22 research outputs found
The laminA/NF-Y protein complex reveals an unknown transcriptional mechanism on cell proliferation
Lamin A is a component of the nuclear matrix that also controls proliferation by
largely unknown mechanisms. NF-Y is a ubiquitous protein involved in cell proliferation
composed of three subunits (-YA -YB -YC) all required for the DNA binding and
transactivation activity. To get clues on new NF-Y partner(s) we performed a mass
spectrometry screening of proteins that co-precipitate with the regulatory subunit
of the complex, NF-YA. By this screening we identified lamin A as a novel putative
NF-Y interactor. Co-immunoprecipitation experiments and confocal analysis confirmed
the interaction between the two endogenous proteins. Interestingly, this association
occurs on euchromatin regions, too. ChIP experiments demonstrate lamin A
enrichment in several promoter regions of cell cycle related genes in a NF-Y dependent
manner. Gain and loss of function experiments reveal that lamin A counteracts NF-Y
transcriptional activity. Taking advantage of a recently generated transgenic reporter
mouse, called MITO-Luc, in which an NF-Y–dependent promoter controls luciferase
expression, we demonstrate that lamin A counteracts NF-Y transcriptional activity
not only in culture cells but also in living animals. Altogether, our data demonstrate
the occurrence of lamin A/NF-Y interaction and suggest a possible role of this protein
complex in regulation of NF-Y function in cell proliferatio
Specific inhibition of NF-Y subunits triggers different cell proliferation defects
Regulated gene expression is essential for a proper progression through the cell cycle. The transcription factor NF-Y has a fundamental function in transcriptional regulation of cell cycle genes, particularly of G2/M genes. In order to investigate common and distinct functions of NF-Y subunits in cell cycle regulation, NF-YA, NF-YB and NF-YC have been silenced by shRNAs in HCT116 cells. NF-YA loss led to a delay in S-phase progression, DNA damage and apoptosis: we showed the activation of the replication checkpoint, through the recruitment of Δp53 and of the replication proteins PCNA and Mcm7 to chromatin. Differently, NF-YB depletion impaired cells from exiting G2/M, but did not interfere with S-phase progression. Gene expression analysis of NF-YA and NF-YB inactivated cells highlighted a common set of hit genes, as well as a plethora of uncommon genes, unveiling a different effect of NF-Y subunits loss on NF-Y binding to its target genes. Chromatin extracts and ChIP analysis showed that NF-YA depletion was more effective than NF-YB in hitting NF-Y recruitment to CCAAT-promoters. Our data suggest a critical role of NF-Y expression, highlighting that the lack of the single subunits are differently perceived by the cells, which activate diverse cell cycle blocks and signaling pathways
Intestinal eosinophils, homeostasis and response to bacterial intrusion
Eosinophils are traditionally considered as end-stage effector cells involved in the pathogenesis of Th2 immune-mediated disorders as well as in the protection against parasite infection. However, this restricted view has recently been challenged by a series of studies revealing the highly plastic nature of these cells and implication in various homeostatic processes. Large numbers of eosinophils reside in the lamina propria of the gastrointestinal tract, at the front line of host defence, where they contribute to maintain the intestinal epithelial barrier function in the face of inflammation-associated epithelial cell damage. Eosinophils confer active protection against bacterial pathogens capable of penetrating the mucosal barrier through the release of cytotoxic compounds and the generation of extracellular DNA traps. Eosinophils also integrate tissue-specific cytokine signals such as IFN-γ, which synergise with bacterial recognition pathways to enforce different context-dependent functional responses, thereby ensuring a rapid adaptation to the ever-changing intestinal environment. The ability of eosinophils to regulate local immune responses and respond to microbial stimuli further supports the pivotal role of these cells in the maintenance of tissue homeostasis at the intestinal interface
Stress-free single-cell transcriptomic profiling and functional genomics of murine eosinophils
ISSN:1750-2799ISSN:1754-218
The GM-CSF-IRF5 signaling axis in eosinophils promotes antitumor immunity through activation of type 1 T cell responses.
The depletion of eosinophils represents an efficient strategy to alleviate allergic asthma, but the consequences of prolonged eosinophil deficiency for human health remain poorly understood. We show here that the ablation of eosinophils severely compromises antitumor immunity in syngeneic and genetic models of colorectal cancer (CRC), which can be attributed to defective Th1 and CD8+ T cell responses. The specific loss of GM-CSF signaling or IRF5 expression in the eosinophil compartment phenocopies the loss of the entire lineage. GM-CSF activates IRF5 in vitro and in vivo and can be administered recombinantly to improve tumor immunity. IL-10 counterregulates IRF5 activation by GM-CSF. CRC patients whose tumors are infiltrated by large numbers of eosinophils also exhibit robust CD8 T cell infiltrates and have a better prognosis than patients with eosinophillow tumors. The combined results demonstrate a critical role of eosinophils in tumor control in CRC and introduce the GM-CSF-IRF5 axis as a critical driver of the antitumor activities of this versatile cell type
Differential regulation of β-catenin-mediated transcription via N- and C-terminal co-factors governs identity of murine intestinal epithelial stem cells
The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/β-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic β-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of β-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with β-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune β-catenin’s transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant “villisation” of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires selective modulation of gene expression by transcriptional co-factors
Repression of the Antiapoptotic Molecule Galectin-3 by Homeodomain-Interacting Protein Kinase 2-Activated p53 Is Required for p53-Induced Apoptosis
Galectin 3 (Gal-3), a member of the β-galactoside binding lectin family, exhibits antiapoptotic functions, and its aberrant expression is involved in various aspects of tumor progression. Here we show that p53-induced apoptosis is associated with transcriptional repression of Gal-3. Previously, it has been reported that phosphorylation of p53 at Ser46 is important for transcription of proapoptotic genes and induction of apoptosis and that homeodomain-interacting protein kinase 2 (HIPK2) is specifically involved in these functions. We show that HIPK2 cooperates with p53 in Gal-3 repression and that this cooperation requires HIPK2 kinase activity. Gene-specific RNA interference demonstrates that HIPK2 is essential for repression of Gal-3 upon induction of p53-dependent apoptosis. Furthermore, expression of a nonrepressible Gal-3 prevents HIPK2- and p53-induced apoptosis. These results reveal a new apoptotic pathway induced by HIPK2-activated p53 and requiring repression of the antiapoptotic factor Gal-3
Single-cell RNA sequencing unveils intestinal eosinophil development and specialization
Eosinophils are an integral part of the gastrointestinal (GI) immune system that contribute to homeostatic and inflammatory processes. Here, we investigated the existence of functional subsets that carry out specialized tasks in health and disease. We used single-cell transcriptomics and high-dimensional flow cytometry to delineate murine eosinophil subpopulations and their ontogenetic relationship in the steady state and during infection and inflammation. Profiling of eosinophils from bone marrow, blood, spleen and several GI tissues revealed five distinct subsets representing consecutive developmental and maturation stages across organs, each controlled by a specific set of transcription factors. Furthermore, we discovered a highly adapted PD-L1+CD80+ eosinophil subset in the GI tract, characterized by its immune regulatory properties, bactericidal activity upon challenge infection and tissue-protective function during inflammation. Our data provide a framework for the characterization of eosinophil subsets in GI diseases and highlight their crucial contribution to homeostasis, immune regulation and host defense