The GIY-YIG Type Endonuclease Ankyrin Repeat and LEM Domain-Containing Protein 1 (ANKLE1) Is Dispensable for Mouse Hematopoiesis

Ankyrin repeat and LEM-domain containing protein 1 (ANKLE1) is a GIY-YIG endonuclease with unknown functions, mainly expressed in mouse hematopoietic tissues. To test its potential role in hematopoiesis we generated Ankle1-deficient mice. Ankle1^Δ/Δ mice are viable without any detectable phenotype in hematopoiesis. Neither hematopoietic progenitor cells, myeloid and lymphoid progenitors, nor B and T cell development in bone marrow, spleen and thymus, are affected in Ankle1^Δ/Δ-mice. Similarly embryonic stress erythropoiesis in liver and adult erythropoiesis in bone marrow and spleen appear normal. To test whether ANKLE1, like the only other known GIY-YIG endonuclease in mammals, SLX1, may contribute to Holliday junction resolution during DNA repair, Ankle1-deficient cells were exposed to various DNA-damage inducing agents. However, lack of Ankle1 did not affect cell viability and, unlike depletion of Slx1, Ankle1-deficiency did not increase sister chromatid exchange in Bloom helicase-depleted cells. Altogether, we show that lack of Ankle1 does neither affect mouse hematopoiesis nor DNA damage repair in mouse embryonic fibroblasts, indicating a redundant or non-essential function of ANKLE1 in mouse.     

Progenitor Stage-Specific Activity of a cis-Acting Double GATA Motif for Gata1 Gene Expression

GATA1 is a master regulator of erythropoiesis, expression of which is regulated by multiple discrete cis-acting elements. In this study, we examine the activity of a promoter-proximal double GATA (dbGATA) motif, using a Gata1 bacterial artificial chromosome (BAC)-transgenic green fluorescent protein (GFP) reporter (G1BAC-GFP) mouse system. Deletion of the dbGATA motif led to significant reductions in GFP expression in hematopoietic progenitors, while GFP expression was maintained in erythroblasts. Consistently, in mice with a germ line deletion of the dbGATA motif (Gata1^ΔdbGATA mice), GATA1 expression in progenitors was significantly decreased. The suppressed GATA1 expression was associated with a compensatory increase in GATA2 levels in progenitors. When we crossed Gata1^ΔdbGATA mice with Gata2 hypomorphic mutant mice (Gata2^fGN/fGN mice), the Gata1^ΔdbGATA::Gata2^fGN/fGN compound mutant mice succumbed to a significant decrease in the progenitor population, whereas both groups of single mutant mice maintained progenitors and survived to adulthood, indicating the functional redundancy between GATA1 and GATA2 in progenitors. Meanwhile, the effects of the dbGATA site deletion on Gata1 expression were subtle in erythroblasts, which showed increased GATA1 binding and enhanced accumulation of active histone marks around the 1st-intron GATA motif of the ΔdbGATA locus. These results thus reveal a novel role of the dbGATA motif in the maintenance of Gata1 expression in hematopoietic progenitors and a functional compensation between the dbGATA site and the 1st-intron GATA motif in erythroblasts.     

MiR-17 Partly Promotes Hematopoietic Cell Expansion through Augmenting HIF-1α in Osteoblasts

Background

Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment, of which osteogenic cells play a crucial role. While evidence is accumulating for an important role of intrinsic miR-17 in regulating HSCs and HPCs, whether miR-17 signaling pathways are also necessary in the cell-extrinsic control of hematopoiesis hereto remains poorly understood.

Methodology/Principal Findings

Using the immortalized clone with the characteristics of osteoblasts, FBMOB-hTERT, in vitro expansion, long-term culture initiating cell (LTC-IC) and non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice repopulating cell (SRC) assay revealed that the ectopic expression of miR-17 partly promoted the ability of FBMOB-hTERT to support human cord blood (CB) CD34⁺ cell expansion and maintain their multipotency. It also seemed that osteoblastic miR-17 was prone to cause a specific expansion of the erythroid lineage. Conversely, deficient expression of miR-17 partly inhibited the hematopoietic supporting ability of FBMOB-hTERT. We further identified that HIF-1α is responsible for, at least in part, the promoted hematopoietic supporting ability of FBMOB-hTERT caused by miR-17. HIF-1α expression is markedly enhanced in miR-17 overexpressed FBMOB-hTERT upon interaction with CB CD34⁺ cells compared to other niche associated factors. More interestingly, the specific erythroid lineage expansion of CB CD34⁺ cells caused by osteoblastic miR-17 was abrogated by HIF-1α knock down.

Conclusion/Significance

Our data demonstrated that CB CD34⁺ cell expansion can be partly promoted by osteoblastic miR-17, and in particular, ectopic miR-17 can cause a specific expansion of the erythroid lineage through augmenting HIF-1α in osteoblasts.     

High-Efficiency Transduction of Primary Human Hematopoietic Stem Cells and Erythroid Lineage-Restricted Expression by Optimized AAV6 Serotype Vectors In Vitro and in a Murine Xenograft Model In Vivo

We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34⁺ cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34⁺ cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease.     

The p53 tumour suppressor inhibits glucocorticoid-induced proliferation of erythroid progenitors

Hypoxia encountered at high altitude, blood loss and erythroleukemia instigate stress erythropoiesis, which involves glucocorticoid-induced proliferation of erythroid progenitors (ebls). The tumour suppressor p53 stimulates hematopoietic cell maturation and antagonizes glucocorticoid receptor (GR) activity in hypoxia, suggesting that it may inhibit stress erythropoiesis. We report that mouse fetal liver ebls that lack p53 proliferate better than wild-type cells in the presence of the GR agonist dexamethasone. An important mediator of GR-induced ebl self-renewal, the c-myb gene, is induced to higher levels in p53^–/– ebls by dexamethasone. The stress response to anemia is faster in the spleens of p53^–/– mice, as shown by the higher levels of colony forming units erythroids and the increase in the CD34/c-kit double positive population. Our results show that p53 antagonizes GR-mediated ebl expansion and demonstrate for the first time that p53–GR cross-talk is important in a physiological process in vivo: stress erythropoiesis.     

Visual vs Fully Automatic Histogram-Based Assessment of Idiopathic Pulmonary Fibrosis (IPF) Progression Using Sequential Multidetector Computed Tomography (MDCT)

Objectives

To describe changes over time in extent of idiopathic pulmonary fibrosis (IPF) at multidetector computed tomography (MDCT) assessed by semi-quantitative visual scores (VSs) and fully automatic histogram-based quantitative evaluation and to test the relationship between these two methods of quantification.

Methods

Forty IPF patients (median age: 70 y, interquartile: 62-75 years; M:F, 33:7) that underwent 2 MDCT at different time points with a median interval of 13 months (interquartile: 10-17 months) were retrospectively evaluated. In-house software YACTA quantified automatically lung density histogram (10^th-90^th percentile in 5^th percentile steps). Longitudinal changes in VSs and in the percentiles of attenuation histogram were obtained in 20 untreated patients and 20 patients treated with pirfenidone. Pearson correlation analysis was used to test the relationship between VSs and selected percentiles.

Results

In follow-up MDCT, visual overall extent of parenchymal abnormalities (OE) increased in median by 5 %/year (interquartile: 0 %/y; +11 %/y). Substantial difference was found between treated and untreated patients in HU changes of the 40th and of the 80th percentiles of density histogram. Correlation analysis between VSs and selected percentiles showed higher correlation between the changes (Δ) in OE and Δ 40^th percentile (r=0.69; p<0.001) as compared to Δ 80^th percentile (r=0.58; p<0.001); closer correlation was found between Δ ground-glass extent and Δ 40^th percentile (r=0.66, p<0.001) as compared to Δ 80^th percentile (r=0.47, p=0.002), while the Δ reticulations correlated better with the Δ 80^th percentile (r=0.56, p<0.001) in comparison to Δ 40^th percentile (r=0.43, p=0.003).

Conclusions

There is a relevant and fully automatically measurable difference at MDCT in VSs and in histogram analysis at one year follow-up of IPF patients, whether treated or untreated: Δ 40^th percentile might reflect the change in overall extent of lung abnormalities, notably of ground-glass pattern; furthermore Δ 80^th percentile might reveal the course of reticular opacities.     

LCR 5′ hypersensitive site specificity for globin gene activation within the active chromatin hub

The DNaseI hypersensitive sites (HSs) of the human β-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human β-globin locus yeast artificial chromosome (β-YAC) lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (β^m), coupled to an intact LCR, a 5′HS3 complete deletion (5′ΔHS3) or a 5′HS3 core deletion (5′ΔHS3c). The 5′ΔHS3c mice expressed β^m-globin throughout development; γ-globin was co-expressed in the embryonic yolk sac, but not in the fetal liver; and wild-type β-globin was co-expressed in adult mice. Although the 5′HS3 core was not required for β^m-globin expression, previous work showed that the 5′HS3 core is necessary for ε-globin expression during embryonic erythropoiesis. A similar phenotype was observed in 5′HS complete deletion mice, except β^m-globin expression was higher during primitive erythropoiesis and γ-globin expression continued into fetal definitive erythropoiesis. These data support a site specificity model of LCR HS-globin gene interaction.     

Analyses of erythropoiesis from embryonic stem cell‐CD34+ and cord blood‐CD34+ cells reveal mechanisms for defective expansion and enucleation of embryomic stem cell‐erythroid cells

Red blood cells (RBCs) generated ex vivo have the potential to be used for transfusion. Human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) possess unlimited self‐renewal capacity and are the preferred cell sources to be used for ex vivo RBC generation. However, their applications are hindered by the facts that the expansion of ES/iPS‐derived erythroid cells is limited and the enucleation of ES/iPS‐derived erythroblasts is low compared to that derived from cord blood (CB) or peripheral blood (PB). To address this, we sought to investigate the underlying mechanisms by comparing the in vitro erythropoiesis profiles of CB CD34⁺ and ES CD34⁺ cells. We found that the limited expansion of ES CD34⁺ cell‐derived erythroid cells was associated with defective cell cycle of erythroid progenitors. In exploring the cellular and molecular mechanisms for the impaired enucleation of ES CD34⁺ cell‐derived orthochromatic erythroblasts (ES‐ortho), we found the chromatin of ES‐ortho was less condensed than that of CB CD34⁺ cell‐derived orthochromatic erythroblasts (CB‐ortho). At the molecular level, both RNA‐seq and ATAC‐seq analyses revealed that pathways involved in chromatin modification were down‐regulated in ES‐ortho. Additionally, the expression levels of molecules known to play important role in chromatin condensation or/and enucleation were significantly lower in ES‐ortho compared to that in CB‐ortho. Together, our findings have uncovered mechanisms for the limited expansion and impaired enucleation of ES CD34⁺ cell‐derived erythroid cells and may help to improve ex vivo RBC production from stem cells.     

In Vivo Regulation of Erythropoiesis by Chemically Inducible Dimerization of the Erythropoietin Receptor Intracellular Domain

Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247–406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.     

Photoreceptor Degeneration in Two Mouse Models for Congenital Stationary Night Blindness Type 2

Light-dependent conductance changes of voltage-gated Ca_v1.4 channels regulate neurotransmitter release at photoreceptor ribbon synapses. Mutations in the human CACNA1F gene encoding the α1F subunit of Ca_v1.4 channels cause an incomplete form of X-linked congenital stationary night blindness (CSNB2). Many CACNA1F mutations are loss-of-function mutations resulting in non-functional Ca_v1.4 channels, but some mutations alter the channels’ gating properties and, presumably, disturb Ca²⁺ influx at photoreceptor ribbon synapses. Notably, a CACNA1F mutation (I745T) was identified in a family with an uncommonly severe CSNB2-like phenotype, and, when expressed in a heterologous system, the mutation was shown to shift the voltage-dependence of channel activation, representing a gain-of-function. To gain insight into the pathomechanism that could explain the severity of this disorder, we generated a mouse model with the corresponding mutation in the murine Cacna1f gene (I756T) and compared it with a mouse model carrying a loss-of-function mutation (ΔEx14–17) in a longitudinal study up to eight months of age. In ΔEx14–17 mutants, the b-wave in the electroretinogram was absent, photoreceptor ribbon synapses were abnormal, and Ca²⁺ responses to depolarization of photoreceptor terminals were undetectable. In contrast, I756T mutants had a reduced scotopic b-wave, some intact rod ribbon synapses, and a strong, though abnormal, Ca²⁺ response to depolarization. Both mutants showed a progressive photoreceptor loss, but degeneration was more severe and significantly enhanced in the I756T mutants compared to the ΔEx14–17 mutants.     

Analysis of Blood Stem Cell Activity and Cystatin Gene Expression in a Mouse Model Presenting a Chromosomal Deletion Encompassing Csta and Stfa2l1

The cystatin protein superfamily is characterized by the presence of conserved sequences that display cysteine protease inhibitory activity (e.g., towards cathepsins). Type 1 and 2 cystatins are encoded by 25 genes of which 23 are grouped in 2 clusters localized on mouse chromosomes 16 and 2. The expression and essential roles of most of these genes in mouse development and hematopoiesis remain poorly characterized. In this study, we describe a set of quantitative real-time PCR assays and a global expression profile of cystatin genes in normal mouse tissues. Benefiting from our collection of DelES embryonic stem cell clones harboring large chromosomal deletions (to be reported elsewhere), we selected a clone in which a 95-kb region of chromosome 16 is missing (Del^16qB3Δ/+). In this particular clone, 2 cystatin genes, namely Csta and Stfa2l1 are absent along with 2 other genes (Fam162a, Ccdc58) and associated intergenic regions. From this line, we established a new homozygous mutant mouse model (Del^{16qB3Δ/16qB3Δ}) to assess the in vivo biological functions of the 2 deleted cystatins. Stfa2l1 gene expression is high in wild-type fetal liver, bone marrow, and spleen, while Csta is ubiquitously expressed. Homozygous Del^{16qB3Δ/16qB3Δ} animals are phenotypically normal, fertile, and not overtly susceptible to spontaneous or irradiation-induced tumor formation. The hematopoietic stem and progenitor cell activity in these mutant mice are also normal. Interestingly, quantitative real-time PCR expression profiling reveals a marked increase in the expression levels of Stfa2l1/Csta phylogenetically-related genes (Stfa1, Stfa2, and Stfa3) in Del^{16qB3Δ/16qB3Δ} hematopoietic tissues, suggesting that these candidate genes might be contributing to compensatory mechanisms. Overall, this study presents an optimized approach to globally monitor cystatin gene expression as well as a new mouse model deficient in Stfa2l1/Csta genes, expanding the available tools to dissect cystatin roles under normal and pathological conditions.     

Assessment of Human Multi-Potent Hematopoietic Stem/Progenitor Cell Potential Using a Single In Vitro Screening System

Hematopoietic stem cells are responsible for the generation of the entire blood system through life. This characteristic relies on their ability to self renew and on their multi-potentiality. Thus quantification of the number of hematopoietic stem cells in a given cell population requires to show both properties in the studied cell populations. Although xenografts models that support human hematopoietic stem cells have been described, such in vivo experimental systems remain restrictive for high throughput screening purposes for example. In this work we developed a conditional tetracycline inducible system controlling the expression of the human NOTCH ligand Delta-like 1 in the murine stromal MS5 cells. We cultured hematopoietic immature cells enriched in progenitor/stem cells in contact with MS5 cells that conditionally express Delta-like 1, in conditions designed to generate multipotential lineage differentiation. We show that upon induction or repression of DL1 expression during co-culture, human immature CD34⁺CD38^−/low(CD45RA⁻CD90⁺) cells can express their B, T, NK, granulo/monocytic and erythroid potentials in a single well, and at the single cell level. We also document the interference of low NOTCH activation with human B and myelo/erythroid lymphoid differentiation. This system represents a novel tool to precisely quantify human hematopoietic immature cells with both lymphoid and myeloid potentials.