Epigenetic rewriting at centromeric DNA repeats leads to increased chromatin accessibility and chromosomal instability
Posted on: 18 April 2021 , updated on: 21 April 2021
Preprint posted on 23 February 2021
Article now published in Epigenetics & Chromatin at http://dx.doi.org/10.1186/s13072-021-00410-x
Categories: cell biology, molecular biology
Context1-5
Centromeres consist of tandem repeated DNA sequences and enable chromosome segregation during mitosis and meiosis. Centromeres are a classic example of epigenetically defined genomic loci that contain histone H3 variant CENP-A nucleosomes. While the core centromeric DNA is wrapped by CENP-A containing nucleosomes interspaced by histone H3 containing nucleosomes, the pericentromeric DNA is wrapped by histone H3 nucleosomes (fig.1).
The core centromeric region seems to be transcriptionally permissive and the pericentromeric region is transcriptionally repressed. This results in differential histone post-translational modifications at the core and pericentromeric regions. Also, post-translational modifications of histone H3 support centromere transcription, integrity, and function. For example, euchromatin histone H3 marks (e.g., H3K36me2/3, H3K9ac) were detected at the core centromeric region. On the other hand, heterochromatin mark histone 3 lysine 9 trimethylation (H3K9me3) is present at the pericentromeric region; removing or preventing the deposition of the H3K9me3 mark induces centromere DNA instability and aneuploidy. However, modifying histone marks (here H3K9me3) specifically at the centromeres without disturbing genome-wide patterns, remains a challenge. Thus, in the current preprint, the authors investigated the impact of H3K9me3 specifically at an endogenous centromere using a targeted histone lysine demethylation.
Experimental system
The authors fused a histone lysine demethylase (hJMJD2B) to the DNA binding domain of a Transcription activator-like effector (TALE6) directed to bind α-satellite repeats present in the centromeric region of human chromosome 7 (or DZ71 array). As controls, they used a catalytically inactive mutant (hJMJD2B-H188A), and a GFP-tagged TALE. They expressed these constructs in U2OS cells either transiently or under a doxycycline-inducible system for 24-48 hrs.
Key findings
- First, they found four hJMJD2B- or H188A- TALE foci in U2OS cells co-stained with CENP-A antibody or DZ71 FISH probe (fluorescence in situ hybridization) as U2OS are mostly tetraploids (fig.2). Thus, they reasoned that both wild-type (hJMJD2B-TALE) and mutant (H188A-TALE) histone lysine demethylase bind to the pericentromeric regions of chromosome 7.
Fig.2: U2OS cells expressing either the TALE-demethylase (top), its point mutant (middle), or the TALE-GFP (bottom). Sketch of the different constructs of TALE fusion proteins used in this study. - Then they found reduced H3K9me3 staining specifically at the DZ71 array in cells expressing the hJMJD2B-TALE but not the H188A-TALE (fig.3a). Additionally, they demonstrated a concomitant increase in H3K9me2 and H3K9me1 staining at the DZ71 array associated with the loss of H3K9me3. Also, they suggest an increase in chromatin accessibility at the DZ71 array followed by hJMJD2B-TALE
- They further report induction of chromosome segregation defects in hJMJD2B-TALE expressing cells as scored by the change in ploidy of chromosome 7 but not of a control chromosome (Chr. 11). Also, they demonstrate reduction of chromosome passenger complex (CPC7) proteins (e.g., INCENP, Aurora B kinase) and heterochromatin protein 1 (HP1α) in the vicinity of hJMJD2B-TALE foci (fig.3b). Thus, they suggest that suboptimal levels of H3K9me3 at the centromere of chromosome 7 specifically induce chromosome segregation defects of that chromosome.
Fig. 3: U2OS cells expressing the TALE-demethylase (top) or the catalytically inactive mutant (bottom). DNA is visualized using Hoechst, the TALE (shown in green), H3K9me3 (a), and Aurora B (b).
Conclusion and perspective
To manoeuvre epigenetic information – histone post-translational modifications and DNA/RNA methylation – specifically at endogenous centromeres without influencing genome-wide gene expression is challenging. The current preprint tries to address this by using a customized genetically engineered histone modifier that can modulate the histone H3 heterochromatin mark at a specific centromere. Also, one could edit DNA methylation at centromeres leveraging cutting-edge CRISPR technologies (here8). Considering that centromeres go through (non-canonical) transcription, it would be also interesting to investigate if and how centromere RNA methylation regulates its function. Future work could build on this to reveal interesting facts about how modulating the epigenetic landscape at the centromeres impact its transcription, replication, and function in a cell-cycle-dependent manner.
Acknowledgments
Thanks to Judith Lopes and Sheldon Decombe to comment on this preLight, and to all the authors for their support.
All figures used in this preLight are taken directly from Decombe S et. al., 2021 under a CC-BY 4.0 international license.
References
- https://doi.org/10.1083/jcb.202005099
- https://doi.org/10.1080/15384101.2017.1325044
- https://doi.org/10.1016/S0092-8674(01)00542-6
- https://doi.org/10.1074/jbc.M505323200
- https://doi.org/10.1007/s10577-016-9539-3
- https://doi.org/10.1126/science.1178817
- https://doi.org/10.1038/nrm3474
- https://doi.org/10.1016/j.cell.2021.03.025
doi: https://doi.org/10.1242/prelights.27981
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