Evidence for low nanocompaction of heterochromatin in living embryonic stem cells.

Despite advances in the identification of chromatin regulators and genome interactions, the principles of higher-order chromatin structure have remained elusive. Here, we applied FLIM-FRET microscopy to analyse, in living cells, the spatial organisation of nanometre range proximity between nucleosomes, which we called "nanocompaction." Both in naive embryonic stem cells (ESCs) and in ESC-derived epiblast-like cells (EpiLCs), we find that, contrary to expectations, constitutive heterochromatin is much less compacted than bulk chromatin. The opposite was observed in fixed cells. HP1α knockdown increased nanocompaction in living ESCs, but this was overridden by loss of HP1ß, indicating the existence of a dynamic HP1-dependent low compaction state in pluripotent cells. Depletion of H4K20me2/3 abrogated nanocompaction, while increased H4K20me3 levels accompanied the nuclear reorganisation during EpiLCs induction. Finally, the knockout of the nuclear cellular-proliferation marker Ki-67 strongly reduced both interphase and mitotic heterochromatin nanocompaction in ESCs. Our data indicate that, contrary to prevailing models, heterochromatin is not highly compacted at the nanoscale but resides in a dynamic low nanocompaction state that depends on H4K20me2/3, the balance between HP1 isoforms, and Ki-67.

Ki-67 regulates global gene expression and promotes sequential stages of carcinogenesis.

Ki-67 is a nuclear protein that is expressed in all proliferating vertebrate cells. Here, we demonstrate that, although Ki-67 is not required for cell proliferation, its genetic ablation inhibits each step of tumor initiation, growth, and metastasis. Mice lacking Ki-67 are resistant to chemical or genetic induction of intestinal tumorigenesis. In established cancer cells, Ki-67 knockout causes global transcriptome remodeling that alters the epithelial-mesenchymal balance and suppresses stem cell characteristics. When grafted into mice, tumor growth is slowed, and metastasis is abrogated, despite normal cell proliferation rates. Yet, Ki-67 loss also down-regulates major histocompatibility complex class I antigen presentation and, in the 4T1 syngeneic model of mammary carcinoma, leads to an immune-suppressive environment that prevents the early phase of tumor regression. Finally, genes involved in xenobiotic metabolism are down-regulated, and cells are sensitized to various drug classes. Our results suggest that Ki-67 enables transcriptional programs required for cellular adaptation to the environment. This facilitates multiple steps of carcinogenesis and drug resistance, yet may render cancer cells more susceptible to antitumor immune responses.

A cyclin-dependent kinase-mediated phosphorylation switch of disordered protein condensation.

Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

Humans from Wuchereria bancrofti endemic area elicit substantial immune response to proteins of the filarial parasite Brugia malayi and its endosymbiont Wolbachia.

BACKGROUND: In the past, immune responses to several Brugia malayi immunodominant antigens have been characterized in filaria-infected populations; however, little is known regarding Wolbachia proteins. We earlier cloned and characterized few B. malayi (trehalose-6-phosphate phosphatase, Bm-TPP and heavy chain myosin, BmAF-Myo) and Wolbachia (translation initiation factor-1, Wol Tl IF-1 and NAD+-dependent DNA ligase, wBm-LigA) proteins and investigated the immune responses, which they triggered in animal models. The current study emphasizes on immunological characteristics of these proteins in three major categories of filarial endemic zones: endemic normal (EN, asymptomatic, amicrofilaraemic; putatively immune), microfilariae carriers (MF, asymptomatic but microfilaraemic), and chronic filarial patients (CP, symptomatic and mostly amicrofilaraemic). METHODS: Immunoblotting and ELISA were carried out to measure IgG and isotype antibodies against these recombinant proteins in various clinical categories. Involvement of serum antibodies in infective larvae killing was assessed by antibody-dependent cellular adhesion and cytotoxicity assay. Cellular immune response was investigated by in vitro proliferation of peripheral blood mononuclear cells (PBMCs) and reactive oxygen species (ROS) generation in these cells after stimulation. RESULTS: Immune responses of EN and CP displayed almost similar level of IgG to Wol Tl IF-1 while other three proteins had higher serum IgG in EN individuals only. Specific IgA, IgG1, IgG3 and IgM to Bm-TPP were high in EN subjects, while BmAF-Myo additionally showed elevated IgG2. Enhanced IgA and IgG3 were detected in both EN and CP individuals in response to Wol Tl IF-1 antigen, but IgG1 and IgM were high only in EN individuals. wBm-LigA and BmAF-Myo exhibited almost similar pattern of antibody responses. PBMC isolated from EN subjects exhibited higher proliferation and ROS generation when stimulated with all three proteins except for Wol Tl IF-1. CONCLUSIONS: Overall, these findings display high immunogenicity of all four proteins in human subjects and revealed that the EN population was exposed to both B. malayi and Wolbachia proteins simultaneously. In addition, immune responses to Wol Tl IF-1 suggest possible role of this factor in Wolbachia-induced pathological responses while immune responses to other three proteins suggest that these can be explored further as vaccine candidates.

Functional attributes of evolutionary conserved Arg45 of Wolbachia (Brugia malayi) translation initiation factor-1.

AIM: Wolbachia is a promising antifilarial chemotherapeutic target. Translation initiation factor-1 (Tl IF-1) is an essential factor in prokaryotes. Functional characterization of Wolbachia's novel proteins/enzymes is necessary for the development of adulticidal drugs. MATERIALS & METHODS: Mutant, Wol Tl IF-1 R45D was constructed by site directed mutagenesis. Fluorimetry and size exclusion chromatography were used to determine the biophysical characteristics. Mobility shift assay and fluorescence resonance energy transfer were used to investigate the functional aspect of Wol Tl IF-1 with its mutant. RESULTS: Both wild and mutant were in monomeric native conformations. Wild exhibits nonspecific binding with ssRNA/ssDNA fragments under electrostatic conditions and showed annealing and displacement of RNA strands in comparison to mutant. CONCLUSION: Point mutation impaired RNA chaperone activity of the mutant and its interaction with nucleotides.

Wolbachia transcription elongation factor "Wol GreA" interacts with α2ßß'σ subunits of RNA polymerase through its dimeric C-terminal domain.

OBJECTIVES: Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for therapy against lymphatic filariasis. Transcription elongation factor GreA is an essential factor that mediates transcriptional transition from abortive initiation to productive elongation by stimulating the escape of RNA polymerase (RNAP) from native prokaryotic promoters. Upon screening of 6257 essential bacterial genes, 57 were suggested as potential future drug targets, and GreA is among these. The current study emphasized the characterization of Wol GreA with its domains. METHODOLOGY/PRINCIPAL FINDINGS: Biophysical characterization of Wol GreA with its N-terminal domain (NTD) and C-terminal domain (CTD) was performed with fluorimetry, size exclusion chromatography, and chemical cross-linking. Filter trap and far western blotting were used to determine the domain responsible for the interaction with α2ßß'σ subunits of RNAP. Protein-protein docking studies were done to explore residual interaction of RNAP with Wol GreA. The factor and its domains were found to be biochemically active. Size exclusion and chemical cross-linking studies revealed that Wol GreA and CTD exist in a dimeric conformation while NTD subsists in monomeric conformation. Asp120, Val121, Ser122, Lys123, and Ser134 are the residues of CTD through which monomers of Wol GreA interact and shape into a dimeric conformation. Filter trap, far western blotting, and protein-protein docking studies revealed that dimeric CTD of Wol GreA through Lys82, Ser98, Asp104, Ser105, Glu106, Tyr109, Glu116, Asp120, Val121, Ser122, Ser127, Ser129, Lys140, Glu143, Val147, Ser151, Glu153, and Phe163 residues exclusively participates in binding with α2ßß'σ subunits of polymerase. CONCLUSIONS/SIGNIFICANCE: To the best of our knowledge, this research is the first documentation of the residual mode of action in wolbachial mutualist. Therefore, findings may be crucial to understanding the transcription mechanism of this α-proteobacteria and in deciphering the role of Wol GreA in filarial development.

Wolbachia translation initiation factor-1 is copiously expressed by the adult, microfilariae and infective larvae of Brugia malayi and competitively inhibited by tetracycline.

The intracellular alphaproteobacteria, Wolbachia, is considered to be a future antimacrofilarial drug target as it is obligatory for filarial endurance. Characterizing wolbachial proteins is necessary to understand wolbachial mechanisms and also for discovering new drug entities. Translation initiation factor-1 (Tl IF-1) is an indispensable prokaryotic factor concerned with bacterial viability. This factor is prioritized as one of the most potent antibacterial drug target. To investigate its role in filarial biology, recombinant Wol Tl IF-1 was purified on metal ion column. The factor was found folded in its monomeric native conformation, and contained a buried fluorophore. Molecular modeling revealed that the factor belonged to the Oligomer Binding family, and consisted of the highly conserved S1 domain with 81.6% of the amino acids occupying the allowed regions in Ramachandran plot. In addition, Wol Tl IF-1 exhibited selective binding to the 30S ribosomal subunit, which declined progressively with tetracycline addition. Tetracycline perturbs interaction of Thr18 and Asn32 of the factor with ribosomal protein S4. The factor was immune-localized in adult, microfilariae (Mf) and infective larvae (L3) of Brugia malayi by immunoblotting. High expression was also observed in Wolbachia within B. malayi Mf, L3 and female adult parasite along the gravid uteri by the confocal microscopy. Therefore, Wol Tl IF-1 appears to be an essential Wolbachia factor whose inhibition leads to extensive cell apoptosis and premature killing of adult worms, validating the antifilarial potential of the factor.