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Architectural RNA is required for heterochromatin organization

Jitendra Thakur, He Fang, Trizia Llagas, Christine M. Disteche, Steven Henikoff

Preprint posted on September 27, 2019 https://www.biorxiv.org/content/10.1101/784835v1

Cut the anchor: CUT&RUN.RNase identifies scaffolding of heterochromatic domains by RNA

Selected by Ramona Jühlen

Background and CUT&RUN.RNase methodology

In eukaryotes, genomic DNA can be found in two main chromatin states. Heterochromatin is highly condensed chromatin that is characterized by gene-poor DNA and repressive histone modifications. Euchromatin is decondensed chromatin that is found to assemble on gene-rich DNA together with RNA polymerase II and is marked with active histone modifications.

Several chromatin modifiers have been identified that orchestrate heterochromatin formation, and interestingly, RNA appears to be a crucial regulator for the heterochromatic state. For example, RNA has been shown to indirectly regulate chromatin organisation by recruiting chromatin modifiers, as in the case of the lncRNA Xist during X-chromosome inactivation. However, to what extent RNA directly influences chromatin organisation is not clear.

Since the resolution of current approaches, such as chromosome conformation capture (Hi-C) assays, is too low for assessing the effects of RNA on chromatin organisation, the authors altered their high-resolution chromatin profiling technique, termed Cleavage Under Targets and Release Using Nuclease (CUT&RUN (1)) with RNaseA treatment (CUT&RUN.RNase). During CUT&RUN antibody-targeted chromatin cleavage releases specific protein-DNA fragments that can be further processed for DNA sequencing. CUT&RUN is performed in situ and thus, preserves the local chromatin environment, thereby surpassing limitations in methods like chromatin immunoprecipitation (ChIP) which require fragmentation of the DNA. By digesting total RNA before the antibody-binding step in CUT&RUN the authors were able to investigate chromatin structure after depletion of RNA (Figure 1).

Figure 1. Schematic representation of the different steps in the CUT&RUN.RNase approach. First, total RNA of intact permeabilised cells is removed by the digestion of RNaseA. Micrococcal nuclease fused to protein-A is targeted to chromatin by an antibody. Upon addition of Ca2+ ions DNA digestion is initiated at the loci of interest and chromatin fragments can be analysed by sequencing. By digesting total RNA of permeabilised cells one can observe the effect of RNA on chromatin structure (modified from preprint).

Key findings

The authors first applied the CUT&RUN.RNase methodology to chromatin surrounding the nucleolus. The nucleolus is rich in nuclear RNA; in particular, the localisation of nucleophosmin (NPM1) to the nucleolus depends on interaction of NPM1 with RNA (2) and depletion of NPM1 results in altered organisation of perinucleolar heterochromatin (3) which consists of major satellite DNA (4). Now, applying CUT&RUN.RNase targeted by NPM1, the authors demonstrated that degradation of RNA leads to depletion of NPM1 on major satellite DNA, whereas formaldehyde cross-linking of the cells before RNA degradation could restore the NPM1 signal. The authors verified their findings also in immunostainings after RNA degradation showing a loss of NPM1 localisation to the nucleolus and redistribution in the whole nucleoplasm. These results confirm previous findings that interaction of NPM1 with RNA indeed targets NPM1 to the nucleolus and perinucleolar heterochromatin.

Further, the authors used CUT&RUN.RNase to assess whether RNA preserves the structural organisation of heterochromatin and euchromatin by targeting repressive (H3K27me3 and H3K9me3, marking heterochromatin) and active (H3K4me3, marking euchromatin) histone modifications. It turned out that RNA depletion lead to a significant loss of repressive histone marks, whereas active histone marks only changed slightly. This suggests that RNA preserves the structural integrity of heterochromatin. Moreover, by using RNase H, which targets RNA in RNA-DNA hybrids, instead of RNase A, which also targets single-stranded and double-stranded RNA, the authors show that RNA-DNA hybrids are minor factors in preserving heterochromatic domains.

So far, the authors applied CUT&RUN.RNase by globally depleting RNA. In order to verify the specificity of RNA preserving heterochromatic structural organisation, they depleted cells for a single lncRNA, termed Firre, which is important for maintaining H3K27me3 marks in X-chromosome inactivation and regulates the expression of some genes involved in RNA processing (5, 6). Cells were depleted for Firre using CRISPR/Cas9, and CUT&RUN was conducted by profiling H3K27me3 and H3K4me3 chromatin marks. Using Firre-depleted cells coupled to CUT&RUN the authors demonstrated that Firre regulates a specific subset of H3K27me3 chromatin marks by comparing these results to CUT&RUN.RNase in wild-type cells. The authors conclude that their observed CUT&RUN.RNase results by globally depleting RNA can be mainly explained by the interaction of various lncRNAs with heterochromatin.

Open questions

How could CUT&RUN.RNase be used to resolve mitotic chromatin compaction in the absence of RNA? It has been found that mitotic chromosomes are only 53–70 % chromatin (7), and it is still to be elucidated how histone post-translational modifications contribute to mitotic chromatin compaction.

Other CUT&RUN approaches

J. Thakur, S. Henikoff, Genes Dev. 32, 20–25 (2018).

D. H. Janssens et al., Epigenetics Chromatin. 11, 74 (2018).

M. P. Meers, T. D. Bryson, J. G. Henikoff, S. Henikoff, Elife. 8 (2019).

References

1. P. J. Skene, S. Henikoff, Elife. 6 (2017).

2. D. M. Mitrea et al., Elife. 5 (2016).

3. K. Holmberg Olausson, M. Nistér, M. S. Lindström, J. Biol. Chem. 289, 34601–34619 (2014).

4. G. Almouzni, A. V. Probst, Nucleus. 2, 332–338 (2011).

5. J. H. Bergmann et al., Genome Res. 25, 1336–1346 (2015).

6. F. Yang et al., Genome Biol. 16, 52 (2015).

7. D. G. Booth et al., Molecular Cell. 64, 790–802 (2016).

Tags: chromatin, cut&run, heterochromatin, histone modification, rna

Posted on: 5th December 2019 , updated on: 6th December 2019

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  • Author's response

    Jitendra Thakur and Steven Henikoff shared

    Thank you for this interesting suggestion. The reduction in volume of mitotic chromosomes results from condensin-mediated topological loop re-organization and cohesin-independent local compaction of the chromatin fiber. Deacetylation of histone tails has been suggested to promote mitotic chromatin condensation by increasing electrostatic interactions between neighboring nucleosomes (1). However, the relative contribution of histone post-translational modifications to overall mitotic chromatin compaction remains unclear. The nucleolus disintegrates in prophase and releases RNA and the proliferation marker protein Ki-67 to form a dense layer on the surface of mitotic chromosomes. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes (2). It remains to be determined if the RNA component of the dense layer contributes to the structural integrity of mitotic chromosomes. Given our findings of a major structural role of RNA in heterochromatin compaction, it is likely that the RNA associated with mitotic chromosomes contributes to the compaction of mitotic chromatin. Since RNA covers the entire surface of a chromosome, we expect RNA-mediated compaction of the mitotic chromatin to be uniform throughout the chromosome unlike the preferential stabilization of the heterochromatic domains during interphase. If RNA indeed compacts mitotic chromosomes, CUT&RUN.RNase should be able to detect the decrease of signals for both active and repressive histone modifications throughout the genome in mitotic cells.

    Jitendra and Steve

    1. B. J. Wilkins et al., Science 343, (2014).

    2. S. Cuylen et al., Nature 535, (2016).

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