The rate of protein synthesis in hematopoietic stem cells is limited partly by 4E-BPs

Here, Signer et al. investigated the mechanism underlying the limited rate of protein synthesis in hematopoietic stem cells (HSCs). The authors found that adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein1 (4E-BP1) and 4E-BP2 as compared with most other hematopoietic progenitors, providing new insight into the mechanism by which HSCs attenuate protein synthesis.

Adult hematopoietic stem cell (HSC) maintenance depends on these cells making less protein per hour as compared with other hematopoietic progenitors (Signer et al. 2014). This does not reflect merely HSC quiescence, as dividing HSCs also make less protein per hour as compared with other hematopoietic progenitors. Several other adult stem cells also exhibit lower rates of protein synthesis as compared with progenitors in the same tissues, including neural stem cells (Llorens-Bobadilla et al. 2015), skeletal muscle stem cells (Zismanov et al. 2016), hair follicle stem cells (Blanco et al. 2016), and Drosophila germline stem cells (Sanchez et al. 2016). Like HSCs, each of these stem cells is depleted by genetic changes that increase their rate of protein synthesis, suggesting that this is a widely shared property of adult stem cells. However, there is limited insight into the mechanisms that suppress protein synthesis in stem cells.
Ribosome biogenesis limits protein synthesis in some stem cells. Quiescent neural stem cells express lower levels of ribosomal subunits and synthesize protein at a lower rate as compared with activated neural stem cells (Llorens-Bobadilla et al. 2015). Increased ribosome biogenesis in Drosophila germline stem cells increases protein synthesis, promoting differentiation and the loss of stem cells (Sanchez et al. 2016). Replication stress in HSCs from old mice transcriptionally silences ribosome genes, reducing ribosome biogenesis and perhaps impairing HSC function during aging (Flach et al. 2014). Runx1-deficient HSCs have impaired ribosome biogenesis, lower protein synthesis, and altered function (Cai et al. 2015). Defects in ribosome biogenesis impair HSC function (Le Bouteiller et al. 2013).
Phosphorylated eukaryotic initiation factor 2α (eIF2α) inhibits translation initiation and is part of the mechanism that limits protein synthesis in some stem cells. eIF2α can be phosphorylated under steady-state circumstances, although phosphorylation is increased by a wide range of stresses, including unfolded protein responses (Wek et al. 2006). Protein synthesis in skeletal muscle stem cells is limited by high levels of phosphorylated eIF2α, which promotes quiescence and stem cell maintenance (Zismanov et al. 2016). However, eIF2α function may vary among stem cells, as HSCs express relatively low levels of phosphorylated eIF2α (Signer et al. 2014), and activation of the unfolded protein response can be deleterious to individual HSCs (van Galen et al. 2014). mTORC1 signaling promotes protein synthesis through phosphorylation and activation of ribosomal protein S6 kinase 1 (Brown et al. 1995) and phosphorylation and inhibition of eIF4E-binding proteins (4E-BPs) (Beretta et al. 1996;Brunn et al. 1997). Three genes (Eif4ebp1, Eif4ebp2, and Eif4ebp3) encode 4E-BPs that negatively regulate translation by binding the cap-binding protein eIF4E, inhibiting eIF4G binding, and preventing eIF4F complex assembly (eIF4E-eIF4G-eIF4A). Phosphorylation of 4E-BPs by mTORC1 weakens their binding to eIF4E, promoting eIF4F assembly and cap-dependent translation (Pause et al. 1994;Gingras et al. 1999).
The synthesis of O-propargyl-puromycin (OP-Puro) has facilitated the quantification of protein synthesis in individual cells in vivo (Liu et al. 2012). OP-Puro enters the acceptor site of ribosomes and is incorporated into nascent polypeptide chains. The amount of protein synthesis per hour in individual cells in vivo can then be quantified based on OP-Puro incorporation (Liu et al. 2012;Signer et al. 2014). In this study, we examined potential mechanisms that might influence differences in protein synthesis between HSCs and restricted hematopoietic progenitors in adult mouse bone marrow.

Little correlation between protein synthesis and cell division or RNA content
We administered the thymidine analog 5-ethynyl-2 ′ -deoxyuridine (EdU) to mice and measured its incorporation by HSCs and restricted progenitors in the bone marrow after 2, 6, and 12-24 h to determine the frequency of cell division in each population (Fig. 1D). We observed a reasonably linear increase in the fraction of EdU + cells over time in each population (Supplemental Fig. S1B-M). We observed a modest correlation between protein synthesis rates and cell division (R = 0.42, P = 0.23) (Fig. 1E). However, when we drove HSCs into cycle by treating them with cyclophosphamide and GCSF, we observed only a limited increase in protein synthesis (Fig. 1E). Therefore, the rate of protein synthesis in HSCs was not determined primarily by the rate at which they divide.
To test whether protein synthesis rates reflected total RNA content (largely ribosomal RNA), we quantified total RNA by nanofluidic electrophoresis using a BioAnalyzer. HSCs had amounts of total RNA similar to those of CMPs and pro-B cells and significantly more total RNA per cell than Gr-1 + , pre-B, and unfractionated bone marrow cells (Supplemental Fig. S1N), each of which had higher protein synthesis rates than HSCs (Fig. 1A). Therefore, we observed little correlation between RefSeq genes in each cell population. Two-sided paired Wilcoxon ranksum test was used for pair-wise comparisons between HSCs and each cell type. (#) P < 2.2 × 10 −16 . (H) Mean protein synthesis (from A) plotted against median total RPKMs (from G). Data represent mean ± SD unless indicated otherwise. The statistical significance of differences relative to HSCs in A, B, and D were assessed using a repeated-measures one-way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons. ( * ) P < 0.05; ( * * ) P < 0.01; ( * * * ) P < 0.001. Regression analyses in C, E, and F were performed excluding HSCs, which were plotted independently. Ninety-five percent confidence intervals (dashed lines) and Pearson's correlation coefficients (R) are shown.
We quantified the amount of mRNA per cell by RNA sequencing (RNA-seq) after adding RNA standards that allow normalization of transcript numbers to cell number ( Fig. 1F; Loven et al. 2012). On average, HSCs had significantly more mRNA per cell as compared with pre-B cells, unfractionated bone marrow, and Gr-1 + cells but less mRNA per cell than CMPs, GMPs, and pro-B cells (Fig. 1G), all of which had higher protein synthesis rates than HSCs (Fig. 1A). Therefore, we observed little correlation between protein synthesis rates and mRNA content in hematopoietic cells (R = 0.48, P = 0.28) (Fig.  1H).
To test the effect of 4E-BP deficiency on HSC function, we performed competitive reconstitution assays in irradiated mice. Bone marrow cells (5 × 10 5 cells) from 4E-BP1 −/− , 4E-BP2 −/− , 4E-BP1 −/− ;4E-BP2 −/− , or wild-type mice (all CD45.2 + ) were transplanted with equal numbers of wild-type recipient bone marrow cells (CD45.1 + ) into irradiated mice (CD45.1 + ). Recipients of 4E-BP-deficient bone marrow cells had significantly higher levels of donor-derived hematopoietic cells in their blood 4-16 wk after transplantation (Fig. 4A-D). To test whether this increased reconstitution reflected increased HSC frequency in the bone marrow of 4E-BP-deficient donors (Fig.  3K) or increased function of 4E-BP-deficient HSCs, we competitively transplanted 10 CD150 + CD48 − LSK HSCs from 4E-BP1 −/− ;4E-BP2 −/− or wild-type donors into irradiated recipient mice along with recipient bone marrow cells. 4E-BP-deficient HSCs gave normal levels of donor cell reconstitution (Fig. 4E-H). The higher level of donor cell reconstitution by 4E-BP-deficient bone marrow cells Frequency of annexin V + HSCs. n = 5-6 mice per genotype in four experiments in L and M. All data represent mean ± SD. The statistical significance of differences relative to wild type was assessed using one-way ANOVAs (repeated-measures in A and B) followed by Dunnett's test for multiple comparisons. ( * ) P < 0.05; ( * * ) P < 0.01; ( * * * ) P < 0.001.  Figure S3A. (E-H) Donor cell engraftment when 10 HSCs from mice of the indicated genotypes were transplanted with 3 × 10 5 recipient bone marrow cells into irradiated mice. (I-L) Donor cell engraftment after serial transplantation of 3 × 10 6 bone marrow cells from primary recipients in A-D into secondary recipient mice. n = 8-11 recipients per genotype from two primary donors from wild type and three primary donors from each of the 4E-BP-deficient genotypes. Data represent mean ± SD. The statistical significance of differences relative to wild-type were assessed with one-way ANOVAs followed by Dunnett's test for multiple comparisons in A-D and I-L and a two-tailed Student's t-test in E-H. ( * ) P < 0.05; ( * * ) P < 0.01; ( * * * ) P < 0.001.

4E-BP hypophosphorylation in HSCs
thus reflects higher HSC frequency in the donor bone marrow rather than increased reconstituting activity by 4E-BP-deficient HSCs.
Although 4E-BP2 −/− and 4E-BP1 −/− ;4E-BP2 −/− mutant mice had increased HSC frequency in their bone marrow as compared with controls (Fig. 3K), the frequency of donor HSCs in primary recipient mice after bone marrow transplantation did not significantly differ between recipients of 4E-BP-deficient cells as compared with control cells (Supplemental Fig. S3B). These data raised the possibility that 4E-BP deficiency may impair HSC self-renewal after transplantation.
To assess HSC self-renewal potential, we performed secondary transplants of 3 × 10 6 bone marrow cells from primary recipient mice with levels of donor cell reconstitution nearest the median values in each treatment. Deficiency for only 4E-BP1 or 4E-BP2 did not significantly affect donor cell reconstitution in secondary recipient mice as compared with control cells. In contrast, secondary recipients of 4E-BP1 −/− ;4E-BP2 −/− cells had significantly less donor cell reconstitution in all lineages 16 wk after transplantation as compared with secondary recipients of wild-type donor cells ( Fig. 4I-L). These data suggest that 4E-BP1 −/− ;4E-BP2 −/− HSCs have reduced self-renewal potential as compared with control HSCs.
4E-BP1 and 4E-BP2 likely regulate the translation of a subset of mRNAs in HSCs. Increased expression of these mRNAs likely increases HSC frequency (Fig. 3K) and reconstituting capacity in the bone marrow of 4E-BP1/2 −/− mice ( Fig. 4A-D) while reducing HSC self-renewal upon serial transplantation (Fig. 4I-L). Nonetheless, other mechanisms must also limit protein synthesis in HSCs, as protein synthesis rates in 4E-BP1/2 −/− HSCs remained significantly lower than in 4E-BP1/2 −/− restricted hematopoietic progenitors. Moreover, both GMPs and megakaryocyte erythroid-restricted progenitors (MEPs) had higher levels of hypophosphorylated 4E-BPs relative to HSCs (Fig. 2D,E) and yet still had higher levels of global protein synthesis (Fig. 1A), perhaps because global protein synthesis in these cells is not as sensitive to 4E-BP1/2 as in HSCs (Fig. 3B).  Bacquer et al. 2007) mice have been described previously. These mice were backcrossed for at least 10 generations onto a C57BL background. C57BL/Ka-Thy-1.1 (CD45.2) mice were used as wild type throughout this study. C57BL/Ka-Thy-1.2 (CD45.1) mice were used as transplant recipients. Male and female mice between 8 and 14 wk old were used in all studies. Cyclophosphamide and GCSF were administered as described (Signer et al. 2014). All mice were housed in the Animal Resource Center at the University of Texas Southwestern Medical Center, and all protocols were approved by the University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee.

Proteasome activity assay
Proteasome activity was determined by measuring the rate of hydrolysis of Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (AMC). This substrate is specific for the proteasome under the assay conditions used here and is hydrolyzed by all proteasome holoenzymes. The activity reported by this assay represents the total enzymatic capacity of the cellular proteasome and is typically dominated by the 26S proteasome (Sasaki et al. 1984;Demartino and Gillette 2007). From each population, 3 × 10 4 cells were double-sorted into 96-well black plates. One-hundred microliters of buffer containing 20 mM Tris-HCl (pH 7.6), 1 mM 2-mercaptoethanol, 5 mM MgCl 2 , 1 mM ATP, and 0.4% NP-40 was added to each well followed immediately by 50 µL of 200 µM Suc-Leu-Leu-Val-Tyr-AMC (Bachem). The fluorescence of free AMC generated by proteasomal hydrolysis of the substrate was monitored continuously (one read per minute for 180 min at 37°C) at 360 ex /460 em using a BioTek Synergy II plate reader. Controls included reactions conducted in the absence of cells, in the presence of 10 mM MG132 (UBPBio), or using purified bovine 26S proteasome instead of cells. Data are expressed as arbitrary fluorescent units per well.

The rate of cell division
Two milligrams of EdU (Thermo Scientific) in PBS was injected intraperitoneally per mouse every 6 h. Mice were analyzed 2, 6, 12, and 24 h after EdU administration. These short labeling times minimized the extent to which results were affected by the entry of labeled cells into each population through the maturation of upstream progenitors, the exit of labeled cells through differentiation into downstream progeny, or saturation of labels in rapidly dividing cell populations. Bone marrow cells (3 × 10 6 cells) were stained with antibodies as described in the Supplemental Material. Cells were fixed and permeabilized, and the azide-alkyne cycloaddition was performed as with OP-Puro detection and analyzed by flow cytometry. For cyclophosphamide-and GCSF-treated mice, BrdU was administered in place of EdU and was detected with the BrdU flow kit.