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Sirt1 Protects Stressed Adult Hematopoietic Stem Cells

Cells lacking Sirt 1 had more DNA double strand break-related aberrations, including chromosome fusions indicated by the arrow.
Cells lacking Sirt 1 had more DNA double strand break-related aberrations, including chromosome fusions indicated by the arrow.

The immune system relies on a stable pool of hematopoietic stem and progenitor cells (HSPCs) to respond properly to injury or stress. Maintaining genomic integrity and appropriate gene expression is essential for HSPC homeostasis, and dysregulation can result in myeloproliferative disorders or loss of immune function. Sirt1 is a histone deacetylase that can protect embryonic stem (ES) cells from accumulating DNA damage and has been linked to hematopoietic differentiation of ES cells. Satyendra Singh, Ph.D., a postdoctoral fellow working with Philipp Oberdoerffer, Ph.D., in CCR’s Laboratory of Receptor Biology and Gene Expression, and their colleagues set out to determine whether Sirt1 could play a similar protective role in adult HSPCs.

The researchers began their studies by generating conditional knockout (KO) mice that lose exon 4 of the Sirt1 gene in adult mice, producing an inactive Sirt1 protein, when treated with the drug 4-hydroxy-tamoxifen (4-OHT). After 4-OHT treatment, they observed increased numbers of multipotent hematopoietic progenitors from the Sirt1 KO mice compared with control animals. The investigators showed that the increase in cell numbers was due to increased proliferation of the Sirt1 KO progenitor cells. However, there was little change in the proliferation of more differentiated hematopoietic cells or non-HSPCs, demonstrating that Sirt1 can alter adult HSPC homeostasis in mice.

In contrast with these results, previous studies of mice lacking the Sirt1 gene revealed no obvious hematopoietic cell phenotype. Since 4-OHT is known to be toxic to cells and the researchers saw a decrease in the total number of bone marrow cells in mice treated with 4-OHT, they wondered whether the increased proliferation of Sirt1 KO cells was due to this stress. To test the idea, the scientists took bone marrow from 4-OHT-treated Sirt1 KO or control mice and transplanted them into mice that were irradiated to remove their bone marrow cells. In these animals, which did not receive 4-OHT, they saw no difference in the number of HSPCs suggesting a stress-dependent role for Sirt1 in HSPCs.

To get away from using 4-OHT to induce Sirt1 KO and, at the same time, cause stress, the investigators generated a second mouse model that eliminated Sirt1 exon 4 in hematopoietic cells (Sirt1Δ). Exposure to 4-OHT or a second stress-inducing chemotherapy drug, 5-fluorouracil, significantly increased the number of Sirt1Δ progenitor cells as compared with cells that express Sirt1.

Excessive proliferation of HSPCs can eventually deplete the progenitor pool. To see whether Sirt1 KO progenitors were affected by their increased proliferation in response to stress, the researchers serially transplanted control or Sirt1 KO cells into irradiated mice. Serial transplantation forces long-term progenitor cells to continually self-renew. With the first transplant, mice that received either 4-OHT-treated Sirt1 KO or control bone marrow survived. However, with the second transplant fewer mice that received Sirt1 KO cells survived. Mice that received control cells only showed reduced survival at the third transplant. These results indicate that loss of Sirt1 leads to long-term progenitor cell exhaustion.

To investigate what could be causing this stress-mediated loss of Sirt1-deficient cells, they assessed the level of DNA damage in the cells. When control or Sirt1 KO bone marrow cells were grown in medium that promotes proliferation, the researchers observed an increase in DNA double strand breaks that was further enhanced in cells lacking Sirt1. Likewise, they saw an increase in chromosome fusions, fragments, and breaks in metaphase spreads from Sirt1 KO cells. These DNA structural changes were accompanied by increased acetylation or activation of the pro-apoptotic p53 gene as well as enhanced Annexin V staining, suggesting that Sirt1 plays an important role in maintaining the genomic stability and survival of HSPCs.

To determine whether Sirt1 plays a similar role in vivo, the investigators transferred 4-OHT-treated Sirt1 KO or control bone marrow cells into irradiated mice. Similar to their in vitro results, the scientists observed increased DNA double strand breaks in Sirt1 KO HSPCs when compared with control cells. The researchers then treated Sirt1Δ or control mice with sublethal doses of radiation to see whether the increased DNA damage has an effect on HSPC function. Sirt1Δ mice were significantly more sensitive to radiation than control animals. These results demonstrate that HSPCs lacking Sirt1 are more sensitive to stress in vivo and that the accumulation of DNA damage promotes the stress-induced loss of these cells.

Finally, the investigators wanted to understand how the loss of Sirt1 led to increased cell proliferation with stress. They conducted gene expression profiling of 4-OHT-treated Sirt1 KO and control bone marrow cells. Focusing on HSPC and hematopoiesis genes, the researchers found that Hoxa9, which is required for efficient proliferation of hematopoietic progenitor cells, showed a greater than two-fold increased expression in Sirt1 KO cells. Using chromatin immunoprecipitation, the scientists showed that Sirt1 directly binds to the Hoxa9 gene. Loss of Sirt1 led to increased acetylation of the Hoxa9 gene and reduced repressive methylation.

Together these studies show that Sirt1 plays a critical role in HSPC homeostasis by protecting HSPCs from stress-induced DNA damage and regulating the expression of Hoxa9 to prevent aberrant proliferation.

Reference
Singh SK, Williams CA, Klarmann K, Burkett SS, Keller JR, Oberdoerffer P. Sirt1 ablation promotes stress-induced loss of epigenetic and genomic hematopoietic stem and progenitor cell maintenance. J Exp Med. April 29, 2013. Pubmed Link