Repetitive Elements Enhance Hematopoietic Regeneration Via Activation of the Innate Immune Receptor MDA5

Hematopoietic stem cells (HSCs) are normally quiescent and have evolved mechanisms to respond to stress. Upon stress like chemotherapy, HSCs are activated, exit quiescence and differentiate to all hematopoietic lineages, features that are highly used in the clinic. Given the clinical relevance of HSCs, understanding the molecular mechanisms governing HSCs under homeostasis and regeneration is critical. Here we evaluated hematopoietic regeneration after chemotherapy using the myeloablative agent 5-fluorouracil (5-FU). We performed chromatin accessibility assays at steady state (D0), 16 hours (H16) and 3 days (D3) after treatment of wild type mice with 5-FU and observed that robust chromatin reorganisation takes place during the early time points of the response. Expression analysis at the same time points revealed that during regeneration, inflammatory signalling is activated. Surprisingly, we also detected increased transcription of repetitive elements (REs) during early recovery. Upregulated REs were located in genomic regions that gain accessibility during regeneration. We reasoned that newly transcribed REs could be responsible for the inflammatory signalling activation by acting as ligands for the innate immune receptor MDA5. Indeed, by sequencing the RNA that is crosslinked to MDA5 upon stress we found several RE transcripts bound to MDA5. Additionally, by performing digital footprinting analysis on our chromatin accessibility data we found increased occupancy of IRF and NF-κB motifs after chemotherapy. These data provided further indication that MDA5 is activated after chemotherapy since IRF and NF-κB factors act downstream of MDA5. We then investigated the role of MDA5 in HSC regeneration in vivo. Cell cycle analysis showed that Mda5^-/- HSCs were more quiescent both at steady state and during regeneration. This quiescent phenotype was validated by metabolic assays and single-cell RNA-seq analysis. Due to their ability to retain quiescence, Mda5^-/- HSCs harbour better repopulating capacity upon bone marrow transplantation, as higher chimerism was observed in secondary recipients when injected with Mda5^-/- cells. In vitro serial Colony Forming Unit-Cell (CFU-C) of wild type and Mda5^-/- HSCs or WT HSCs treated with TBK1 inhibitor, a kinase downstream of MDA5 and essential for signal mediation, verified our transplantation experiments. We reasoned that the quiescent phenotype of Mda5^-/- HSCs could be due to the inability of Mda5^-/- HSCs to upregulate either REs or inflammatory signaling upon regeneration. To answer this question we performed chromatin accessibility and expression assays in Mda5^-/- HSCs at several time points after chemotherapy and compared them to wild type cells. Indeed, robust chromatin accessibility and upregulation of REs takes place in Mda5^-/- HSCs but inflammatory signaling is impaired. Concomitantly we observed less phosho-IRF3 and less translocation of NF-κB to the nucleus in Mda5^-/- HSCs thus verifying our expression analysis results. To prove that REs can indeed activate MDA5 leading to increased inflammatory signaling that facilitates HSCs to exit quiescence, we overexpressed two of these elements in wild type and Mda5^-/- HSCs. Indeed overexpression of both elements in wild type HSCs led to enhanced exit from quiescence but this effect was abrogated in Mda5^-/- HSCs. Our step-by-step model proposes that during regeneration, robust chromatin rearrangement and RE upregulation take place. Upregulated RE transcripts bind and activate the innate immune receptor MDA5 that in turn, stimulates the inflammatory signalling necessary for HSCs to exit quiescence and regenerate the hematopoietic system. Thus, using hematopoiesis as an exemplary system, our work uncovers a novel function of REs in tissue regeneration.