Retinoid X receptor promotes hematopoietic stem cell fitness and quiescence and preserves hematopoietic homeostasis.

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain hematopoietic fitness throughout life. In steady-state conditions, HSC exhaustion is prevented by the maintenance of most HSCs in a quiescent state, with cells entering the cell cycle only occasionally. HSC quiescence is regulated by retinoid and fatty-acid ligands of transcriptional factors of the nuclear retinoid X receptor (RXR) family. Here, we show that dual deficiency for hematopoietic RXRa and RXRb induces HSC exhaustion, myeloid cell/megakaryocyte differentiation, and myeloproliferative-like disease. RXRa and RXRb maintain HSC quiescence, survival, and chromatin compaction; moreover, transcriptome changes in RXRa;RXRb-deficient HSCs include premature acquisition of an aging-like HSC signature, MYC pathway upregulation, and RNA intron retention. Fitness loss and associated RNA transcriptome and splicing alterations in RXRa;RXRb-deficient HSCs are prevented by Myc haploinsufficiency. Our study reveals the critical importance of RXRs for the maintenance of HSC fitness and their protection from premature aging.We thank the members of the J.A.C. and M.R. laboratories for extensive discussions and critiques of the manuscript. We thank Daniel Metzger (Université de Strasbourg, France) for Rxrbf/f 418 mice, Juan Carlos Zúñiga-Pflücker (Sunnybrook Health Sciences Centre, Canada) for OP9-NL1 cells, Daniel Jiménez-Carretero (CNIC) for t-SNE analysis, the CRG (Barcelona, Spain) Genomics Unit for ATACseq sequencing, and S. Bartlett (CNIC) for editorial assistance. We also thank the staff of the CNIC Cellomics and Animal facilities for technical support. This study was supported by grants from the Spanish Ministerio de Ciencia e Innovación (MICIN) (SAF2017-90604-REDT-NurCaMein, RTI2018- 095928-B100, and PID2021-122552OB-I00), La Marató de TV3 Foundation (201605-32), and the Comunidad de Madrid (MOIR-B2017/BMD-3684) to M.R and from the Formación de Profesorado Universitario (FPU17/01731) program (MICIN) to J.P. The project also received funding from the US National Institutes of Health (R01 DK124115, P01 HL158688, R01 HL147536, R01 CA237016 and U54 DK126108 to J.A.C). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN), and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033).S

Retinoid X receptor promotes hematopoietic stem cell fitness and quiescence and preserves hematopoietic homeostasis

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain hematopoietic fitness throughout life. In steady-state conditions, HSC exhaustion is prevented by the maintenance of most HSCs in a quiescent state, with cells entering the cell cycle only occasionally. HSC quiescence is regulated by retinoid and fatty-acid ligands of transcriptional factors of the nuclear retinoid X receptor (RXR) family. Herein, we show that dual deficiency for hematopoietic RXRα and RXRβ induces HSC exhaustion, myeloid cell/megakaryocyte differentiation, and myeloproliferative-like disease. RXRα and RXRβ maintain HSC quiescence, survival, and chromatin compaction; moreover, transcriptome changes in RXRα;RXRβ-deficient HSCs include premature acquisition of an aging-like HSC signature, MYC pathway upregulation, and RNA intron retention. Fitness loss and associated RNA transcriptome and splicing alterations in RXRα;RXRβ-deficient HSCs are prevented by Myc haploinsufficiency. Our study reveals the critical importance of RXRs for the maintenance of HSC fitness and their protection from premature aging

Widespread variation in molecular interactions and regulatory properties among transcription factor isoforms.

Most human Transcription factors (TFs) genes encode multiple protein isoforms differing in DNA binding domains, effector domains, or other protein regions. The global extent to which this results in functional differences between isoforms remains unknown. Here, we systematically compared 693 isoforms of 246 TF genes, assessing DNA binding, protein binding, transcriptional activation, subcellular localization, and condensate formation. Relative to reference isoforms, two-thirds of alternative TF isoforms exhibit differences in one or more molecular activities, which often could not be predicted from sequence. We observed two primary categories of alternative TF isoforms: "rewirers" and "negative regulators", both of which were associated with differentiation and cancer. Our results support a model wherein the relative expression levels of, and interactions involving, TF isoforms add an understudied layer of complexity to gene regulatory networks, demonstrating the importance of isoform-aware characterization of TF functions and providing a rich resource for further studies

STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function

<div><p>Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. <i>In vivo</i>, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. <i>In vitro</i> binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the <i>in vivo</i> separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The <i>in vitro</i> studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress.</p></div

Sequence of oligonucleotides used in DNA binding assays.

<p>Sequence of oligonucleotides used in DNA binding assays.</p

<i>In vivo</i> expression of STN1-OBM causes anaphase bridges.

<p>(A) Western blot showing levels of STN1 in HeLa cells expressing non-target shRNA (shNT) or STN1 shRNA (shSTN1) and shSTN1 cells with sh-resistant mutant STN1 (STN1-OBM) or wild type STN1 (STN1-Res). Blot was probed with antibody to STN1 or to actinin for a loading control. (B) Left; representative images of DAPI-stained anaphase cells with/ without bridges. Anaphase cells with no bridge in shNT (top) and with bridges in shSTN1 and STN1-OBM cells (middle and bottom). Right; quantification of bridges (200 anaphases counted per cell line per experiment. n = 3 experiments, mean ± S.E.M, p-values are indicated above bars).</p

Analysis of CST DNA binding parameters.

<p>(A) Binding isotherms used to determine apparent dissociation constants for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates. Data were obtained by filter binding assay and fit to a one site specific binding model. Mean ± SEM, n = 3 independent experiments each with a different protein preparation. (B) Dissociation kinetics for CST bound to the indicated substrates The fraction of labeled DNA remaining bound was determined by filter binding at the indicated times. Data were fit to a one phase exponential decay equation to obtain the dissociation rate (t½)). Mean ± SEM, n = 3 independent experiments. (C) Table summarizing Kd(app) and t½ for CST(WT) or CST(STN1-OBM) and the indicated ssDNA substrates.—undetectable binding, ND: not determined.</p

STN1-OBM rescues viability and restores origin firing after HU treatment.

<p>(A) MTT assay showing viability after HU treatment. Cells were treated with 2 mM HU for the indicated times and harvested for MTT assay 24 hrs later. Values are relative to untreated cells of the same cell type. Each time point was assayed in triplicate and the data are shown as the mean ± S.D from 3 independent experiments. For each cell line, the value of the untreated sample was set at 1. (B-D) DNA fiber analysis of origin firing following release from 2 mM HU. (B) Left: schematic showing timing of IdU and CldU labeling relative to HU treatment. Right: types of replication event scored. (C) Representative images of DNA tracks. Red, IdU; Green, CldU. (D) Graph indicating the percent of DNA tracks showing new origin firing (green-only tracks) (n = 7 experiments, mean ± S.E.M, p-values are indicated above bars).</p

STN1-OBM is competent for C-strand fill-in and TPP1 and pol α interaction.

<p>(A-C) Analysis of C-strand fill-in. (A) FACS analysis of DNA content showing synchrony of STN1-OBM cells used in (B). (B) Representative gels showing in-gel hybridization of (TA₂C₃)₄ probe to DNA from cells harvested at the indicated times after release from G1/S block. (C) Quantification of G-overhang abundance. Cell types were analyzed in pairs, n = 3 experiments for shSTN1 + STN1-OBM, mean ± S.E.M.; n = 2 experiments for STN1-OBM + STN1-Res, error bars show min/max values). (D) Western blot showing co-immunoprecipitation of TPP1 with STN1 or STN1-OBM. Cells were transfected with FLAG-STN1 or FLAG-STN1-OBM plus HA-mCherry-TPP1 expression constructs. TPP1 was precipitated with antibody to HA. (E) Co-immunoprecipitation of DNA pol α with CST. Cells were transfected with FLAG-STN1 or FLAG-STN1-OBM, Myc-CTC1 and TEN1. CST was precipitated with FLAG beads.</p

STN1-OBM causes multiple telomere signals (MTS) but does not affect G-overhang maintenance.

<p>(A) Telomere FISH of STN1-OBM or shSTN1 cells. Left; representative images of single metaphase chromosomes. White arrows, MTS; green, FITC-(C₃TA₂)₃ probe; blue, DAPI. Right; Quantification of MTS (n = 4 experiments mean ± S.E.M.). Individual chromosomes were scored positive for MTS if they had MTS at one or more telomeres. (B-C) G-overhang abundance in asynchronous cells monitored by in-gel hybridization with (A₂TC₃)₄ probe. (B) Representative gels showing hybridization to genomic DNA from the indicated cells under native or denaturing conditions. (C) Quantification of G-overhang abundance in asynchronous cells (n = 3 experiments, mean ± S.E.M., p-values are indicated above bars).</p