24 research outputs found
The transcription factor STAT6 mediates direct repression of inflammatory enhancers and limits activation of alternatively polarized macrophages
The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1β production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli
Class IHLA oligomerization at the surface of B cells is controlled by exogenous βâ-microglobulin: implications in activation of cytotoxic T lymphocytes
Submicroscopic molecular clusters (oligomers) of class I HLA have been detected by physical techniques [e.g. fluorescence resonance energy transfer (FRET) and single particle tracking of molecular diffusion] at the surface of various activated and transformed human cells, including B lymphocytes. Here, the sensitivity of this homotypic association to exogenous βâ-microglobulin (βâm) and the role of free heavy chains (FHC) in class I HLA oligomerization were investigated on a B lymphoblastoid cell line, JY. Scanning near-field optical microscopy and FRET data both demonstrated that FHC and class I HLA heterodimers are co-clustered at the cell surface. Culturing the cells with excess βâm resulted in a reduced co-clustering and decreased molecular homotypic association, as assessed by FRET. The decreased HLA clustering on JY target cells (antigen-presenting cells) was accompanied with their reduced susceptibility to specific lysis by allospecific CD8âş cytotoxic T lymphocytes (CTL). JY B cells with reduced HLA clustering also provoked significantly weaker T cell activation signals, such as lower expression of CD69 activation marker and lower magnitude of TCR down-regulation, than did the untreated B cells. These results together suggest that the actual level of βâm available at the cell surface can control CTL activation and the subsequent cytotoxic effector function through regulation of the homotypic HLA-I association. This might be especially important in some inflammatory and autoimmune diseases where elevated serum βâm levels are reported
Class IHLA oligomerization at the surface of B cells is controlled by exogenous βâ-microglobulin: implications in activation of cytotoxic T lymphocytes
Submicroscopic molecular clusters (oligomers) of class I HLA have been detected by physical techniques [e.g. fluorescence resonance energy transfer (FRET) and single particle tracking of molecular diffusion] at the surface of various activated and transformed human cells, including B lymphocytes. Here, the sensitivity of this homotypic association to exogenous βâ-microglobulin (βâm) and the role of free heavy chains (FHC) in class I HLA oligomerization were investigated on a B lymphoblastoid cell line, JY. Scanning near-field optical microscopy and FRET data both demonstrated that FHC and class I HLA heterodimers are co-clustered at the cell surface. Culturing the cells with excess βâm resulted in a reduced co-clustering and decreased molecular homotypic association, as assessed by FRET. The decreased HLA clustering on JY target cells (antigen-presenting cells) was accompanied with their reduced susceptibility to specific lysis by allospecific CD8âş cytotoxic T lymphocytes (CTL). JY B cells with reduced HLA clustering also provoked significantly weaker T cell activation signals, such as lower expression of CD69 activation marker and lower magnitude of TCR down-regulation, than did the untreated B cells. These results together suggest that the actual level of βâm available at the cell surface can control CTL activation and the subsequent cytotoxic effector function through regulation of the homotypic HLA-I association. This might be especially important in some inflammatory and autoimmune diseases where elevated serum βâm levels are reported
Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA
By using a microscopic approach, field inversion single-cell gel electrophoresis, we show that preformed single-strand discontinuities are present in the chromatin of resting and proliferating mammalian and yeast cells. These single-strand breaks are primarily nicks positioned at â50-kbp intervals throughout the entire genome that could be efficiently labeled in situ by DNA polymerase I holoenzyme but not by Klenow fragment and terminal transferase unless after ribonucleolytic treatments. The RNA molecules involved appear to comprise R-loops, recognized by the S9.6 RNA/DNA hybrid-specific antibody. By using the breakpoint cluster region of the Mixed Lineage Leukemia (MLL) gene as a model, we have found that the number of manifest nicks detected by FISH performed after field inversion single-cell gel electrophoresis depends on epigenetic context, but the difference between germ-line and translocated MLL alleles is abolished by protease treatment. Our data imply that the double-stranded genomic DNA is composed of contiguous rather than continuous single strands and reveal an aspect of higher-order chromatin organization with ribonucleoprotein-associated persistent nicks defining â50-kbp domains