Regulation of immunoglobulin diversification by checkpoint signalling

The primary immunoglobulin (Ig) repertoire is further diversified during secondary Ig diversification in germinal centre B cells. The secondary Ig diversification via somatic hypermutation and class switch recombination in humans and mice or by Ig gene conversion in some farm animals like chickens is based on the induction of DNA damage by activation-induced cytidine deaminase (AID), an enzyme that deaminates cytidine to uracil. These DNA lesions can either be repaired in an error-prone mode in the Ig loci or in an error-free manner, which is favoured in other cellular genes. However, the molecular mechanism of this differential repair fidelity is unknown to date. Maintenance of the integrity of the entire genome is essential for cancer prevention and activation of two different checkpoint signalling axes plays an important role in triggering cell cycle arrest and improved DNA repair. In case of long single stranded regions as a result of stalled replication forks or single strand breaks, ATR is activated, which phosphorylates the checkpoint kinase 1 (Chk1). On the other hand, DNA double strand breaks activate ATM, which phosphorylates the checkpoint kinase 2 (Chk2). Given the extensive DNA damage caused by AID during Ig diversification, activation of checkpoint control is likely, but not much is known about the effects of checkpoint signalling on secondary Ig diversification processes. In this work, we could show that the two checkpoint kinases are involved and oppose each other in the regulation of somatic hypermutation, Ig gene conversion and class switch recombination. Our results suggest that Ig diversification depends on a highly regulated interplay of the two checkpoint kinases, as Chk2 is required for efficient somatic hypermutation and class switch recombination, while Chk1 activity may contribute to the prevention of B cell lymphomas

Rapid Genome-wide Recruitment of RNA Polymerase II Drives Transcription, Splicing, and Translation Events during T Cell Responses

Summary: Activation of immune cells results in rapid functional changes, but how such fast changes are accomplished remains enigmatic. By combining time courses of 4sU-seq, RNA-seq, ribosome profiling (RP), and RNA polymerase II (RNA Pol II) ChIP-seq during T cell activation, we illustrate genome-wide temporal dynamics for ∼10,000 genes. This approach reveals not only immediate-early and posttranscriptionally regulated genes but also coupled changes in transcription and translation for >90% of genes. Recruitment, rather than release of paused RNA Pol II, primarily mediates transcriptional changes. This coincides with a genome-wide temporary slowdown in cotranscriptional splicing, even for polyadenylated mRNAs that are localized at the chromatin. Subsequent splicing optimization correlates with increasing Ser-2 phosphorylation of the RNA Pol II carboxy-terminal domain (CTD) and activation of the positive transcription elongation factor (pTEFb). Thus, rapid de novo recruitment of RNA Pol II dictates the course of events during T cell activation, particularly transcription, splicing, and consequently translation. : Davari et al. visualize global changes in RNA Pol II binding, transcription, splicing, and translation. T cells change their functional program by rapid de novo recruitment of RNA Pol II and coupled changes in transcription and translation. This coincides with fluctuations in RNA Pol II phosphorylation and a temporary reduction in cotranscriptional splicing. Keywords: RNA Pol II, cotranscriptional splicing, T cell activation, ribosome profiling, 4sU, H3K36, Ser-5 RNA Pol II, Ser-2 RNA Pol II, immune response, immediate-early gene

Electron transfer pathways in a light, oxygen, voltage (LOV) protein devoid of the photoactive cysteine

Blue-light absorption by the flavin chromophore in light, oxygen, voltage (LOV) photoreceptors triggers photochemical reactions that lead to the formation of a flavin-cysteine adduct. While it has long been assumed that adduct formation is essential for signaling, it was recently shown that LOV photoreceptor variants devoid of the photoactive cysteine can elicit a functional response and that flavin photoreduction to the neutral semiquinone radical is sufficient for signal transduction. Currently, the mechanistic basis of the underlying electron- (eT) and proton-transfer (pT) reactions is not well understood. We here reengineered pT into the naturally not photoreducible iLOV protein, a fluorescent reporter protein derived from the Arabidopsis thaliana phototropin-2 LOV2 domain. A single amino-acid substitution (Q489D) enabled efficient photoreduction, suggesting that an eT pathway is naturally present in the protein. By using a combination of site-directed mutagenesis, steady-state UV/Vis, transient absorption and electron paramagnetic resonance spectroscopy, we investigate the underlying eT and pT reactions. Our study provides strong evidence that several Tyr and Trp residues, highly conserved in all LOV proteins, constitute the eT pathway for flavin photoreduction, suggesting that the propensity for photoreduction is evolutionary imprinted in all LOV domains, while efficient pT is needed to stabilize the neutral semiquinone radical