Biology subjects

Mikulski, P.

Publications and source records attributed to Mikulski, P..

7 recordsLinked to original sources

bioRxiv2025

Heterochromatin boundaries maintain centromere position, size and number

Centromeres are chromosomal loci that ensure proper chromosome segregation by providing a platform for kinetochore assembly and spindle force transduction during cell division. Human centromeres are defined primarily by a unique chromatin domain featuring the histone H3 variant, Centromere Protein A (CENP-A), that marks a single active centromere locus per chromosome. CENP-A chromatin typically occupies a small subregion of low DNA methylation within multi-megabase arrays of hypermethylated alpha-satellite repeats and constitutive pericentric heterochromatin. However, the mechanisms defining and maintaining precise centromere position and domain size, and the role of the underlying alpha satellite DNA sequence, are poorly characterised. Using an experimentally-induced neocentromere in RPE1 cells, we discovered that the SUV39H1 and H2 methyltransferases tri-methylate H3K9 at neocentromere boundaries to maintain CENP-A domain size independent of DNA methylation or satellite sequences. Furthermore, we found that the CENP-A domain at canonical alpha-satellite-based centromeres is characterized by local depletion of H3K9me3-mediated heterochromatin, coinciding with the DNA methylation dip region. We identified the SETDB1 methyltransferase as key to maintaining H3K9me3 within flanking active higher order alpha satellite arrays while SUV39s and SUZ12 contribute to globally heterochromatinize both alpha satellites and neighbouring repeats. Loss of this heterochromatin boundary results in the progressive expansion of the primary CENP-A domain, erosion of DNA methylation, and the nucleation of new centromeres across alpha satellite sequences. Our study identifies the functional specialization of different H3K9 methyltransferases across centromeric and pericentric domains, crucial for maintaining centromere domain size and number.

genomics↗

bioRxiv2023

Transcriptional memory is conferred by combined heritable maintenance and local removal of selective chromatin modifications

Interferon-{gamma} (IFN{gamma}) transiently activates genes involved in inflammation and innate immunity. A subset of targets maintain a mitotically heritable memory of prior IFN{gamma} exposure resulting in hyperactivation upon reexposure. Here we discovered that the active chromatin marks H3K4me1, H3K14Ac and H4K16Ac are established during IFN{gamma} priming and selectively maintained on a cluster of GBP genes for at least 7 days in dividing cells in the absence of transcription. The histone acetyltransferase KAT7 is required for the accelerated GBP reactivation upon reexposure to IFN{gamma}. In naive cells, we find the GBP cluster is maintained in low-level repressive chromatin marked by H3K27me3 limiting priming in a PRC2-dependent manner. Unexpectedly, IFN{gamma} results in transient accumulation of this repressive mark but is then selectively depleted from primed GBP genes during the memory phase facilitating hyperactivation of primed cells. Furthermore, we identified a cis-regulatory element that makes transient, long-range contacts across the GBP cluster and acts as a repressor, primarily to curb the hyperactivation of previously IFN{gamma}-primed cells. Combined our results identify the putative chromatin basis for long-term transcriptional memory of interferon signalling that may contribute to enhanced innate immunity.

molecular biology↗

bioRxiv2022

STAT1 is required to establish but not maintain IFNγ-induced transcriptional memory

Exposure of human cells to interferon-{gamma} (IFN{gamma}) results in a mitotically heritable yet reversible state called long-term transcriptional memory. We previously identified the clustered GBP genes as strongly primed by IFN{gamma}. Here we discovered that in primed cells, both interferon-responsive transcription factors STAT1 and IRF1 target chromatin with accelerated kinetics upon re-exposure to IFN{gamma}, specifically at promotors of primed genes. Priming does not alter the degree of IFN{gamma}-induced STAT1 activation or nuclear import, indicating that memory does not alter upstream JAK-STAT signalling. We found STAT1 to be critical to establish transcriptional memory but in a manner that is independent of mere transcription activation. Interestingly, while Serine 727 phosphorylation of STAT1 was maintained during the primed state, STAT1 is not required for the heritability of GBP gene memory. Our results suggest that memory of interferon exposure constitutes a STAT1-mediated, heritable state that is established during priming. This renders GBP genes poised for subsequent STAT1 and IRF1 binding and accelerated gene activation upon a secondary interferon exposure.

molecular biology↗

bioRxiv2021

Chlamydomonas reinhardtii exhibits stress memory in the accumulation of triacylglycerols induced by nitrogen deprivation.

Stress memory is a phenomenon whereby exposure to initial stress event influences a response to subsequent stress exposures. Studying stress memory is important to understand the cellular behaviour in dynamic environment, especially nowadays, in times with growing environmental instability. Stress memory has been characterized in vascular plants but its occurrence in non-vascular plant species has been rarely investigated. We hypothesized that stress memory occurs in non-vascular plants in relation to metabolic stress. We sought to test it using accumulation of lipids (triacylglycerols) in model green alga Chlamydomonas reinhardtii subjected to nitrogen deprivation stress as a model system. Here, we established stress memory protocol on C. reinhardtii cells. Using a blend of microscopy and gas chromatography methods, we showed that the cells exposed to recurrent stress show differential accumulation of triacylglycerols on the quantitative level without qualitative changes in lipid composition, comparing to single stress controls. Overall, our results suggest that metabolic stress memory does occur in non-vascular plant C. reinhardtii and provides a starting point to characterize metabolic stress memory mechanism. Due to the commercial potential of algae, our findings are relevant for basic science, as well as industrial production of algae-derived compounds.

molecular biology↗

bioRxiv2021

VAL1 as an assembly platform co-ordinating co-transcriptional repression and chromatin regulation at Arabidopsis FLC

Polycomb (PcG) silencing is crucial for development across eukaryotes, but how PcG targets are regulated is still incompletely understood. The slow timescale of cold-induced PcG silencing at Arabidopsis thaliana FLOWERING LOCUS C (FLC) makes it an excellent system to dissect this mechanism. Binding of the DNA binding protein VAL1 to an FLC intronic RY motif within the PcG nucleation region is an early step in the silencing process. VAL1 interacts with APOPTOSIS AND SPLICING ASSOCIATED PROTEIN (ASAP) complex and POLYCOMB REPRESSIVE COMPLEX 1 (PRC1). Here, we show that ASAP and PRC1 function as co-repressors that quantitatively regulate FLC transcription. Upon the shift to cold PRC1-mediated H2Aub accumulates only at the nucleation region, is transiently maintained after transfer back to warm, but unlike the PRC2-delivered H3K27me3 does not spread across the locus. H2K27me3 thus provides long-term epigenetic silencing, whereas H2Aub is a transient repression signal. Overall, our work highlights how a DNA sequence-specific binding protein can act as an assembly platform co-ordinating the co-transcriptional repression and chromatin regulation necessary for Polycomb silencing.

molecular biology↗

bioRxiv2017

PWO1 interacts with PcG proteins and histones to regulate Arabidopsis flowering and development.

Polycomb-group (PcG) proteins mediate epigenetic gene regulation by setting H3K27me3 via Polycomb Repressive Complex 2 (PRC2). In plants, it is largely unclear how PcG proteins are recruited to their target genes.\n\nHere, we identified the PWWP-DOMAIN INTERACTOR OF POLYCOMBS1 (PWO1) protein which interacts with all three Arabidopsis PRC2 histone methyltransferases and is required for keeping full H3 occupancy at several Arabidopsis genes. PWO1 localizes and recruits CLF to nuclear speckles in tobacco nuclei, suggesting a role in spatial organization of PcG regulation. PWO1 belongs to a gene family with three members acting redundantly: pwo1 pwo2 pwo3 triple mutants are seedling lethal and show shoot and root meristem arrest, while pwo1 single mutants are early flowering. Interestingly, PWO1s PWWP domain confers binding to histones, which is reduced by a point mutation in a highly conserved residue of this domain and blocked by phosphorylation of H3S28. PWO1 carrying this mutation is not able to fully complement the pwo1 pwo2 pwo3 triple mutant, indicating the requirement of this domain for PWO1 in vivo activity. Thus, the PWO family may present a novel class of histone readers which are involved in recruiting PcG proteins to subnuclear domains and in promoting Arabidopsis development.

plant biology↗

bioRxiv2017

PWWP INTERACTOR OF POLYCOMBS (PWO1) links PcG-mediated gene repression to the nuclear lamina in Arabidopsis.

Polycomb group (PcG) proteins facilitate chromatin-mediated gene repression through the modification of histone tails in a wide range of eukaryotes, including plants and animals. One of the PcG protein complexes, Polycomb Repressive Complex 2 (PRC2), promotes repressive chromatin formation via tri-methylation of lysine-27 on histone H3 (H3K27me3). The animal PRC2 is implicated in impacting subnuclear distribution of chromatin as its complex components and H3K27me3 are functionally connected with the nuclear lamina (NL) - a peripheral protein mesh that resides underneath the inner nuclear membrane (INM) and consists of lamins and lamina-associated proteins. In contrast to animals, NL in plants has an atypical structure and its association with PRC2-mediated gene repression is largely unknown. Here, we present a connection between lamin-like protein, CROWDED NUCLEI 1 (CRWN1), and a novel PRC2-associated component, PWWP INTERACTOR OF POLYCOMBS 1 (PWO1), in Arabidopsis thaliana. We show that PWO1 and CRWN1 proteins associate physically with each other, act in the same pathway to maintain nuclear morphology and control expression of similar set of target genes. Moreover, we demonstrate that PWO1 proteins form speckle-like foci located partially at the subnuclear periphery in Nicotiana benthamiana and Arabidopsis thaliana. Ultimately, as CRWN1 and PWO1 are plant-specific, our results argue that plants developed an equivalent, rather than homologous, mechanism of linking PRC2-mediated chromatin repression and nuclear lamina.

molecular biology↗