Increased γH2AX Foci in Old Hematopoietic Stem Cells Are Independent of the DNA Damage Response and Linked to Inefficient DNA Replication/Transcription

Abstract 1207

Hematopoietic stem cells (HSCs) are responsible for the life-long production of all blood cells. Changes in the biological function of old HSCs have been directly linked to the occurrence of age-related blood defects including immunosenescence, anemia and the development of a broad spectrum of hematological disorders (i.e., myeloproliferative neoplasms, leukemia, bone marrow failure). Gene expression studies and analysis of genetically modified mice have also suggested that error-prone DNA repair as well as a decrease in genomic stability are one of the driving forces for the reduced functional capacity of old HSCs. Here, we used HSCs (Lin^-/c-Kit⁺/Sca-1⁺/Flk2^-/CD48^-/CD150⁺) isolated from the bone marrow of old (20–24 months old) and young (6–12 weeks old) C57Bl/6 mice to directly investigate the DNA damage response of old HSCs.

Using immunofluorescence, we first confirmed that freshly isolated quiescent old HSCs have an increased number of γH2AX foci, which is a well-established indicator for DNA double-strand breaks. In addition, we found that these intrinsically occurring γH2AX foci specifically co-localized with nucleolar markers (i.e., UBF, fibrillarin and nucleolin) and were a cell-intrinsic feature of old HSCs as demonstrated by transplantation experiments of old HSCs into young recipient mice. However, we could not demonstrate that nucleolar γH2AX foci in old HSCs represent sites of DNA damage. Neither did known DNA repair-associated markers like 53BP1 co-localize with nucleolar γH2AX foci, nor were other DNA damage markers such as phospho-ATM or PARP1 increased in old HSCs. In addition, none of the other methods we used to measure DNA fragmentation such as TUNEL or COMET assays revealed elevated levels of DNA damage in old HSCs, and spectral karyotyping (SKY) analysis of in vitro cultured old HSCs did not provide evidence for DNA damage-associated chromosomal alterations. We then used 2Gy ionizing radiation (IR) to directly induce DNA double-strand breaks and measured the DNA repair capacity of old HSCs. Strikingly, we observed a similar DNA damage response and DNA repair kinetics in young and old HSCs. These results provide evidence that old HSCs can respond adequately to DNA damage and that accumulation of γH2AX at the nucleolus is not the consequence of an activated DNA damage response.

The nucleolus consists of a highly regulated repetitive sequence of rDNA units and is the site of ribosomal DNA transcription. Both young and old quiescent HSCs have well-formed nucleoli as shown by electron microscopy analyses. Strikingly, we found a complete disappearance of nucleolar γH2AX foci when old HSCs are forced into cell cycle upon in vitro culture. Furthermore, we observed a significant delay in the onset of the first cell division and timing of nucleolar reformation following mitosis in old HSCs. In addition, cycling old HSCs displayed higher levels of DNA replication/transcription-associated γH2AX foci compared to cycling young HSCs. We are currently investigating how defects in the DNA replication machinery could contribute to the nucleolar γH2AX foci and cell cycle features of old HSCs, and whether old HSCs maintain similar levels of rRNA transcription as compared to young HSCs.

Taken together, our results demonstrate that the increased numbers of γH2AX foci observed in old HSCs is not caused by an accumulation of DNA damage. Instead, nucleolar γH2AX foci appear to be a hallmark feature of quiescent old HSCs that could reflect epigenetic changes in rDNA chromatin structure linked to inefficient DNA replication/transcription.