Biology subjects

Borso, M.

Publications and source records attributed to Borso, M..

3 recordsLinked to original sources

bioRxiv2025

A time-resolved atlas of histone modifications during mitotic entry

Mitotic chromosome formation is essential for faithful chromosome segregation in metazoans. While condensin complexes are critical for the formation of rod-shaped mitotic chromosomes, additional mechanisms--particularly those involving phosphorylation and deacetylation of specific histone residues--have been proposed to contribute a further 2- to 4-fold reduction in mitotic chromatin volume. In this study, we employ high-resolution mass spectrometry to determine the kinetics of histone modifications in cell cultures undergoing a highly synchronous mitotic entry at 2.5-minute resolution. Our analysis reveals three different programmes of histone H3 phosphorylation on T3, S10 and S28. These modifications are consistent with methyl-phos switches regulating the association of readers with chromatin other than at promoters. Mass spectrometry and quantitative ChIP-Seq reveal that H3 T3 phosphorylation is a general marker of heterochromatin and not specifically centromeres as previously suggested. Finally, we show that histone acetylation undergoes only modest changes as rod-shaped chromosomes form during unperturbed mitotic entry. Thus, previously reported reductions in acetylation associated with chromosome formation were apparently attributable to delays in mitotic exit used as part of mitotic synchronisation protocols. The mechanism of condensin-independent chromatin compaction in mitosis remains unexplained.

cell biology↗

bioRxiv2025

Intrinsic specificity of a 'core' Tip60 acetyltransferase complex in Drosophila

The lysine acetyltransferase Tip60 (KAT5) regulates gene expression through acetylation of histone N-terminal tail domains. We determined the intrinsic substrate selectivity of a recombinant, 4-subunit TIP60 core module from Drosophila melanogaster with synthetic nucleosome arrays. We compared matched arrays of nucleosomes containing either the replication-dependent histone H2A, or the variant H2A.V (H2A.Z in mammals), a prominent substrate of Tip60. Targeted mass spectrometry allowed to quantify acetylation of individual lysines in histones H2A, H2A.V and H4. Overall, H4 and H2A/H2A.V were equally well acetylated. The analysis comprehensively identified selected sites of acetylation, their relative acetylation levels, diacetylation patterns and revealed surprisingly different acetylation rates of individual lysines. We also applied this defined acetylation system to evaluate the effectiveness and selectivity of a TIP60 inhibitor, NU9056. Remarkably, the inhibitor shows variable effectiveness at different acetylation sites. Knowledge about the intrinsic substrate selectivity of Tip60 is a prerequisite for a mechanistic understanding of the enzymes mode of action.

biochemistry↗

bioRxiv2023

The role of RNA in the maintenance of chromatin domains as revealed by antibody mediated proximity labelling coupled to mass spectrometry

Eukaryotic chromatin is organised in individual domains, which are defined by their location within the nuclear space, the molecular interactions within each other and their distinct proteomic compositions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To identify proteins associated with a specific chromatin domain and to better understand how those domains are established and maintained, we used a new method, which we termed AMPL-MS (Antibody mediated proximity labelling mass spectrometry). This method is based on antibodies and does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize chromatin domains containing specific signature proteins or histone modifications. We used AMPL-MS to characterize the composition of chromocenter as well as the chromosome territory containing the hyperactive X-chromosome in Drosophila. As an outcome of this method, we show the importance of RNA in maintaining the integrity of these domains as treatment with RNAse alters their proteomic composition. Our data demonstrate the power of AMPL-MS to characterize the composition of non-membranous nuclear domains.

biochemistry↗