Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments

Martina Begnis, Julien Duc, Sandra Offner, Delphine Grun, Shaoline Sheppard, Olga Rosspopoff, Didier Trono

Posted on: 10 April 2024

Preprint posted on 4 January 2024

Article now published in Science Advances at http://dx.doi.org/10.1126/sciadv.ado1662

Find out how genome loci are anchored to the nucleolus! Exciting new study from @Trono_Lab shows that ZNF274 competes with CTCF to keep H3K9me3 silenced genome at the nucleolus periphery, fine-tuning cell-specific gene expression

Selected by Silvia Carvalho

Categories: cell biology, molecular biology, systems biology

Background

Inside the mammalian nucleus, gene transcription is tightly regulated at different spatial scales, from the linear DNA to nucleosomes compaction, up to the level of chromatin looping and nuclear compartmentalization. DNA molecules can be decorated with different histone marks according to their transcription status, creating accessible or compacted chromatin environments. Typically, accessible genomic regions are found in the centre of the nucleus or closer to specific compartments, whereas compacted regions meet at the nuclear periphery or at the surface of the nucleolus. The interconnection between the different dimensions of transcription regulation and the mechanisms that contribute to it remain poorly understood¹.

The nucleolus is the most prominent nuclear compartment and its main function is ribosomal biogenesis. As a central hub of heterochromatin domains, the nucleolus also has roles in shaping the 3D genome². However, little is known about how the genome domains at the nucleolus periphery are tethered and how its nuclear positioning influences transcription. The transcriptional repressor zinc finger protein ZNF274 accumulates predominately at the nucleolus³ and promotes the deposition of the repressive H3K9me3 mark⁴, but its roles in shaping the 3D genome have remained unexplored. This preprint proposes ZNF274 as an anchor of silenced genome to the nucleolus, promoting long-range interactions between heterochromatin domains. The study thereby sheds light on the intertwined relationship of genome positioning within the 3D nuclear space with genome folding and transcription.

Key findings

1 – ZNF274 represses genes clusters’ expression by mediating heterochromatin formation and tethering it to the nucleolus

Begnis and colleagues started by evaluating the impact of ZNF274 on gene expression by using ZNF274 homozygous knockout (KO) cells. The authors found that ZNF274 promotes the transcriptional repression of Krüppel-associated box (KRAB)-containing zinc finger proteins (KZFP) and Protocadherins (PCDHs). Both KZFP and PCDH are organized in gene clusters, implying a role for ZNF274 in the structural organization of the genome. ZNF274 promoted the deposition of H3K9me3 onto KZFP and PCDH, in line with previous evidence⁴. When ZNF274 was depleted, KZFP and PCDH were transcribed and decorated with the active marks, H3K4me3 and H3K36me3.

PCDH genes are predominantly expressed in the developing nervous system and have the particularity that different PCDH isoforms are uniquely expressed in individual cells^5–7. Single-cell data showed that ZNF274 depletion in neuron progenitor cells increased the number of PCDH and KZFP isoforms expressed per cell. The authors propose that the ZNF274-mediated fine-tuning of isoform diversity is important for cell identity.

2 – ZNF274 occupancy hampers CTCF binding and promotes genome compartmentalization

To explore how ZNF274 impacts genome folding, the authors produced Hi-C, 4C and CTCF ChIP data. Depletion of ZNF274 led to a disruption in long-range DNA contacts within the PCDH domain with an increase in local interactions and CTCF binding, which is known to promote enhancer-promoter interactions in this locus^8–11. Moreover, Hi-C data identified a ZNF274-mediated long-range contact between KZFP and PCDH, implying that these genomic regions share the same nuclear space. The inverse correlation between H3K9me3 and CTCF was not restricted to the PCDH locus but to most of the regions that lost this histone mark upon ZNF274 depletion.

3 – ZNF274 SCAN domain bridges DNA to nucleoli to promote repressive chromatin

Next, the authors confirmed the presence of ZNF274 at the nucleolus and its interaction with several nucleolus-associated proteins³. By expressing several mutated versions of ZNF274, the authors showed that the SCAN domain of ZNF274 is necessary to tether ZNF274 to the nucleolus and to nucleate H3K9me3, but not sufficient to spread this histone mark to the same levels found in wild-type cells. Due to the SCAN domain’s properties on mediating homo and hetero-oligomerization, the authors propose that “ZNF274 would function as a homodimer accumulating around nucleoli and bringing bound loci in spatial proximity”. To test this, the authors performed co-immunoprecipitation experiments and demonstrated the ability of ZNF274 to form homodimers and that the SCAN domain was essential for nucleoli association. Then, the authors revealed that nucleoli-associated genomic contacts between rRNA are disrupted in ZNF274 KO cells and that the KZFP cluster is frequently found at the nucleolus. Moreover, the expression of ectopic ZNF274 in ZNF274 KO cells recovers partially the long-range contact between KZFP and PCDH clusters.

Conclusions

This preprint proposes that ZNF274 homodimerization by the SCAN domain competes with CTCF to promote genome repression, by seeding H3K9me3 and anchoring silenced genome at the nucleoli, promoting genomic interactions between DNA located at the periphery of nucleoli. This mechanism can modulate cell specialization by modulating transcriptional signatures, such as the specific activation of PCDH.

Figure 5F of the preprint: “Model. (Top) Nucleolar tethering of ZNF274-bound clusters is functional to establish silencing of extended genomic regions and segregate them in repressive domains (in dark pink) that modulate access of CTCF and cohesin complexes to enable selective gene expression. (Bottom) Ablation of ZNF274 triggers loss of repressive chromatin marks, de novo CTCF binding and altered 3D spatial organization of the same genomic neighborhoods (in light pink), thus precluding the cell-specific fine-tuning of isoform diversity. The figure panel was created with BioRender.com“.

What I like about the preprint and why I think this new work is important

By focusing on the role of ZNF274 in the nucleolus, this study expands our understanding of how gene expression mechanisms work across the different layers of gene regulation. I believe that holistic perspectives on gene regulation are essential to better understand gene expression dynamics.

Questions to the authors

1 – Genome-wide exploration of H3K9me3 and CTCF enrichment upon ZNF274 KO identified two distinct clusters, one losing H3K9me3 while gaining CTCF and the other showing the opposite behaviour. I am curious about the relationship of these groups to the nucleolus and the type of genes present in these groups. Did you identify different features for these clusters?

2 – By overexpressing ZNF274-SCAN mutants in KO cells, you showed that the SCAN domain of ZNF274 is necessary to maintain the association of genomic regions with the nucleolus. Do you have any evidence supporting the idea that this domain is sufficient to tether a genomic region back to the nucleolus?

3 – The study shows how ZNF274 mediates the deposition of H3K9me3, gene silencing and genomic tethering to the nucleolus, but you do not explore if the gene repression happens as a cause or consequence of ZNF274-nucleoli-tethering. What are your thoughts about the possible order of events for the studied loci?

4 – I wonder if ZNF274 depletion led to any partial disruption of the nucleolus structure and if the cell differentiation efficiency was impaired. Did you identify nucleoli anomalies in the imaging data?

References

Willemin, A., Szabó, D. & Pombo, A. Epigenetic regulatory layers in the 3D nucleus. Mol Cell 84 (2024).
Bersaglieri, C. & Santoro, R. Genome Organization in and around the Nucleolus. Cells 8(2019).
Yano, K. et al. Identification and characterization of human ZNF274 cDNA, which encodes a novel Kruppel-type zinc-finger protein having nucleolar targeting ability. Genomics 65 (2000).
Frietze, S., O’Geen, H., Blahnik, K. R., Jin, V. X. & Farnham, P. J. ZNF274 Recruits the Histone Methyltransferase SETDB1 to the 3′ Ends of ZNF Genes. PLoS One 5 (2010).
Esumi, S. et al. Monoallelic yet combinatorial expression of variable exons of the protocadherin-alpha gene cluster in single neurons. Nat Genet 37 (2005).
Kaneko, R. et al. Allelic gene regulation of Pcdh-alpha and Pcdh-gamma clusters involving both monoallelic and biallelic expression in single Purkinje cells. J Biol Chem 281 (2006).
Hirano, K. et al. Single-neuron diversity generated by Protocadherin-β cluster in mouse central and peripheral nervous systems. Front Mol Neurosci 5 (2012).
Guo, Y. et al. CTCF/cohesin-mediated DNA looping is required for protocadherin α promoter choice. Proc Natl Acad Sci U S A 109 (2012).
Guo, Y. et al. CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function. Cell 162 (2015).
Jiang, Y. et al. The methyltransferase SETDB1 regulates a large neuron-specific topological chromatin domain. Nat Genet 49 (2017).
Canzio, D. et al. Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice. Cell 177 (2019).

doi: https://doi.org/10.1242/prelights.37095

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Author's response

Martina Begnis shared

R: Only the chromatin marked by a loss of H3K9me3 and gain of CTCF upon KO of ZNF274 contains genes organised in clusters, while we did not detect the enrichment of a particular category of genes for the group showing opposite behaviour (loss of CTCF and gain of H3K9me3).

R: We do not have a direct proof that the SCAN domain is sufficient to tether a genomic region back to the nucleolus, but it is definitely something we are planning to look at. However, we do have some evidence that presence of ZNF274’s SCAN domain enhances the repressor ability of tools used for transcriptional silencing such as the dCas9-KRAB system.

R: It is known that during early embryonic development the nucleolus provides a platform for the establishment of heterochromatin and proper chromosome segregation. In particular, in mouse nucleolar maturation is required for the silencing of DUX4, the transcription factor priming human embryonic genome activation (EGA), thus promoting embryonic progression. Therefore, we do like to think that ZNF274-mediated nucleolar tethering might have some active role in promoting transcriptional silencing and is not a mere consequence of repression.

R: Again, this is something we are curious to investigate in the next future. Our confocal imaging, which was not designed to specifically address the nucleolar structure, did not show any dramatic nucleolar anomalies. Still, we plan to invest on a thorough acquisition and analysis of the imaging data for this purpose, which could potentially reveal significant changes in nucleolar functioning upon KO of ZNF274.

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