RNAi-dependent H3K27 methylation is required for heterochromatin formation and DNA elimination in Tetrahymena

ABSTRACTExtensive DNA elimination occurs as part of macronuclear differentiation duringTetrahymenasexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway.LIA4knockout (ΔLIA4) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, withoutLIA4, these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus duringTetrahymenamacronuclear differentiation.

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Faculty Opinions recommendation of RNAi-dependent H3K27 methylation is required for heterochromatin formation and DNA elimination in Tetrahymena.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1087505.540557 ◽

2007 ◽

Author(s):

Maarten van Lohuizen

Keyword(s):

Heterochromatin Formation ◽

H3k27 Methylation ◽

Dna Elimination

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Tetrahymena thermophila JMJD3 Homolog Regulates H3K27 Methylation and Nuclear Differentiation

Eukaryotic Cell ◽

10.1128/ec.05290-11 ◽

2012 ◽

Vol 11 (5) ◽

pp. 601-614 ◽

Cited By ~ 16

Author(s):

Pei-Han Chung ◽

Meng-Chao Yao

Keyword(s):

Gene Expression ◽

Tetrahymena Thermophila ◽

Critical Role ◽

Content Type ◽

Chromatin Dynamics ◽

H3k27 Methylation ◽

Dna Elimination ◽

Severe Reduction ◽

Nuclear Development ◽

Important Regulator

ABSTRACT Histone H3K27me3 modification is an important regulator for development and gene expression. In Tetrahymena thermophila , the complex chromatin dynamics of H3K27me3 marks during nuclear development suggested that an H3K27me3 demethylase might exist. Here, we report an H3K27me3 demethylase homolog, JMJ1 , in Tetrahymena . During conjugation, JMJ1 expression is upregulated and the protein is localized first in the parental macronucleus and then in the new macronucleus. In conjugating cells, knockdown of JMJ1 expression resulted in a severe reduction in the production of progeny, suggesting that JMJ1 is essential for Tetrahymena conjugation. Furthermore, knockdown of JMJ1 resulted in increased H3K27 trimethylation in the new macronucleus and reduced transcription of genes related to DNA elimination, while the DNA elimination process was also partially blocked. Knockdown of the H3K27 methyltransferase EZL2 but not that of EZL1 partially restored progeny production in JMJ1 -knockdown cells and reduced abnormal H3K27me3 accumulation in the new macronucleus. Taken together, these results demonstrate a critical role for JMJ1 in regulating H3K27me3 during conjugation and the importance of JMJ1 in regulating gene expression in the new macronucleus but not in regulating the formation of heterochromatin associated with programmed DNA deletion.

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Telomere repeats induce domains of H3K27 methylation in Neurospora

10.1101/189258 ◽

2017 ◽

Author(s):

Kirsty Jamieson ◽

Kevin J. McNaught ◽

Tereza Ormsby ◽

Neena A. Leggett ◽

Shinji Honda ◽

...

Keyword(s):

Genome Organization ◽

Chromosomal Rearrangements ◽

Histone H3 ◽

Facultative Heterochromatin ◽

Large Domain ◽

Heterochromatin Formation ◽

H3k27 Methylation ◽

And Control ◽

The Relationship ◽

Insight Into

ABSTRACTDevelopment in higher organisms requires selective gene silencing, directed in part by di-/tri-methylation of lysine 27 on histone H3 (H3K27me2/3). Knowledge of the cues that control formation of such repressive Polycomb domains is extremely limited. We exploited natural and engineered chromosomal rearrangements in the fungus Neurospora crassa to elucidate the control of H3K27me2/3. Analyses of H3K27me2/3 in strains bearing chromosomal rearrangements revealed both position-dependent and position-independent facultative heterochromatin. We found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive, subtelomeric H3K27me2/3. We ascertained that such telomere-proximal facultative heterochromatin requires native telomere repeats and found that a short array of ectopic telomere repeats, (TTAGGG)17, can induce a large domain (∼225 kb) of H3K27me2/3. This provides an example of a cis-acting sequence that directs H3K27 methylation. Our findings provide new insight into the relationship between genome organization and control of heterochromatin formation.

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snRNA and Heterochromatin Formation Are Involved in DNA Excision during Macronuclear Development in Stichotrichous Ciliates

Eukaryotic Cell ◽

10.1128/ec.4.11.1934-1941.2005 ◽

2005 ◽

Vol 4 (11) ◽

pp. 1934-1941 ◽

Cited By ~ 33

Author(s):

Stefan A. Juranek ◽

Sina Rupprecht ◽

Jan Postberg ◽

Hans J. Lipps

Keyword(s):

Dna Sequences ◽

Chromatin Immunoprecipitation ◽

De Novo ◽

Histone H3 ◽

Heterochromatin Formation ◽

Macronuclear Development ◽

Dna Elimination ◽

Histone H3 Variant ◽

Specific Sequences

ABSTRACT Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant “X” is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates.

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Two GW Repeat Proteins Interact with Tetrahymena thermophila Argonaute and Promote Genome Rearrangement

Molecular and Cellular Biology ◽

10.1128/mcb.00076-09 ◽

2009 ◽

Vol 29 (18) ◽

pp. 5020-5030 ◽

Cited By ~ 37

Author(s):

Janna Bednenko ◽

Tomoko Noto ◽

Leroi V. DeSouza ◽

K. W. Michael Siu ◽

Ronald E. Pearlman ◽

...

Keyword(s):

Genome Rearrangement ◽

Developmental Stages ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Nuclear Bodies ◽

Interacting Proteins ◽

Heterochromatin Formation ◽

Dna Elimination ◽

Somatic Genome ◽

Argonaute Family

ABSTRACT In conjugating Tetrahymena thermophila, massive DNA elimination occurs upon the development of the new somatic genome from the germ line genome. Small, ∼28-nucleotide scan RNAs (scnRNAs) and Twi1p, an Argonaute family member, mediate H3K27me3 and H3K9me3 histone H3 modifications, which lead to heterochromatin formation and the excision of the heterochromatinized germ line-limited sequences. In our search for new factors involved in developmental DNA rearrangement, we identified two Twi1p-interacting proteins, Wag1p and CnjBp. Both proteins contain GW (glycine and tryptophan) repeats, which are characteristic of several Argonaute-interacting proteins in other organisms. Wag1p and CnjBp colocalize with Twi1p in the parental macronucleus early in conjugation and in the new developing macronucleus during later developmental stages. Around the time DNA elimination occurs, Wag1p forms multiple nuclear bodies in the developing macronuclei that do not colocalize with heterochromatic DNA elimination structures. Analyses of ΔWAG1, ΔCnjB, and double ΔWAG1 ΔCnjB knockout strains revealed that WAG1 and CnjB genes need to be deleted together to inhibit the downregulation of specific scnRNAs, the formation of DNA elimination structures, and DNA excision. Thus, Wag1p and CnjBp are two novel players with overlapping functions in RNA interference-mediated genome rearrangement in Tetrahymena.

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RNA-mediated nucleosome depletion is required for elimination of transposon-derived DNA.

10.1101/2022.01.04.474918 ◽

2022 ◽

Author(s):

Aditi Singh ◽

Xyrus X. Maurer-Alcalá ◽

Therese Solberg ◽

Silvan Gisler ◽

Michael Ignarski ◽

...

Keyword(s):

Transposable Elements ◽

Chromatin Remodeling ◽

Dna Cleavage ◽

Small Rnas ◽

Heterochromatin Formation ◽

Dna Accessibility ◽

Dna Elimination ◽

Nucleosome Density ◽

Somatic Genome ◽

Genomic Regions

Small RNAs are known to mediate silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. Post-zygotic development of the Paramecium somatic genome requires elimination of thousands of transposon remnants (IESs) and transposable elements that are scattered throughout the germline genome (Garnier et al. 2004). The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries (Bouhouche et al. 2011; Furrer et al. 2017). Previous research suggests that small RNAs induce heterochromatin formation within IESs, which, in turn, is required for DNA elimination (Liu et al. 2007). Here we show that IES recognition and precise excision is facilitated by recruitment of a homolog of a chromatin remodeler ISWI, which depletes target genomic regions of nucleosomes, making the chromatin accessible for DNA cleavage. ISWI knockdown in Paramecium leads to pronounced inhibition of DNA elimination. Furthermore, nucleosome profiling indicates that ISWI is required for IES elimination in nucleosome-dense genomic regions, while other IESs do not require small RNAs or ISWI for excision. ISWI silencing notably also reduces DNA elimination precision, resulting in aberrant excision at alternative IES boundaries. In summary, we demonstrate that chromatin remodeling that increases DNA accessibility together with small RNAs are necessary for efficient and precise DNA elimination in Paramecium.

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Telomere repeats induce domains of H3K27 methylation in Neurospora

eLife ◽

10.7554/elife.31216 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 14

Author(s):

Kirsty Jamieson ◽

Kevin J McNaught ◽

Tereza Ormsby ◽

Neena A Leggett ◽

Shinji Honda ◽

...

Keyword(s):

Genome Organization ◽

Chromosomal Rearrangements ◽

Histone H3 ◽

Facultative Heterochromatin ◽

Heterochromatin Formation ◽

Cis Acting ◽

H3k27 Methylation ◽

And Control ◽

The Relationship ◽

Insight Into

Development in higher organisms requires selective gene silencing, directed in part by di-/trimethylation of lysine 27 on histone H3 (H3K27me2/3). Knowledge of the cues that control formation of such repressive Polycomb domains is extremely limited. We exploited natural and engineered chromosomal rearrangements in the fungus Neurospora crassa to elucidate the control of H3K27me2/3. Analyses of H3K27me2/3 in strains bearing chromosomal rearrangements revealed both position-dependent and position-independent facultative heterochromatin. We found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive, subtelomeric H3K27me2/3. We ascertained that such telomere-proximal facultative heterochromatin requires native telomere repeats and found that a short array of ectopic telomere repeats, (TTAGGG)17, can induce a large domain (~225 kb) of H3K27me2/3. This provides an example of a cis-acting sequence that directs H3K27 methylation. Our findings provide new insight into the relationship between genome organization and control of heterochromatin formation.

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