Haspin kinase modulates nuclear architecture and Polycomb-dependent gene silencing

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Open Access

Research Article

Ujué Fresán, 

Maria A. Rodríguez-Sánchez, 

Oscar Reina, 

Victor G. Corces, 

M. Lluisa Espinàs


Published: August 4, 2020


?This is an uncorrected proof.

AbstractHaspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. Here we report that haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. Our data reveal that haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. We show that haspin is a suppressor of position-effect variegation involved in heterochromatin organization. Our results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation.
Author summary
Haspin is a highly conserved kinase in eukaryotes involved in chromosome organization during mitosis. In this study we demonstrated that haspin is also required to maintain proper chromatin organization during interphase. Our analyses showed that Drosophila haspin is necessary for insulator activity, nuclear architecture, heterochromatin organization and pairing-sensitive gene silencing. We further found that haspin modulates Pds5-cohesin association with chromatin and it is required for robust Polycomb-mediated homeotic gene silencing. Overall our findings reveal that haspin kinase is a key element in chromatin organization and thereby regulates gene expression.

Citation: Fresán U, Rodríguez-Sánchez MA, Reina O, Corces VG, Espinàs ML (2020) Haspin kinase modulates nuclear architecture and Polycomb-dependent gene silencing. PLoS Genet 16(8):

https://doi.org/10.1371/journal.pgen.1008962Editor: Kami Ahmad, Fred Hutchinson Cancer Research Center, UNITED STATESReceived: October 10, 2019; Accepted: June 29, 2020; Published: August 4, 2020Copyright: © 2020 Fresán et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All relevant data are within the manuscript and its Supporting Information files except ChIP-seq files that are available from the NCBI GEO database (accession number GSE98223).Funding: This work was supported by the Spanish Ministerio de Economia y Competitividad (BFU2013-48712-P) and the Generalitat de Catalunya (SGR2017-475). U. F. and M. R-S. acknowledge receipt of doctoral fellowships from Consejo Superior de Investigaciones Cientificas and Ministerio de Economia y Competitividad respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.

IntroductionGenome organization in the cell nucleus plays an important role in the regulation of gene expression during cellular differentiation and development [1,2]. Insulator or architectural proteins are essential components of the three-dimensional organization of chromatin by mediating long-range interactions between distant sites in the genome. Current results suggest that architectural complexes have two inter-related functions: to organize the genome in domains and to facilitate the interaction between regulatory elements [3,4,5]. Several architectural proteins have been characterized in Drosophila melanogaster, including DNA-binding proteins (CTCF, SuHw, BEAF-32, GAGA, DREF, TFIIIC, Z4, Elba, ZIPIC, Ibf1 and Ibf2) that recruit accessory factors (CP190, mod(mdg4), Rad21, Cap-H2, Fs(1)h-L, L(3)mbt and chromator) to mediate chromatin interactions [6]. A small number of architectural proteins have been characterized in mammals, and among them, CTCF and cohesin, have also been shown to support interactions between distant sites in the genome [5].
Long-range gene regulation also involves epigenetic components such as the Polycomb group proteins (PcG) [7,8,9,10,11]. In Drosophila, homeotic (Hox) genes, which encode evolutionary conserved master regulators of development, are the most prominent PcG targets. Precise spatiotemporal expression of Hox genes involves an intricate collection of enhancers, promoters, polycomb response elements (PREs) and insulators. It has been demonstrated, both by fluorescent in situ hybridization and Chromosome Conformation Capture approaches, that chromatin organization of the Abdominal-B (Abd-B) locus in the bithorax complex (BX-C) is a critical determinant of the regulation of the expression of the gene. Several reports have shown that insulators and PREs interact with the Abd-B promoter in tissues where the gene is not expressed, and Polycomb and the insulator/architectural proteins CTCF and CP190 are required for these interactions [12,13,14,15].
Histone modifications and the enzymes responsible for them are also important players in the regulation of chromatin organization. Haspin, a highly conserved kinase in eukaryotes, is responsible for phosphorylation of histone H3T3 during mitosis [16]. Haspin kinase has been shown to be involved in sister chromatid cohesion [17] and to be necessary to localize the Chromosomal Passenger Complex (CPC) on mitotic chromatin at centromeres to activate Aurora B that regulates kinetochore-microtubule attachments [18,19,20]. In fission yeast and mammalian cells, haspin has been shown to bind the cohesin-associated protein Pds5 at centromeres and to antagonize the cohesin-unloading factor Wapl [21,22,23]. H3T3ph dephosphorylation upon exit from M phase has been shown to be necessary for chromosome decondensation and nuclear envelope reformation [24]. Haspin kinase has been reported to be strongly activated by Cdk1 and Polo-like kinase in mitosis [25,26]. However, haspin contains an atypical protein kinase domain, which is conserved from yeast to humans, that does not require phosphorylation on the activation loop for activity, suggesting that it could be partially active all along the cell cycle. We show here that haspin is necessary for insulator activity, position-effect variegation (PEV) and pairing-sensitive silencing (PSS) modulating nuclear architecture in interphase. We also demonstrate that haspin is required for robust Polycomb-dependent homeotic gene silencing. Altogether our results indicate that Drosophila haspin kinase is involved, not only in chromosome organization during mitosis, but also in genome organization in interphase cells playing a functional role in gene regulation.

Haspin kinase is necessary for insulator activity
In order to identify new proteins with insulator activity we performed a mutagenesis screen in Drosophila by random transposition of a P element. New insertions were analyzed for changes in reporter gene expression in enhancer-blocking assays using a transgenic line that contain the Fab7 boundary/insulator element of the BX-C between the white enhancer and the mini-white reporter gene (B727.1, S1 Fig), which blocks promoter activation by the distal enhancer. Any relief of insulator activity should allow communication between enhancer and promoter of the white reporter gene increasing eye color. Among all the lines showing a significant relief of enhancer-blocking we further studied line 86 (Fig 1A). The P element insertion in this line was mapped to the first exon in the 5’UTR of the gene haspin (S2A Fig). We analyzed haspin expression in the homozygous line, and we observed a strong reduction in transcript levels indicating that haspin86 is either a null or a strong hypomorph haspin alelle (S2B Fig). By mobilazing the P element in line 86 we obtained line 128 that harbors a partial deletion of the P element, the first and second exons and part of the second intron of the gene haspin (S2A Fig), likely rendering the gene non functional. Haspin128 homozygous mutant flies are viable, they show a decrease in adult longevity which is stronger in females than males (S2C Fig), and fertility of both sexes is clearly affected (S2D Fig).
Fig 1. Insulator activity of haspin.A) Eye color of representative flies of enhancer-blocking assays using the transgenic line B727.1 that contains the Fab7 insulator element in wild-type and heterozygous line 86 background. B) Eye color of representative flies of enhancer-blocking assays using the transgenic line F72.5 that contains the Fab7 insulator and PRE elements in haspin RNAi mutant background. C) Quantitative analysis of eye pigment in flies with the F72.5 construct and the genotypes indicated. n=3, significant differences between wild-type and the different mutant backgrounds as determined by Student’s t-test (p38, p-value Read More

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