DNA damage reduces heterogeneity and coherence of chromatin motions.

Chromatin motions depend on and may regulate genome functions, in particular the DNA damage response. In yeast, DNA double-strand breaks (DSBs) globally increase chromatin diffusion, whereas in higher eukaryotes the impact of DSBs on chromatin dynamics is more nuanced. We mapped the motions of chromatin microdomains in mammalian cells using diffractive optics and photoactivatable chromatin probes and found a high level of spatial heterogeneity. DNA damage reduces heterogeneity and imposes spatially defined shifts in motions: Distal to DNA breaks, chromatin motions are globally reduced, whereas chromatin retains higher mobility at break sites. These effects are driven by context-dependent changes in chromatin compaction. Photoactivated lattices of chromatin microdomains are ideal to quantify microscale coupling of chromatin motion. We measured correlation distances up to 2 µm in the cell nucleus, spanning chromosome territories, and speculate that this correlation distance between chromatin microdomains corresponds to the physical separation of A and B compartments identified in chromosome conformation capture experiments. After DNA damage, chromatin motions become less correlated, a phenomenon driven by phase separation at DSBs. Our data indicate tight spatial control of chromatin motions after genomic insults, which may facilitate repair at the break sites and prevent deleterious contacts of DSBs, thereby reducing the risk of genomic rearrangements.

A guide for single-particle chromatin tracking in live cell nuclei.

The emergence of labeling strategies and live cell imaging methods enables the imaging of chromatin in living cells at single digit nanometer resolution as well as milliseconds temporal resolution. These technical breakthroughs revolutionize our understanding of chromatin structure, dynamics and functions. Single molecule tracking algorithms are usually preferred to quantify the movement of these intranucleus elements to interpret the spatiotemporal evolution of the chromatin. In this review, we will first summarize the fluorescent labeling strategy of chromatin in live cells which will be followed by a systematic comparison of live cell imaging instrumentation. With the proper microscope, we will discuss the image analysis pipelines to extract the biophysical properties of the chromatin. Finally, we expect to give practical suggestions to broad biologists on how to select methods and link to the model properly according to different investigation purposes.

The Impact of Pro-Inflammatory Cytokines on Alternative Splicing Patterns in Human Islets.

Alternative splicing (AS) within the ß cell has been proposed as one potential pathway that may exacerbate autoimmunity and unveil novel immunogenic epitopes in type 1 diabetes (T1D). We employed a computational strategy to prioritize pathogenic splicing events in human islets treated with IL-1ß + IFN-γ as an ex vivo model of T1D and coupled this analysis with a k-mer based approach to predict RNA binding proteins involved in AS. In total, 969 AS events were identified in cytokine-treated islets, with the majority (44.8%) involving a skipped exon. ExonImpact identified 129 events predicted to impact protein structure. AS occurred with high frequency in MHC Class II-related mRNAs, and targeted qPCR validated reduced inclusion of Exon5 in the MHC Class II gene HLA-DMB. Single molecule RNA FISH confirmed increased HLA-DMB splicing in pancreatic sections from human donors with established T1D and autoantibody positivity. Serine and Arginine Rich Splicing Factor 2 was implicated in 37.2% of potentially pathogenic events, including Exon5 exclusion in HLA-DMB. Together, these data suggest that dynamic control of AS plays a role in the ß cell response to inflammatory signals during T1D evolution.

The Impact of Pro-Inflammatory Cytokines on Alternative Splicing Patterns in Human Islets.

Alternative splicing (AS) within the ß cell has been proposed as one potential pathway that may exacerbate autoimmunity and unveil novel immunogenic epitopes in type 1 diabetes (T1D). We employed a computational strategy to prioritize pathogenic splicing events in human islets treated with IL-1ß + IFN-γ as an ex vivo model of T1D and coupled this analysis with a k-mer based approach to predict RNA binding proteins involved in AS. In total, 969 AS events were identified in cytokine-treated islets, with the majority (44.8%) involving a skipped exon. ExonImpact identified 129 events predicted to impact protein structure. AS occurred with high frequency in MHC Class II-related mRNAs, and targeted qPCR validated reduced inclusion of Exon5 in the MHC Class II gene HLA-DMB. Single molecule RNA FISH confirmed increased HLA-DMB splicing in pancreatic sections from human donors with established T1D and autoantibody positivity. Serine and Arginine Rich Splicing Factor 2 was implicated in 37.2% of potentially pathogenic events, including Exon5 exclusion in HLA-DMB. Together, these data suggest that dynamic control of AS plays a role in the ß cell response to inflammatory signals during T1D evolution.