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Securin controls hematopoietic progenitor function
in their susceptibility to HU treatment (Figure 4A, right panel). Similarly, upon downregulation of Pttg1 in progeni- tor cells from line A and D2 mice, we observed a trend towards reduced HU sensitivity (Online Supplementary Figure S4D). These data confirm a causative role for distinct levels of expression of Pttg1 for the susceptibility of HSPC to short-term HU treatment, and thus strongly imply that Pttg1 is the QTL gene within the QTL locus.
Ultimately, the question remains whether the locus also accounts for a variation in life span. Previously, the methy- lation status of CpG sites within the genes Prima1, Hsf4, Kcns1 was shown to qualify as a reliable predictor of chronological age of B6 mice.10 This same study also revealed enhanced epigenetic aging of the D2 strain in accordance with its general reduced mean life span, sup- porting the possibility that the panel might also serve as a marker for the biological age in mice. Applying this B6- trained marker panel to our (congenic) experimental strains, we observed that epigenetic age predictions corre- lated with chronological age in B6 (R2=0.93) and line A mice (R2=0.89). Notably, epigenetic aging was clearly accelerated in line A mice compared to B6 (Figure 4B and C). We have previously demonstrated that in D2 mice the same epigenetic age predictor significantly accelerated epi- genetic age predictions that rather follow a logarithmic regression,10 which, however, line K did not deviate from (Figure 4B and C). More in depth analyses for line K would warrant the development of an improved age predictor that is adjusted to more control samples of D2, as the ini- tial marker panel was trained on B6. However, the data are consistent with a possible role of the QTL in affecting lifespan at least of line A mice, which will need to be test- ed in longevity studies of larger cohorts of animals.
Discussion
Forward genetic approaches in BXD RI strains have been shown to allow for the identification of QTL linked to lifespan and changes in various tissues and cells upon aging.22,23 We previously reported the likely linkage of a locus on the distal part of murine chromosome 11 to two phenotypes, regulation of lifespan as well the susceptibil- ity of HSPC to short-term treatment with HU. While this finding implies a common mechanism of regulation for the two phenotypes, speculations on the mechanistic con- nection between these two phenotypes remains difficult without the identification of the gene within the locus regulating at least one of the phenotypes. Here, by gener- ating and analyzing reciprocal strains congenic for the interval on chromosome 11 (B6 onto D2 and D2 onto B6), we verify the initial linkage analysis by demonstrating that this locus indeed controls the susceptibility of HSPC to HU. Other loci than the chromosome 11 locus may at least in part also contribute to the HU response pheno- type, as line A and K mice are also congenic for other loci in addition to the locus on chromosome 11 (Online Supplementary Figure S1). The proximal locus on chromo- some 11, which spans about 18.6 Mb, is, however, the only region which is identical between both congenic mouse strains, making a substantial contribution of other loci less likely (Online Supplementary Table S2). Unexpectedly, elevated sensitivity of HSPC to HU is not linked to altered cell cycle activity and thus elevated num- bers of HSPC in S-phase, nor to apoptosis, senescence or
enhanced replication fork stalling as might be anticipated by previously reported outcomes to HU exposure. The precise mechanism that confers elevated susceptibility thus still remains to be further investigated. Our data strongly support Pttg1/Securin to be the QTL gene in that interval, as elevated levels of its expression conferred by the D2 allele result in increased HU susceptibility of HSPC. Recently, Pttg1 overexpression was reported to restrict BrdU incorporation and cause enhanced levels of senescence and DNA damage in proliferating human fibroblasts,24 a feature which is not mirrored in HSPC according to our data. Thus, these mechanistic differences illustrate the unique properties of HSPC with respect to cell cycle regulation and DNA damage response, as also demonstrated recently.25-27 The initial linkage data also imply a role for Pttg1 in regulating lifespan. The primary role of Pttg1 is an inhibition of Separase. This cysteine pro- tease opens cohesin rings to allow for transition from metaphase to anaphase.28 Pttg1 is thus seen primarily as a target of the anaphase promoting complex (APC/C) to ini- tiate chromosome segregation, although other additional roles have been described in the literature, such as a cen- tral role in pituitary tumor formation when over- expressed.29 Interestingly, the APC/C is directly involved in regulating lifespan in yeast and results in dysregulation of rDNA biology,30 while likely dominant negative muta- tions in cohesin genes have been recently identified as novel contributors to the initiation of acute myeloid leukemia through modulation of chromatin accessibility in HSPC and subsequent inhibition of differentiation by recruiting “stemness” transcription factors to the daughter cells upon division. Extended presence of cohesin, in the case of elevated levels of Pttg1, might thus contribute to loss of HSPC potential, which would be consistent with our phenotype (Online Supplementary Figure S4B). Hence, the two phenotypes might be mechanistically connected via alterations in the epigenetic landscape rather than changes in chromatid cohesion itself. This interpretation is supported by the finding that age-associated DNA methy- lation changes are acquired at a different pace in congenic mouse strains. It is thus possible that HU treatment inter- feres with epigenetic parameters regulated by Pttg1/Securin.
Acknowledgments
We thank the FACS core at Ulm University, especially Ali Gawanbacht-Ramhormose and Sarah Warth for cell sorting and the Central Animal Facility of Ulm University as well as the Comprehensive Mouse and Cancer Core at CCHMC for help with mouse experiments. We are grateful to José Cancelas for providing FBMD-1 stromal feeder cells. We thank Karin Müller from the Internal Medicine III Department for assistance with the GloMax 96 luminometer, Sebastian Iben from the Dermatology Department for helpful advice regarding the promotor studies and all lab members for fruitful discussions.
Funding
Work in the laboratory of HG is supported by the Deutsche Forschungsgemeinschaft SFB 1074, the RTG 1789, and FOR 2674. AB was supported by a Bausteinprogramm of the Medical Faculty of Ulm University. WW was supported by the Else Kröner-Fresenius-Stiftung (2014_A193); by the Interdisciplinary Center for Clinical Research within the faculty of Medicine at the RWTH Aachen University (O3-3); by the Deutsche Forschungsgemeinschaft (WA 1706/8-1 and WA1706/11 1).
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