Nicolls, M. R. | Search bioRxiv

bioRxiv2018

Inducible expression of immediate early genes is regulated through dynamic chromatin association by NF45/ILF2 and NF90/ILF3

Immediate early gene (IEG) transcription is rapidly activated by diverse stimuli without requiring new protein synthesis. This transcriptional regulation is assumed to involve constitutively expressed nuclear factors that are targets of signaling cascades initiated at the cell membrane. NF45 and its heterodimeric partner NF90 are chromatin-interacting proteins that are constitutively expressed and localized predominantly in the nucleus. Previously, NF90 chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in K562 erythroleukemia cells revealed its enriched association with chromatin at active promoters and strong enhancers. NF90 specifically occupied the promoters of IEGs. Here, ChIP in serum-starved HEK293 cells demonstrated that NF45 and NF90 pre-exist and specifically occupy the promoters of IEG transcription factors EGR1, FOS and JUN. Cellular stimulation with phorbol myristyl acetate increased NF90 occupancy, while decreasing NF45 occupancy at promoters of EGR1, FOS and JUN. In HEK293 cells stably transfected with doxycycline-inducible shRNA vectors targeting NF90 or NF45, doxycycline-mediated knockdown of NF90 or NF45 attenuated the inducible expression of EGR1, FOS, and JUN at the levels of mRNA and protein. NF90 and NF45 operate as constitutively-expressed transcriptional regulators of IEGs. Dynamic chromatin association of NF45 and NF90 at IEG promoters are observed upon stimulation, and NF45 and NF90 contribute to inducible expression of IEGs. NF45 and NF90 operate as chromatin regulators of the immediate early response.

cell biology↗

bioRxiv2017

Dynamics Of The Human Antibody Repertoire Following B-cell Depletion In Systemic Sclerosis

Systemic sclerosis with pulmonary arterial hypertension (SSc-PAH) is a debilitating and frequently lethal disease of unknown cause lacking effective treatment options. Lymphocyte anomalies and autoantibodies observed in systemic sclerosis have suggested an autoimmune character. Here we study the clonal structure of the B-cell repertoire in SSc-PAH using immunoglobulin heavy-chain sequencing before and after B-cell depletion. We found SSc-PAH to be associated with anomalies in B-cell development, namely altered VDJ rearrangement frequencies (reduced IGHV2-5 segment usage) and an increased somatic mutation-fixation probability in expanded B-cell lineages. SSc-PAH was also characterized by anomalies in B-cell homeostasis, namely an expanded IgD+ proportion with reduced mutation loads and an expanded proportion of highly antibody-secreting cells. Disease signatures pertaining to IGHV2-5 segment usage, IgD proportions and mutation loads were temporarily reversed after B-cell depletion. Analyzing the time course of B-cell depletion, we find that the kinetics of naive replenishment are predictable from baseline measurements alone, that release of plasma cells into the periphery can precede naive replenishment and that modes of B-cell receptor diversity are highly elastic. Our findings shed light on the humoral immune basis of SSc-PAH and provide insights into the effect of B-cell depletion on the antibody repertoire.\n\nAbbreviations

immunology↗