Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation
Morgan Jones, … , Catherine E. Keegan, Ivan Maillard
Morgan Jones, … , Catherine E. Keegan, Ivan Maillard
Published December 9, 2013
Citation Information: J Clin Invest. 2014;124(1):353-366. https://doi.org/10.1172/JCI67871.
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Research Article Hematology

Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation

Abstract

The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors.

Authors

Morgan Jones, Gail Osawa, Joshua A. Regal, Daniel N. Weinberg, James Taggart, Hande Kocak, Ann Friedman, David O. Ferguson, Catherine E. Keegan, Ivan Maillard

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Figure 1

Reduced Acd expression results in phenotypic abnormalities and G2/M arrest in fetal hematopoietic progenitors.

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Reduced Acd expression results in phenotypic abnormalities and G2/M arre...
(A) Structure of the hypomorphic acd allele, a mutant Acd allele arising from an intron 3 G→A transition and leading to aberrant splicing. acd homozygosity decreased the abundance of normal transcripts to approximately 2% of WT (33). (B) Fetal liver cellularity in E13.5 mice homozygous for the acd hypomorphic allele. (C) GM-CFU analysis demonstrating preserved GM progenitor activity in E13.5 acd fetal liver (representative of three experiments with triplicate colony formation assays; mean ± SEM). (D) Preserved overall frequency of phenotypically defined long-term hematopoietic stem cells (LT-HSC), defined as CD150+CD48– Lin–Sca-1hicKithi (LSK) cells (n ≥9 mice per group from five independent experiments; mean ± SEM). (E) Increased Sca-1 expression in acd LSK progenitors. acd LSK cells were also larger and more granular by forward (FSC-A) and side scatter (SSC-A) characteristics, respectively (n ≥9 mice per group from five independent experiments). Gray shading shows data from control littermates, and white shading represents acd progenitors. (F) Cell cycle analysis with BrdU incorporation (12-hour pulse) and intracellular DAPI (icDAPI) staining for DNA content in E13.5 acd Lin–cKithi hematopoietic progenitors showing accumulation in G2/M phases of the cell cycle (n = 3 mice per group from three independent experiments; mean ± SD). Representative flow cytometry plots are shown. Numbers indicate the percentage of cells in each gate. *P < 0.05; **P < 0.01; ***P < 0.001.