Bub1 is a multi-task protein kinase required for proper chromosome segregation in eukaryotes. Impairment of Bub1 in humans may lead to chromosomal instability (CIN) or tumorigenesis. Yet, the primary ...cellular substrate of Bub1 has remained elusive. Here, we show that Bub1 phosphorylates the conserved serine 121 of histone H2A in fission yeast Schizosaccharomyces pombe. The h2a-SA mutant, in which all cellular H2A-S121 is replaced by alanine, phenocopies the bub1 kinase-dead mutant (bub1-KD) in losing the centromeric localization of shugoshin proteins. Artificial tethering of shugoshin to centromeres largely restores the h2a-SA or bub1-KD-related CIN defects, a function that is evolutionally conserved. Thus, Bub1 kinase creates a mark for shugoshin localization and the correct partitioning of chromosomes.
In recent years, our knowledge of the epigenetic functions regulated by post-translational modifications (PTMs) of histones, and their role in various diseases, has expanded rapidly, opening the way ...to novel therapeutic strategies that treat epigenetic abnormalities. Many of the current approaches have been focusing on the chemical inhibition of histone-modifying enzymes to modulate histone PTM states for attaining therapeutic effects. However, recent developments in chemistry and molecular biology have contributed to the emergence of new methods that introduce histone PTMs entirely through artificial means, without reliance on endogenous enzymes. In this review article, we summarize several state-of-the-art approaches for the introduction of synthetic epigenetic modifications in cells, and discuss both their therapeutic potential and the possible challenges in developing novel therapeutic strategies utilizing them.
All cellular proteins are synthesized by ribosomes, whose biogenesis in eukaryotes is a complex multi-step process completed within minutes. Several chemical inhibitors of ribosome function are ...available and used as tools or drugs. By contrast, we lack potent validated chemical probes to analyze the dynamics of eukaryotic ribosome assembly. Here, we combine chemical and genetic approaches to discover ribozinoindoles (or Rbins), potent and reversible triazinoindole-based inhibitors of eukaryotic ribosome biogenesis. Analyses of Rbin sensitivity and resistance conferring mutations in fission yeast, along with biochemical assays with recombinant proteins, provide evidence that Rbins’ physiological target is Midasin, an essential ∼540-kDa AAA+ (ATPases associated with diverse cellular activities) protein. Using Rbins to acutely inhibit or activate Midasin function, in parallel experiments with inhibitor-sensitive or inhibitor-resistant cells, we uncover Midasin’s role in assembling Nsa1 particles, nucleolar precursors of the 60S subunit. Together, our findings demonstrate that Rbins are powerful probes for eukaryotic ribosome assembly.
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•Ribozinoindoles are potent chemical inhibitors of eukaryotic ribosome assembly•Activity of four of Mdn1’s six ATPase sites is likely needed for cell growth•Ribozinoindoles inhibit recombinant full-length Mdn1’s ATPase activity in vitro•Assembly of Nsa1 particles, precursors of the 60S subunit, depends on Mdn1
Selective potent inhibitors of eukaryotic ribosome biogenesis are found through a chemical synthetic lethal screen.
Abstract
Life emerges from a network of biomolecules and chemical reactions catalyzed by enzymes. As enzyme abnormalities are often connected to various diseases, a chemical catalyst promoting ...physiologically important intracellular reactions in place of malfunctional endogenous enzymes would have great utility in understanding and treating diseases. However, research into such small-molecule chemical enzyme surrogates remains limited, due to difficulties in developing a reactive catalyst capable of activating inert cellular metabolites present at low concentrations. Herein, we report a small-molecule catalyst,
m
BnA, as a surrogate for a histone acetyltransferase. A hydroxamic acid moiety of suitable electronic characteristics at the catalytic site, paired with a thiol-thioester exchange process, enables
m
BnA to activate endogenous acyl-CoAs present in low concentrations and promote histone lysine acylations in living cells without the addition of exogenous acyl donors. An enzyme surrogate utilizing cellular metabolites will be a unique tool for elucidation of and synthetic intervention in the chemistry of life and disease.
Meiosis comprises a pair of specialized nuclear divisions that produce haploid germ cells. To accomplish this, sister chromatids must segregate together during the first meiotic division (meiosis I), ...which requires that sister chromatid cohesion persists at centromeres. The factors that protect centromeric cohesion during meiosis I have remained elusive. Here we identify Sgo1 (shugoshin), a protector of the centromeric cohesin Rec8 in fission yeast. We also identify a homologue of Sgo1 in budding yeast. We provide evidence that shugoshin is widely conserved among eukaryotes. Moreover, we identify Sgo2, a paralogue of shugoshin in fission yeast, which is required for faithful mitotic chromosome segregation. Localization of Sgo1 and Sgo2 at centromeres requires the kinase Bub1, identifying shugoshin as a crucial target for the kinetochore function of Bub1. These findings provide insights into the evolution of meiosis and kinetochore regulation during mitosis and meiosis.
Chemical modifications of histones, such as lysine acetylation and ubiquitination, play pivotal roles in epigenetic regulation of gene expression. Methods to alter the epigenome thus hold promise as ...tools for elucidating epigenetic mechanisms and as therapeutics. However, an entirely chemical method to introduce histone modifications in living cells without genetic manipulation is unprecedented. Here, we developed a chemical catalyst, PEG-LANA-DSSMe 11, that binds with nucleosome's acidic patch and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells. The size of polyethylene glycol in the catalyst was a critical determinant for its in-cell metabolic stability, binding affinity to histones, and high activity. The synthetic acetylation promoted by 11 without genetic manipulation competed with and suppressed physiological H2B ubiquitination, a mark regulating chromatin functions, such as transcription and DNA damage response. Thus, the chemical catalyst will be a useful tool to manipulate epigenome for unraveling epigenetic mechanisms in living cells.
Shugoshin family proteins are involved in various aspects of chromatin regulations, such as chromosome segregation, chromatin structure, and gene expression. In growing yeast and mammalian cells, ...C-terminal phosphorylation of histone H2A by Bub1 kinase is essential for the localization of Shugoshin proteins to chromatin. Here, we show that in stationary-phase cells, Bub1-mediated H2A phosphorylation is not necessary for chromatin localization of the Shugoshin paralog Sgo2 in Schizosaccharomyces pombe, or for Sgo2-dependent suppression of gene expression in subtelomeric regions. The conserved C-terminal basic domain of Sgo2, which directly binds with phosphorylated H2A, is also dispensable for the localization of Sgo2 to chromatin at stationary phase. Instead, we found that the conserved N-terminal coiled-coil domain and the uncharacterized medial region of Sgo2 are required for Bub1-independent localization of Sgo2. Moreover, Set2-mediated H3K36 methylation was important for the regulation. Intriguingly, the chromatin localization of Sgo2 in the absence of Bub1 was also observed when cells were grown in low-glucose medium. These findings suggest a novel mechanism between nutrient availability and regulation of chromatin by Shugoshin proteins.
Posttranslational modifications (PTMs) of histones play an important role in the complex regulatory mechanisms governing gene transcription, and their dysregulation can cause diseases such as cancer. ...The lack of methods for site-selectively modifying native chromatin, however, limits our understanding of the functional roles of a specific histone PTM, not as a single mark, but in the intertwined PTM network. Here, we report a synthetic catalyst DMAP-SH (DSH), which activates chemically stable thioesters (including acetyl-CoA) under physiological conditions and transfers various acyl groups to the proximate amino groups. Our data suggest that DSH, conjugated with a nucleosome ligand, such as pyrrole-imidazole-polyamide and LANA (latency-associated nuclear antigen)-peptide, promotes both natural (including acetylation, butyrylation, malonylation, and ubiquitination) and non-natural (azido- and phosphoryl labeling) PTMs on histones in recombinant nucleosomes and/or in native chromatin, at lysine residues close to the DSH moiety. To investigate the validity of our method, we used LANA-DSH to promote histone H2B lysine-120 (K120) acylation, the function of which is largely unknown. H2BK120 acetylation and malonylation modulated higher-order chromatin structures by reducing internucleosomal interactions, and this modulation was further enhanced by histone tail acetylation. This approach, therefore, may have versatile applications for dissecting the regulatory mechanisms underlying chromatin function.
Summary
Psoriasis is characterized by excessive growth and aberrant differentiation of epidermal keratinocytes due to persistent inflammation. However, the underlying mechanism that triggers immune ...activation in psoriasis is not clear. In this study, we explored excessive DNA as a potential trigger of psoriasis using cultured human keratinocytes and psoriatic skin tissues. We demonstrated that human genomic DNA fragments induced tumour necrosis factor (TNF)‐α expression, hyperproliferation and over‐expression of heparin‐binding epidermal‐like growth factor (HB‐EGF) and transforming growth factor (TGF)‐α, accompanied by defective expression of keratins 1 and 10 in cultured normal human epidermal keratinocytes, which have a similar phenotype to that of keratinocytes in psoriatic skin lesions. In psoriatic lesions, we found high levels of double‐stranded (ds)DNA fragments, accompanying keratinocytes expressing Ki‐67, HB‐EGF and TNF‐α. In addition, we showed that 1,25‐dihydroxyvitamin D3 inhibited genomic DNA fragment‐induced TNFA and interleukin‐1β (IFNB) expression in human keratinocytes, and an intact function of cathelicidin anti‐microbial peptide (CAMP) was required for this effect. These results suggest that excessive dsDNA fragments probably act as a risk factor for immune activation in psoriasis, and the active form of vitamin D can prevent genomic DNA‐mediated skin inflammation via CAMP.
Cytosolic dsDNA fragments upregulated inflammatory cytokines (TNF‐α, IFN‐β) and growth factors (HB‐EGF, TGF‐α) in cultured human primary keratinocytes, accompanied by increased proliferation and impaired differentiation. Aberrant dsDNA fragments were found in psoriatic skin lesions, but not in unaffected areas, in association with keratinocytes overexpressing Ki‐67, HB‐EGF and TNF‐α. Active vitamin D suppressed dsDNA fragment‐inducible TNF‐α and IFN‐β in a cathelicidin antimicrobial peptide‐dependent manner in human primary keratinocytes. These results suggest that excessive dsDNA are potential triggers of skin inflammation, and vitamin D creams can exert therapeutic effects on psoriasis by suppressing dsDNA fragment‐inducible skin inflammation via cathelicidin antimicrobial peptide.
Chromatin structure and gene expression are dynamically regulated by posttranslational modifications of histones. Recent advance in mass spectrometry has identified novel types of lysine acylations, ...such as butyrylation and malonylation, whose functions and regulations are likely different from those of acetylation. Sirtuins, nicotinamide adenine dinucleotide (NAD
)-dependent histone deacetylases, catalyze various deacylations. However, it is poorly understood how distinct sirtuins regulate the histone acylation states of nucleosomes that have many lysine residues. Here, we provide mass spectrometry-based quantitative information about the acyl group- and site-selectivity of all human sirtuins on acylated nucleosomes. The acyl group- and site-selectivity of each sirtuin is unique to its subtype. Sirt5 exclusively removes negatively-charged acyl groups, while Sirt1/2/3/6/7 preferentially remove hydrophobic acyl groups; Sirt1 and Sirt3 selectively remove acetyl group more than butyryl group, whereas Sirt2 and Sirt6 showed the opposite selectivity. Investigating site-selectivity for active sirtuins revealed acylated lysines on H4 tails to be poor substrates and acylated H3K18 to be a good substrate. Furthermore, we found Sirt7 to be a robust deacylase of H3K36/37, and its activity reliant on nucleosome-binding at its C-terminal basic region. All together, our quantitative dataset provides a useful resource in understanding chromatin regulations by histone acylations.
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