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C. Leveau et al.
Furthermore, TTC7A reportedly interacts with EFR3 homolog B and phosphatidylinositol 4-kinase alpha, which is known to catalyze the production of phos- phatidylinositol 4-phosphate at the plasma membrane in yeast and human cells.17,18 This observation emphasizes the conservation, at least in part, of the functions of Ttc7a during evolution. However, data on TTC7A’s biological function(s) are still scarce.
Inadequate proliferation of peripheral hematopoietic lineages has been reported in several modified murine models; this impairment is ultimately associated with the exhaustion of the hematopoietic stem cell (HSC) pool.19 Indeed, the production of blood cells requires HSC to leave their quiescent state and differentiate into functional progeny. An excessive requirement for hematopoietic cell production biases HSC function toward differentiation, at the expense of self-renewal.20 Various intrinsic and extrin- sic factors influence HSC fate, i.e. quiescence or prolifera- tion. Endoplasmic reticulum (ER) stress has recently been highlighted as an important regulator of HSC function.21 This stress is triggered by various stimuli and leads to the accumulation of unfolded proteins in the lumen of the ER, and induction of the unfolded protein response (UPR). The chaperone BIP (Hspa5/GRP78) is the main inducer of the UPR.22 This response results in enhanced expression of chaperone proteins (heat shock proteins, Hsp), phospho- diesterase (Pdi), and other proteins such as calreticulin that, together with BIP, boost protein folding capacities. Depending on the intensity of the ER stress, UPR activa- tion can lead to apoptosis or survival.23
In the present study, we found that Ttc7a regulates murine HSC self-renewal and hematopoietic reconstitu- tion potential and controls the sensitivity of these cells to stress. Loss of Ttc7a consistently enhanced HSC stemness, since Ttc7a-deficient HSC displayed a greater proliferation capacity than control counterparts in response to ER stress in vitro, and after myeloablative stress in vivo. Hence, our results reveal a new role for Ttc7a as a regulator of self- renewal and response to stress in HSC.
Methods
Mice
Heterozygous Balb/cByJ fsn (CByJ.A-Ttc7fsn/J) mice and Balb/cByJ CD45.1 (CByJ.SJL(B6)-Ptprca/J) mice were obtained from the Jackson Laboratory. All mice were maintained in specific pathogen-free conditions and handled according to national and institutional guidelines.
Repopulations assays
Bone marrow (BM) cells were transferred into CD45.1+ control recipient mice upon irradiation and then 30,000 Lin- Sca1+ cKit+ (LSK) donor cells were injected into the irradiated recipient mice. For serial transplantations, recipients were reconstituted with 107 BM cells. To perform competitive repopulation assays, 1,000 LSK cells were injected with 2 x 106 unfractionated CD45.1+ BM cells. Twelve weeks after transfer, mice were treated with a single dose of 5-fluorouracil (5-FU, 150 mg/kg).
Flow cytometry and isolation of hematopoietic stem cells
Splenocytes and peripheral blood cells were incubated with conjugated antibodies and viability exclusion dyes. The antibodies used are listed in Online Supplementary Table S2. Stained cells were
quantified using a Gallios flow cytometer (Beckman Coulter), and analyzed with FlowJo software (Treestar). HSC and LSK cells were isolated by depleting Lin+ cells using the Lineage Cell Depletion Kit according to the manufacturer’s protocol (Miltenyi Biotec), stained with a Lin- antibody cocktail, and antibodies against CD117, Sca-1, CD150 and CD48, and sorted with FACS AriaTM (BD Biosciences).
Cell culture
Lin- cells were cultured in StemSpan medium (StemCell Technologies) supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin, recombinant human thrombopoietin (100 mg/mL), recombinant murine stem cell factor (100 mg/mL) and recombinant murine FLT3 ligand (100 mg/mL). Tunicamycin (Cayman Chemical) was added (0.6 or 1.2 mg/mL) for 24 or 48 h.
RNA-sequencing
RNA was extracted using the ZR-RNA MicroPrepTM isolation kit (Proteinegene). cDNA libraries were generated using the Ovation SoLo RNA-seq system (NuGEN). The libraries were controlled with a High Sensitivity DNA Analysis Kit and Bioanalyzer (Agilent). NextSeq 500 (Illumina) was used for sequencing. FASTQ files were mapped to the ENSEMBL MM38 reference using Hisat2 and counts were produced with feature Counts. Read count nor- malization and group comparisons were performed by DESeq2, edgeR, and LimmaVoom. Heatmaps were made with R and imaged by Java Treeview software. Differentially expressed genes were examined with gene set enrichment analysis (GSEA) for functional enrichment in gene ontology (GO) terms using normal- ized expression values of LimmaVoom.
Western blot
Lin- cells were cultured for 3 days and HSC were sorted directly into 10% trichloroacetic acid. Proteins were extracted and solubi- lized as previously described.24
Statistical analysis
Data were analyzed with GraphPad Prism 6 software. Statistical analyses were performed using two-tailed Student t-test. Differences were considered to be statistically significant when P<0.05 (indicated as *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001).
Data availability
The data are available at the Sequence Read Analysis (SRA) database under accession number SRA139913.
Results
Ttc7a is required for the maintenance of immune homeostasis
It has previously been shown that adult Ttc7a-deficient (fsn) mice (aged 8 to 10 weeks) develop an imbalance in hematopoiesis, characterized by leukocytosis and anemia.7 To gain insight into the change over time in the fsn mice’s pathology, we analyzed the different hematopoietic lineages in the blood and the spleen at 3, 6 and 12 weeks of age. Fsn mice had a considerably higher circulating leukocyte count than control littermates (ctrl) at all time points (Figure 1A). The spleen was much larger in fsn mice than in ctrl mice, twice as large at 3 weeks and ten times larger at 12 weeks (Figure 1B). The splenic architec- ture in fsn mice became increasingly disorganized, with an age-related expansion of red and white pulp (Figure 1C).
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haematologica | 2020; 105(1)