Page 84 - 2020_02-Haematologica-web

A. Brown et al.
the regulation of both phenotypes. To verify the linkage, and identify the underlying quantitative trait gene, we generated B6 as well as D2 mice that are reciprocally con- genic for this locus on chromosome 11.
Methods
Mice
Laboratory C57BL/6J (B6), DBA/2J (D2) and BXD inbred mice were obtained from Janvier Labs (France). All mice were fed acid- ified water and food ad libitum, and housed under pathogen-free conditions at the University of Kentucky, Division of Laboratory Animal Resource, the animal facility at CCHMC. Mouse experi- ments were performed in compliance with the German Law for Welfare of Laboratory Animals and were approved by the Regierungspräsidium Tübingen or approved by the IACUCs of the University of Kentucky and CCHMC.
Quantitative trait locus mapping
Linkage analysis and determination of the likelihood ratio sta- tistic values for suggestive linkage were performed as described by using WebQTL (http://www. genenetwork.org/ webqtl/ main.py? FormID=submitSingleTrait), identifying the restrictive chromosome 11 locus, among others, correlating to mean life span and HU sen- sitivity.3-6
Generation of congenic mice
Congenic animals were generated in five generations by a marker-assisted backcrossing strategy as described3,5,7-9 (Figure 1C). The particular DBA/2J genomic region was derived from BXD31, one of the BXD recombinant inbred strains used in the quantitative trait locus (QTL) mapping and which phenotypically best demonstrated the decline in HSC in old age and the HU sensitivity.3
Preparation of hematopoietic tissue and cells
For the isolation of total bone marrow (BM), tibiae, femur and hips of mice were isolated and flushed using a syringe and a G21 needle. Mononuclear (low density bone marrow, LDBM) cells were isolated by Histopaque low-density centrifugation (#10831, Sigma). Lineage depletion was performed using the mouse lineage cell depletion Kit (#130-090-858, Miltenyi Biotec) according to their protocol.
Cobblestone area forming cell assay
Cobblestone area forming cell (CAFC) assay was performed as described.1 Briefly, 1,000 FBMD-1 cells, a stromal cell line, were seeded in each well of 96-well plates. Plates were incubated at 33°C in 5% C02, and used seven days later for CAFC assay. BM cells were either treated with 200 μg/mL HU or its solvent (PBS) and seeded onto the pre-established stromal layers in six dilutions, serially in 3-fold increments from 333 to up to 81,000 cells/well (12 wells per dilution). At this time, the medium was switched from 5% horse serum and 10% fetal bovine serum to 20% horse serum. Alternatively, mice were treated with HU in vivo as indicated fol- lowing bone marrow isolation and seeding. After seven days, all wells were evaluated for the presence or absence of cobblestone areas and the frequency of the appearance of a colony calculated using L-Calc software (STEMCELL Technologies).
Analysis of the epigenetic aging signature
Analysis of DNA methylation levels was analyzed at three age- associated CG dinucleotides (CpG) as described previously.10 Briefly, genomic DNA was isolated from blood samples, bisulfite converted, and DNA methylation was analyzed within the three
genes (Prima1, Hsf4, Kcns1) by pyrosequencing. The DNA methy- lation results at these sites can be integrated into a multivariable model for epigenetic age predictions in B6 mice, which clearly cor- relate with the chronological age.10
Statistical analysis
All statistical analyses were performed using Student’s t-test or two-way Anova, when appropriate with GraphPad Prism 6 soft- ware. For Figure 4C, linear and non-linear regression was calculat- ed. The number of biological repeats (n) is indicated in the figure legends. Error bars are Standard Error of Mean (SEM).
Results
Hematopoietic stem and progenitor cells from BXD RI strains show highly divergent reactions when exposed to HU as judged by their ability to form cobblestones on stromal feeder layers in the CAFC assay after seven days of culture (CAFC day 7 assay).9 Re-analyzing the initial phenotypic data based on the most recent marker map (New Genotypes 2017 dataset) provided for BXD RI strains, we verified the initially identified locus on chro- mosome 11 (35-75 Mb) linked (with a suggestive thresh- old of 10.53/10.88) to both HU susceptibility of HSPC as well as mean lifespan of the analyzed mice (Figure 1A and B and Online Supplementary Tables S1A and B, and S3). We used a marker assisted speed congenic approach to obtain a reciprocal set of mice congenic for the chromosome 11 locus (Figure 1C). These novel mouse lines were named line A (D2 onto B6) and K (B6 onto D2). We performed whole genome SNP mapping of our congenic mouse strains to identify the length of the congenic intervals transferred as well as the overlap between the reciprocal strains. Ultimately, the common region transferred in line A and line K spans an 18.6 Mb (8.3 cM) region on chromo- some 11 from rs26900200, 37,929,686 bp to rs3088940, 56,516,067 bp with no other transferred intervals stem- ming from the donor strains that are identical between the two congenic strains. The SNP analysis further revealed a small set of additional congenic regions in both line A and K animals, though not covering identical regions (Figure 1D, Online Supplementary Table S2 and Online Supplementary Figure S1). This interval contains about 130 protein coding genes (Online Supplementary Table S3).
Next, based on the CAFC assay, we tested whether the genotype of the locus conferred in the congenic strains correlated with the magnitude of our phenotype of HSPC susceptible to HU. HU treatment efficiently suppresses BrdU incorporation and thus active S-Phase in freshly iso- lated Lin-cKit+ (LK) cells from all strains (Online Supplementary Figure S2A). Indeed, HSPC isolated from B6 or line K (B6 onto D2) mice presented with a lower fre- quency of dysfunctional HSPC in response to short-term in vivo as well as to ex vivo treatment with HU, while inversely, D2 and line A (D2 onto B6) HSPC were more sensitive to HU (Figure 2A and Online Supplementary Figure S2B). These data confirm that the interval on chro- mosome 11 shared among the congenic strains confers this phenotype and might thus contain a gene regulating the response of HSPC to HU.
Since HU inhibits dNTP synthesis,11 and a lack of dNTP causes replication fork stalling and thus DNA damage and apoptosis,12 it is believed that the frequency of cells sus- ceptible to HU treatment is an indirect measurement for
318
haematologica | 2020; 105(2)