HETEROchromatin and RETROelements: devils of aging and leukemic development – vadeRETRO

To investigate the impact of heterochromatin alterations and RTE derepression on the transcriptome and function of HSCs after IR and/or in the context of loss of TET2 function, we performed ChIP-seq or CUT&Tag experiments with an antibody recognizing the H3K9me3 mark, and RNA-seq experiments. In both cases, we bioinformatically analyzed the enrichment of H3K9me3 at, or the expression of, genes and RTEs, and validated the data obtained on some candidates targets by ChIP- and RT-qPCR.
We validated the impact of intronic L1Md on gene expression in cis after IR by deleting these LINE-1s via the CRISPR-Cas9 system and guide RNAs targeting each end of LINE-1.
We also tested the impact of TNF-a treatment on HSC function after IR by transplantation experiments of bone marrow cells sorted from mice after IR and with or without TNF-a treatment.

We show that IR leads to a loss of H3K9me3, mainly at L1Md, the most recent subfamilies of LINE-1. We also show that IR leads to a strong deregulation of the HSC transcriptome, with a loss of the TNF-a via NF-?B signaling pathway, and a loss of HSC signatures. Loss of H3K9me3 at specific L1Md, enriched in binding site motifs for NF-?B factors, and located in the introns of HSC genes, leads to the repression in cis of these genes, and loss of the HSC signature, both in the short term after IR in vitro, and in the long term in vivo. We thus show for the first time that irradiation directly modifies HSC heterochromatin, and suggest that these alterations could explain the long-term effect of IR on HSC function.
These effects are correlated with a loss of the NFKB1 repressor factor. We show that TNF-a treatment prevents loss of NFKB1 during IR, loss of H3K9me3 at L1Md, and loss of HSC signatures, both in vitro and in vivo. We also show that TNF-a treatment prevents loss of HSC function after IR, independent of the level of DNA damage in HSCs. This study is now published in Journal of experimental medicine (Pelinski et al. JEM 2022).

Our objectives are now to characterize the molecular mechanisms involved in L1Md-mediated gene repression. Our data suggest that gene repression may not be due to intronic L1Md transcription. Our hypothesis is that the loss of H3K9me3 in the gene body, by affecting the elongation rate of Pol II, leads to splicing defects and transcript instability.

We also plan to validate the impact of L1Md as a causative agent in the loss of function of HSCs.

Finally, we plan to characterize the impact of TET2 loss on the heterochromatin, transcriptome and function of HSC before and after IR.

Pelinski, Y. et al. NF-?B signaling controls H3K9me3 levels at intronic LINE-1 and hematopoietic stem cell genes in cis. Journal of Experimental Medicine 219, e20211356 (2022).

Understanding how exposure to ionizing radiations (IR) induces premature aging and installs a pre-leukemic state is a major public health concern.
Hematopoietic stem cells (HSC) give rise to all blood cell lineages. Maintenance of their integrity throughout life is thus crucial. HSC are particularly sensitive to Ionizing Radiations (IR). The persistence of panhematopoietic defects as late as 50 years after initial IR exposure in Japanese atomic bomb survivors suggests that HSC are the primary targets of IR. IR and aging induce similar defects in HSCs: DNA damage accumulation, loss of self-renewal, and a biased differentiation towards the myeloid lineage leading to increased myeloid cell counts and decline of the adaptive immune response. These changes are likely contributing to many IR-induced premature aging disorders and to the high risk of developing therapy-related myeloid neoplasms (t-MN). T-MN are associated with very poor survival and are one of the most devastating consequences of treatments applied to otherwise curable cancers. This has become a major public concern in the last decades as the number of long-term survivors of cancer treatments is continuously growing, consequently increasing the incidence of t-MN. However, the mechanisms governing HSC loss of function upon IR are unknown.
IR induces DNA double strand breaks. DNA damage is thought to be one of the main driving forces of aging. However a direct link between DSBs and long-lasting defects in HSCs function induced by IR is still lacking. On another hand, IR has been shown to induce epigenetic alterations in different tissues. Epigenetic factors controlling DNA and histone methylation are key regulators of HSC function and are often mutated in leukemia. These mutations also increase with age in otherwise healthy individuals where they confer clonal hematopoiesis. Thus epigenetic defects may be the first molecular events leading to HSC loss of function and leukemia.
Alteration of heterochromatin, that represses lineage inappropriate genes and retrotransposable elements (RTEs) expression, was reported in aging. RTE are repetitive sequences elements that can spread in the genome through a copy/paste mechanism. They constitute a great source of genomic instability. RTE uncontrolled expression is also involved in major transcriptomic changes. Recently, our team uncovered a new molecular mechanism involved in IR-induced HSC defects involving RTE. We showed that RTE are overexpressed upon IR. This is linked to persistent DNA-damage. The team also showed increased RTE expression with age in mouse and human HSCs. However, the impact of RTE derepression on HSC transcriptome and function is unknown.
We propose to explore a novel concept: that IR directly affects HSC heterochromatin. Heterochromatin alterations, by unleashing RTE, will rewire the transcriptome and act as the driving force behind HSC loss of function and the establishment of a preleukemic state.
Loss of function mutations in TET2 are among the most frequent mutations observed in clonal hematopoiesis, upon aging or in hematopoietic malignancies, and are associated with an increased risk of t-MN. WE also propose to investigate the consequences of loss of function mutations in TET2 on HSC
We will finally investigate the hypothesis that IR-induced heterochromatin alterations and TET2 loss of function may cooperate to synergistically deregulate common and/or different sets of genes/RTE finally increasing and accelerating the risk of developing t-MN.
Together, this research proposal should provide novel insights in the molecular mechanisms involved in HSC loss of function upon irradiation or aging, and the development of t-MN. It should also provide some clues on the first events involved in leukemogenesis.
The final aim of this project is to identify pathways that could be targeted therapeutically in order to alleviate the effects of genotoxic therapies and aging.