RESUMO
Genome sequencing has identified hundreds of histone post-translational modifications (PTMs) that define an open or compact chromatin nanostructure at the level of nucleosome proximity, and therefore serve as activators or repressors of gene expression. Direct observation of this epigenetic mode of transcriptional regulation in an intact single nucleus, is however, a complex task. This is because despite the development of fluorescent probes that enable observation of specific histone PTMs and chromatin density, the changes in nucleosome proximity regulating gene expression occur on a spatial scale well below the diffraction limit of optical microscopy. In recent work, to address this research gap, we demonstrated that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labelled histones core to the nucleosome, is a readout of chromatin nanostructure that can be multiplexed with immunofluorescence (IF) against specific histone PTMs. Here from application of this methodology to gold standard gene activators (H3K4Me3 and H3K9Ac) versus repressors (e.g., H3K9Me3 and H3K27Me), we find that while on average these histone marks do impart an open versus compact chromatin nanostructure, at the level of single chromatin foci, there is significant spatial heterogeneity. Collectively this study illustrates the importance of studying the epigenetic landscape as a function of space within intact nuclear architecture and opens the door for the study of chromatin foci sub-populations defined by combinations of histone marks, as is seen in the context of bivalent chromatin.
Assuntos
Cromatina , Histonas , Cromatina/genética , Histonas/metabolismo , Nucleossomos , Transferência Ressonante de Energia de Fluorescência , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/genética , Epigênese GenéticaRESUMO
The thermal tolerance of symbiodiniacean photo-endosymbionts largely underpins the thermal bleaching resilience of their cnidarian hosts such as corals and the coral model Exaiptasia diaphana. While variation in thermal tolerance between species is well documented, variation between conspecific strains is understudied. We compared the thermal tolerance of three closely related strains of Breviolum minutum represented by two internal transcribed spacer region 2 profiles (one strain B1-B1o-B1g-B1p and the other two strains B1-B1a-B1b-B1g) and differences in photochemical and non-photochemical quenching, de-epoxidation state of photopigments, and accumulation of reactive oxygen species under rapid short-term cumulative temperature stress (26-40 °C). We found that B. minutum strains employ distinct photoprotective strategies, resulting in different upper thermal tolerances. We provide evidence for previously unknown interdependencies between thermal tolerance traits and photoprotective mechanisms that include a delicate balancing of excitation energy and its dissipation through fast relaxing and state transition components of non-photochemical quenching. The more thermally tolerant B. minutum strain (B1-B1o-B1g-B1p) exhibited an enhanced de-epoxidation that is strongly linked to the thylakoid membrane melting point and possibly membrane rigidification minimizing oxidative damage. This study provides an in-depth understanding of photoprotective mechanisms underpinning thermal tolerance in closely related strains of B. minutum.
Assuntos
Fotossíntese , Dinoflagellida/fisiologia , Resposta ao Choque Térmico , Temperatura AltaRESUMO
Non-homologous end-joining (NHEJ) stands as a pivotal DNA repair pathway crucial for the survival and persistence of Mycobacterium tuberculosis (Mtb) during its dormant, non-replicating phase, a key aspect of its long-term resilience. Mycobacterial NHEJ is a remarkably simple two-component system comprising the rate-limiting DNA binding protein Ku (mKu) and Ligase D. To elucidate mKu's role in NHEJ, we conducted a series of in silico and in vitro experiments. Molecular dynamics simulations and in vitro assays revealed that mKu's DNA binding stabilizes both the protein and DNA, while also shielding DNA ends from exonuclease degradation. Surface plasmon resonance (SPR) and electrophoretic mobility shift assays (EMSA) demonstrated mKu's robust affinity for linear double-stranded DNA (dsDNA), showing positive cooperativity for DNA substrates of 40 base pairs or longer, and its ability to slide along DNA strands. Moreover, analytical ultracentrifugation, size exclusion chromatography, and negative stain electron microscopy (EM) unveiled mKu's unique propensity to form higher-order oligomers exclusively with DNA, suggesting a potential role in mycobacterial NHEJ synapsis. This comprehensive characterization sheds new light on mKu's function within the Mtb NHEJ repair pathway. Targeting this pathway may thus impede the pathogen's ability to persist in its latent state within the host for prolonged periods.
RESUMO
The Hippo pathway is an important regulator of organ growth and cell fate. The major mechanism by which Hippo is known to control transcription is by dictating the nucleo-cytoplasmic shuttling rate of Yorkie, a transcription co-activator, which promotes transcription with the DNA binding protein Scalloped. The nuclear biophysical behavior of Yorkie and Scalloped, and whether this is regulated by the Hippo pathway, remains unexplored. Using multiple live-imaging modalities on Drosophila tissues, we found that Scalloped interacts with DNA on a broad range of timescales, and enrichment of Scalloped at sites of active transcription is mediated by longer DNA dwell times. Further, Yorkie increased Scalloped's DNA dwell time, whereas the repressors Nervous fingers 1 (Nerfin-1) and Tondu-domain-containing growth inhibitor (Tgi) decreased it. Therefore, the Hippo pathway influences transcription not only by controlling nuclear abundance of Yorkie but also by modifying the DNA binding kinetics of the transcription factor Scalloped.
Assuntos
Cromatina , Proteínas de Drosophila , Drosophila melanogaster , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Transativadores , Transcrição Gênica , Proteínas de Sinalização YAP , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Cromatina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Proteínas de Sinalização YAP/metabolismo , Transativadores/metabolismo , Transativadores/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ligação Proteica , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA/metabolismo , DNA/genéticaRESUMO
In this work, we examine the use of environment-sensitive fluorescent dyes in fluorescence lifetime imaging microscopy (FLIM) biosensors. We screened merocyanine dyes to find an optimal combination of environment-induced lifetime changes, photostability, and brightness at wavelengths suitable for live-cell imaging. FLIM was used to monitor a biosensor reporting conformational changes of endogenous Cdc42 in living cells. The ability to quantify activity using phasor analysis of a single fluorophore (e.g., rather than ratio imaging) eliminated potential artifacts. We leveraged these properties to determine specific concentrations of activated Cdc42 across the cell.
Assuntos
Técnicas Biossensoriais , Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Técnicas Biossensoriais/métodosRESUMO
Variants in the poorly characterised oncoprotein, MORC2, a chromatin remodelling ATPase, lead to defects in epigenetic regulation and DNA damage response. The C-terminal domain (CTD) of MORC2, frequently phosphorylated in DNA damage, promotes cancer progression, but its role in chromatin remodelling remains unclear. Here, we report a molecular characterisation of full-length, phosphorylated MORC2, demonstrating its preference for binding open chromatin and functioning as a DNA sliding clamp. We identified a phosphate interacting motif within the CTD that dictates ATP hydrolysis rate and cooperative DNA binding. The DNA binding impacts several structural domains within the ATPase region. We provide the first visual proof that MORC2 induces chromatin remodelling through ATP hydrolysis-dependent DNA compaction, regulated by its phosphorylation state. These findings highlight phosphorylation of MORC2 CTD as a key modulator of chromatin remodelling, presenting it as a potential therapeutic target.