of genes affected by clr4Δ at 18 °C. Notice the high correlation between RNA processing mutant mtl1-1 and cells depleted for iron or lacking Clr4. (c) Density plots comparing transcripts (n = 6380) in mtl1-1 cells grown at 30 °C and wild-type cells treated with 250 µM Dip and grown at 30 °C (upper) or 18 °C (lower). Pearson's correlation coefficients (r) and the P values of the linear regressions are indicated. Source Data for Supplementary Fig. 8a are available with the paper online.

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Supplementary Figure 9 Iron homeostasis is important for epigenetic genome control.

(Upper) Model showing regulation of iron transporter genes by Fep1, Ssn6-Tup11/12 and Clr6 histone deacetylase complex. In the presence of Fep1, Ssn6-Tup11/12 and Clr6 complex localize to iron transporter loci and regulate gene expression (left panel). In fep1 mutant cells, Ssn6 and likely Clr6 complex are de-localized, allowing for increased expression of iron transporter genes (right panel). The thickness of the arrows indicates expression levels of iron transporter genes. In addition to iron transporters, Clr6 regulates other loci at 30 °C that are regulated by Clr4 at 18 °C. (Lower) Model showing the requirement for intracellular iron in facultative heterochromatin assembly and proper gene regulation in adaptive genome control. In cells grown under suboptimal growth conditions, Clr4 is recruited to genomic locations in a transcription- and RNA-dependent mechanism involving RNA processing and termination factors, such as cleavage factors (CF) and Dhp1/Xrn2, for facultative heterochromatin assembly and serves as a rheostat to prevent hyper-elevation of transcripts (right panel). Cells depleted of intracellular iron show defects in facultative heterochromatin assembly and display aberrant gene expression in response to changing environmental growth conditions (left panel).

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List of strains and oligos used in this study

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Gallagher, P.S., Larkin, M., Thillainadesan, G. et al. Iron homeostasis regulates facultative heterochromatin assembly in adaptive genome control. Nat Struct Mol Biol 25, 372–383 (2018). https://doi.org/10.1038/s41594-018-0056-2

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