Stat and interferon genes identified by network analysis differentially regulate primitive and definitive erythropoiesis

BACKGROUND: Hematopoietic ontogeny is characterized by overlapping waves of primitive, fetal definitive, and adult definitive erythroid lineages. Our aim is to identify differences in the transcriptional control of these distinct erythroid cell maturation pathways by inferring and analyzing gene-interaction networks from lineage-specific expression datasets. Inferred networks are strongly connected and do not fit a scale-free model, making it difficult to identify essential regulators using the hub-essentiality standard. RESULTS: We employed a semi-supervised machine learning approach to integrate measures of network topology with expression data to score gene essentiality. The algorithm was trained and tested on the adult and fetal definitive erythroid lineages. When applied to the primitive erythroid lineage, 144 high scoring transcription factors were found to be differentially expressed between the primitive and adult definitive erythroid lineages, including all expressed STAT-family members. Differential responses of primitive and definitive erythroblasts to a Stat3 inhibitor and IFNγ in vitro supported the results of the computational analysis. Further investigation of the original expression data revealed a striking signature of Stat1-related genes in the adult definitive erythroid network. Among the potential pathways known to utilize Stat1, interferon (IFN) signaling-related genes were expressed almost exclusively within the adult definitive erythroid network. CONCLUSIONS: In vitro results support the computational prediction that differential regulation and downstream effectors of STAT signaling are key factors that distinguish the transcriptional control of primitive and definitive erythroid cell maturation

Zeb2 is essential for terminal differentiation of multiple hematopoietic lineages

The differentiation of hematopoietic stem cells (HSC) into specialized blood cells is tightly controlled by a complex network of transcription factors (TF). The zinc finger E-Box binding TF Zeb2 is known to govern the epithelial to mesenchymal transition (EMT) during embryonic development and tumor progression and metastasis in an adult organism. Our previous work showed that deletion of Zeb2 within hematopoietic compartment resulted in early embryonic lethality due to differentiation and migration defects. In order to identify the role of Zeb2 in adult hematopoiesis, the Mx1-Cre based inducible Zeb2 knockout model was used. We found drastic reduction of B-lymphocytes, monocytes, platelets and erythrocytes in peripheral blood while accumulation of hematopoietic stem and progenitor cells in the bone marrow after Zeb2 deletion. In this study, we firstly demonstrated that impaired terminal differentiation of the erythroid lineage takes place at the transition of CD71highTER119+ to CD71medTER119+ population upon Zeb2 ablation. This may be caused by the dysregulation of several transcription regulators, including Klf1 and Fli1 and Gata1. In addition, the most prominent differentiation block was observed within B lymphopoiesis at the Prepro-B to Pro-B cell transition. The early B-cell receptor IL-7R as well as fingerprint transcription factors such as Runx1, E2A, EBF1 and Pax5 were markedly changed in Zeb2Δ/ΔMx1-Cre Prepro-B cells. Moreover, the expression level of DNA methylation related genes and histone modifications were significantly changed in Zeb-deficient early B-cells, suggesting the B-cell differentiation block due to altered epigenetic regulation. A set of higher or lower methylated key HSC genes were discovered by reduced representation bisulfite sequencing (RRBS) analysis between Zeb2 WT and Zeb2Δ/ΔMx1-Cre HSCs. Gene ontology (GO) enrichment analysis highlights altered protein kinases (including phosphorylation, Serine/threonine protein kinase, tyrosine protein kinase) after Zeb2 deletion. This was further confirmed by abnormal JAK/STAT activity in Zeb2Δ/ΔMx1-Cre cells. In summary, Zeb2 is essential for multilineage differentiation at different stages of hematopoiesis. Ablation of Zeb2 in adult hematopoiesis results in defective epigenetic regulation, a causative event in impaired transcriptional network and JAK/STAT activation that account for multilineage defects

Dynamic Modeling of the JAK2/STAT5 Signal Transduction Pathway to Dissect the Specific Roles of Negative Feedback Regulators

Erythropoietin (Epo) acts as the key regulator of red blood cell development in mammals. During erythropoiesis, Epo initiates the JAK2/STAT5 signal transduction pathway that elicits pro-survival signals in erythroid progenitor cells. Therefore, the tight regulation of JAK2/STAT5 signaling is crucial for the fine-tuned balance of erythrocyte production. Recently, several factors regulating Epo-induced JAK2/STAT5 signaling have been identified. However, their relative contribution in controlling the dynamic behavior of JAK2/STAT5 signaling is poorly understood. To elucidate the specific roles of these negative regulators in attenuating the pathway, data-based mathematical modeling was employed. In this study, standardized protocols were established facilitating the generation of quantitative time-resolved data of Epo-induced JAK2/STAT5 pathway activation in primary erythroid progenitor cells and the hematopoietic cell line BaF3-EpoR, which is a frequently used model system to study EpoR signaling. For the fine-tuned overexpression of negative regulators in hematopoietic cells, an inducible Tet-On retroviral vector system was developed. Systematic comparison of stoichiometries and activation dynamics of Epo-induced JAK2/STAT5 signaling in CFU-E and BaF3-EpoR cells revealed fundamental differences between both cell types, emphasizing the importance of the use of primary cells in the investigation of EpoR signaling. Genome-wide expression profiling identified potential feedback regulators of Epo-induced JAK2/STAT5 signaling in CFU-E cells. To dissect the complex roles of negative regulators that employ different mechanisms to attenuate JAK2/STAT5 signaling, a data-based dynamic pathway model was established. Calibration of the mathematical model was performed using multiple experimental data sets of Epo-induced JAK2/STAT5 signaling monitored under different conditions. The estimated parameters were fully identifiable and displayed small confidence intervals, which are required for accurate simulations. Comprehensive model analysis identified the rapid recruitment of the phosphatase SHP-1 as major mechanism controlling the early-phase kinetics of pathway activation, while the two transcriptionally induced regulators SOCS3 and CIS were elucidated as modulators of the STAT5 steady-state phosphorylation level. Furthermore, global sensitivity analysis uncovered the concentrations of SHP-1 and JAK2 as well as the parameter SOCS3 expression as critical to control the integral signal strength of nuclear phosphorylated STAT5, which is proportionally linked to the survival of erythroid progenitor cells. In conclusion, by combining mathematical modeling with experimental data, the crucial regulators enabling the tight control of Epo-induced JAK2/STAT5 signaling were elucidated. The detailed understanding of the molecular processes and regulatory mechanisms of Epo-induced signaling during normal erythropoiesis can be further exploited to gain insights into alterations promoting erythroleukemia and related malignant hematopoietic diseases

Dissecting the function of B cell lymphoma 6A (BCL6A) protein in zebrafish

This study investigated the normal function of the BCL6A cancer gene in normal growth and development, particularly of immune cells, using zebrafish as a model. BCL6A was shown to be highly conserved, with an essential role in immune function and growth

The Activity of the JAK-STAT Pathway in Infantile Haemangioma and the Haemogenic Potential of Infantile Haemangioma Explant Derived Cells

Background: Stem cells have been identified within proliferating infantile haemangioma (IH), the most common tumour of infancy, and have been demonstrated to play a critical role in the rapid proliferation and gradual involution of this tumour. There is accumulating evidence showing that IH is caused by aberrant proliferation and differentiation of a haemogenic endothelium (HE). This HE possesses a functional capacity to undergo primitive erythropoiesis in vitro. Short chain fatty acid (SCFA) derivatives have been shown to stimulate cell proliferation and induce STAT-5 activation in various haematopoietic cell lines. Aims: The aims of this study were to investigate (1) the activity of the components of JAK-STAT pathway within the three phases of IH development; (2) the haematopoietic capacity of IH in vitro; and (3) the effects of SCFAs, butyric acid and propionic acid, to induce erythroid differentiation of explant-derived cells (IHEDCs) in culture. Methods: The presence of pSTAT proteins in proliferating, involuted and involuting IH were investigated using 3,3'-diaminobenzidine (DAB) and immunofluorescent (IF) immunohistochemical (IHC) staining, 1-DE Western Blotting, and NanoString analysis. Proliferating IH explants were cultured using an in vitro model and the IHEDCs emanating from the explants were harvested. Cell suspension of volume equivalent to 5x105 live cells was plated on Matrigel and incubated in 0.05-1mM butyric acid, RPMI and 0.05-1mM propionic acid, and 0.05M DMSO (positive control) in each of RPMI media only, RPMI enriched with iron and MCDB media. Media was changed daily and cells were extracted and quantified following 24-72 hours in culture. Differentiated IHEDCs were characterised by IF immunocytochemical (ICC) staining with glycophorin-A. Results: Protein and genomic data reveal the expression of STATs 1, 3 and 5 which are activated in IH, particularly in the proliferative phase, with expression tapering as the lesion involutes. pSTAT3 is expressed most abundantly with pSTAT5 the least abundant. Low concentrations of both butyric acid and propionic acid significantly increased proliferation and differentiation of IHEDCs into blast colonies and the production of bi-concave cells within 72 hours in culture. These enucleated bi-concave cells expressed the erythrocyte-specific marker, glycophorin-A. Conclusion: The findings of SCFAs promoting proliferation and differentiation of IHEDCs into blast colonies and differentiated erythrocytes reveal a novel role for SCFAs in human haematopoietic differentiation, possibly via pSTAT-5 signalling. IH offers a simple and novel in vitro model for generating haematopoietic precursors and production of human erythrocytes

SRP54 mutations induce Congenital Neutropenia via dominant-negative effects on XBP1 splicing

Heterozygous de novo missense variants of SRP54 were recently identified in patients presenting with Congenital Neutropenia (CN) or its syndromic form Shwachman-Diamond Syndrome (SDS). Ever since its discovery as a driver of CN and SDS, SRP54 has been increasingly studied in the context of disease and is nowadays considered the second most common cause of CN. Despite its hitherto unknown prevalence, the molecular mechanisms leading to the development of the disease are still largely unknown and patient treatments are far from specific. In this thesis, I aimed to investigate the underlying mechanisms and processes contributing to the pathophysiology of SRP54 deficiencies. To follow this aim, I characterized and established a transgenic srp54 KO zebrafish as the first in vivo model of srp54-driven disease. Interestingly, srp54-/- zebrafish show early embryonic mortality and suffer from severe neutropenia and developmental defects affecting multiple organs. srp54+/- zebrafish on the other hand are viable and only display mild neutropenia and no overt other defects. However, when injecting srp54+/- fish with human mRNA of three mutated SRP54 variants (T115A, T117Δ and G226E) identified in patients, the neutropenia intensified, and pancreatic defects developed – a phenotype accurately mimicking the characteristics of SDS patients. Of note, the induced phenotypes showed mutation-specific differences, indicating that different SRP54 lesions exert unique dominant-negative effects on the functionality of the residual wildtype SRP54 protein. Consistent with these findings, overexpression of SRP54 missense variants in human promyelocytic HL60 cells as well as in healthy CD34+ cord blood cells impaired granulocytic maturation. Mechanistically, we found that SRP54 defects significantly reduce the efficiency of the unconventional splicing of the transcription factor X-box binding protein 1 (XBP1), which is one of the major regulators of the unfolded protein response (UPR). Vice-versa, xbp1 morphant zebrafish recapitulate phenotypes observed in srp54 mutant fish, and the injection of spliced xbp1 but not unspliced xbp1 rescues the neutropenia in srp54+/- embryos. In order to identify additional mechanisms contributing to the pathophysiology of SRP54 deficient patients, we performed single cell RNA sequencing of srp54-mutated zebrafish. Sequencing analysis revealed several differentially expressed genes with most of them converging on the major signaling branches of the UPR, indicating the cell’s efforts to circumvent the impaired XBP1 activity aiming to alleviate unresolved ER-stress

Genetic studies of hereditary myeloproliferative disorders

More than 300 billion blood cells are being replaced daily in a process called hematopoiesis. Hematopoiesis is orchestrated by hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs produce multipotent and lineage-restricted progenitors, that are responsible for the supply of mature blood cells. Production of blood cells is governed by hematopoietic growth factors that are required for the survival and proliferation of blood cells at all stages of development. Mutations in genes responsible for the regulation of this fine-tuned system cause aberrant proliferation of different blood compartments. Myeloproliferative neoplasms (MPN) are characterized by the abnormal expansion of erythroid, megakaryocytic, and myeloid lineages, that is caused either by somatic mutations or by germline mutations transmitted through Mendelian inheritance within the family. The main topic of my doctoral research was the investigation of two distinct pedigrees diagnosed with erythrocytosis and thrombocytosis, respectively. Erythrocytosis occurred in ten individuals of Norwegian family that presented elevated hemoglobin and erythropoietin (EPO) serum levels. We performed genome-wide linkage analysis using SNP arrays coupled with targeted sequencing and identified a heterozygous single base deletion (ΔG) in exon 2 of the EPO gene as the sole candidate gene mutation in affected family members. EPO stimulates the proliferation of erythrocyte progenitors and prevents their apoptosis in order to produce mature erythrocytes. Surprisingly, ΔG introduces a frame-shift that generates a novel, 51-residue long polypeptide, which would predict a loss of erythropoietin function, and is at odds with the erythrocytosis phenotype. To elucidate the mechanism by which the loss-of-function mutation causes gain-off function phenotype, we utilized the CRISPR/Cas9 genome editing to introduce the ΔG mutation into Hep3B cells, a human hepatoma cell line that expresses EPO. We found that cells with ΔG mutation produce excessive amounts of biologically active EPO and reproduces the observation form the affected family members. On the molecular level, in addition to the known transcript originating from the physiologic promoter (P1), we identified novel transcripts that initiate in intron 1 of EPO from a putative alternative promoter (P2). Further functional analysis of P2 mRNAs revealed an alternative translational start site in exon 2 that P2 transcripts use to produce a biologically active EPO protein, by fusing a novel N-terminus to the EPO coding sequence through the ΔG single base deletion. Our data demonstrate for the first time, that a mutation in EPO cause familial erythrocytosis and explain how the ΔG mutation results in a gain-function phenotype. I also investigated a pedigree with autosomal-dominant. Targeted sequencing identified a novel activating mutation in exon 3 of the thrombopoietin (THPO) gene, a single nucleotide G->T substitution. Thrombopoietin stimulates the production of platelets from megakaryocytes. THPO expression is regulated on the translational level by seven upstream open reading frames (uORF1-7) in the exons 1-3 of THPO mRNA, that are interfering with the translation of TPO. G>T mutation maps to the Kozak sequence of the uORF7, the most critical negative regulator of TPO translation. We performed TPO overexpression and in vitro translation experiments to demonstrate that the G>T mutation disrupts the negative regulation governed by uORF7 and allows for increased translation of THPO protein coding sequence, ultimately causing thrombocytosis. Collectively, in both studies we identified novel gain-of-function mutations in hematopoietic growth factors, that act at different steps of gene expression and result in the dysregulated production of EPO and TPO, causing erythrocytosis and thrombocytosis in respective pedigrees

Dynamic regulation of GATA transcription factors in hematopoiesis

The hematopoietic system is composed of a variety of cells, whose activity is essential for the normal functioning of an organism. Erythrocytes, or red blood cells, transport oxygen and carbon dioxide throughout the body, platelets are essential for coagulation and white blood cells (lymphocytes, granulocytes and macrophages) are responsible for the protection of the organism against pathogens. All these different cells originate from a single cell type, the hematopoietic stem cell (HSC), through a process denominated hematopoiesis. To understand how the HSC can originate so many different cell type has been the aim of many scientists over the years. Advances in molecular biology tools allowed the gathering of vast amounts of information about the hematopoietic system and the process of hematopoiesis. However, many questions remain without answers. The HSC gives rise to the different hematopoetic cell lineages via a series of steps. HSCs are rare cells that have the capacity to duplicate themselves (self-renewal) as well as to give rise to all the different hematopoietic cell types (pluripotency). The descendants of the HSC are still able to give rise to all hematopoietic lineages but they lose the ability to self-renew. These cells will further differentiate into other cells that can give rise to an increasingly restricted number of hematopoietic lineages until they reach a stage were they can only differentiate into a single lineage. Such process is called lineage-commitment and its accuracy is essential for the normal function of the hematopoietic system. How this lineage commitment occurs is as yet not clear. It is known that it is dependent on environmental cues as