A novel role for Lyl1 in primitive erythropoiesis

© 2018. Published by The Company of Biologists Ltd. Stem cell leukemia (Scl or Tal1) and lymphoblastic leukemia 1 (Lyl1) encode highly related members of the basic helix-loop-helix family of transcription factors that are co-expressed in the erythroid lineage. Previous studies have suggested that Scl is essential for primitive erythropoiesis. However, analysis of single-cell RNA-seq data of early embryos showed that primitive erythroid cells express both Scl and Lyl1. Therefore, to determine whether Lyl1 can function in primitive erythropoiesis, we crossed conditional Scl knockout mice with mice expressing a Cre recombinase under the control of the Epo receptor, active in erythroid progenitors. Embryos with 20% expression of Scl from E9.5 survived to adulthood. However, mice with reduced expression of Scl and absence of Lyl1 (double knockout; DKO) died at E10.5 because of progressive loss of erythropoiesis. Gene expression profiling of DKO yolk sacs revealed loss of Gata1 and many of the known target genes of the SCL-GATA1 complex. ChIP-seq analyses in a human erythroleukemia cell line showed that LYL1 exclusively bound a small subset of SCL targets including GATA1. Together, these data show for the first time that Lyl1 can maintain primitive erythropoiesis

Modeling Reveals Bistability and Low-Pass Filtering in the Network Module Determining Blood Stem Cell Fate

Combinatorial regulation of gene expression is ubiquitous in eukaryotes with multiple inputs converging on regulatory control elements. The dynamic properties of these elements determine the functionality of genetic networks regulating differentiation and development. Here we propose a method to quantitatively characterize the regulatory output of distant enhancers with a biophysical approach that recursively determines free energies of protein-protein and protein-DNA interactions from experimental analysis of transcriptional reporter libraries. We apply this method to model the Scl-Gata2-Fli1 triad—a network module important for cell fate specification of hematopoietic stem cells. We show that this triad module is inherently bistable with irreversible transitions in response to physiologically relevant signals such as Notch, Bmp4 and Gata1 and we use the model to predict the sensitivity of the network to mutations. We also show that the triad acts as a low-pass filter by switching between steady states only in response to signals that persist for longer than a minimum duration threshold. We have found that the auto-regulation loops connecting the slow-degrading Scl to Gata2 and Fli1 are crucial for this low-pass filtering property. Taken together our analysis not only reveals new insights into hematopoietic stem cell regulatory network functionality but also provides a novel and widely applicable strategy to incorporate experimental measurements into dynamical network models

Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys.

Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic

The non-coding RNA landscape of human hematopoiesis and leukemia

© The Author(s) 2017. Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs-such as LINC00173 in granulocytes-and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy

Characterization of transcriptional networks in blood stem and progenitor cells using high-throughput single-cell gene expression analysis

Cellular decision-making is mediated by a complex interplay of external stimuli with the intracellular environment, in particular transcription factor regulatory networks. Here we have determined the expression of a network of 18 key haematopoietic transcription factors in 597 single primary blood stem and progenitor cells isolated from mouse bone marrow. We demonstrate that different stem/progenitor populations are characterized by distinctive transcription factor expression states, and through comprehensive bioinformatic analysis reveal positively and negatively correlated transcription factor pairings, including previously unrecognized relationships between Gata2, Gfi1 and Gfi1b. Validation using transcriptional and transgenic assays confirmed direct regulatory interactions consistent with a regulatory triad in immature blood stem cells, where Gata2 may function to modulate cross-inhibition between Gfi1 and Gfi1b. Single-cell expression profiling therefore identifies network states and allows reconstruction of network hierarchies involved in controlling stem cell fate choices, and provides a blueprint for studying both normal development and human disease

Delivery of PEGylated liposomal doxorubicin by bispecific antibodies improves treatment in models of high-risk childhood leukemia

High-risk childhood leukemia has a poor prognosis because of treatment failure and toxic side effects of therapy. Drug encapsulation into liposomal nanocarriers has shown clinical success at improving biodistribution and tolerability of chemotherapy. However, enhancements in drug efficacy have been limited because of a lack of selectivity of the liposomal formulations for the cancer cells. Here, we report on the generation of bispecific antibodies (BsAbs) with dual binding to a leukemic cell receptor, such as CD19, CD20, CD22, or CD38, and methoxy polyethylene glycol (PEG) for the targeted delivery of PEGylated liposomal drugs to leukemia cells. This liposome targeting system follows a "mix-and-match" principle where BsAbs were selected on the specific receptors expressed on leukemia cells. BsAbs improved the targeting and cytotoxic activity of a clinically approved and low-toxic PEGylated liposomal formulation of doxorubicin (Caelyx) toward leukemia cell lines and patient-derived samples that are immunophenotypically heterogeneous and representative of high-risk subtypes of childhood leukemia. BsAb-assisted improvements in leukemia cell targeting and cytotoxic potency of Caelyx correlated with receptor expression and were minimally detrimental in vitro and in vivo toward expansion and functionality of normal peripheral blood mononuclear cells and hematopoietic progenitors. Targeted delivery of Caelyx using BsAbs further enhanced leukemia suppression while reducing drug accumulation in the heart and kidneys and extended overall survival in patient-derived xenograft models of high-risk childhood leukemia. Our methodology using BsAbs therefore represents an attractive targeting platform to potentiate the therapeutic efficacy and safety of liposomal drugs for improved treatment of high-risk leukemia

VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

Angiogenesis is the process by which new blood vessels arise from existing ones by the budding out of endothelial cell capillaries from the luminal side of blood vessels. Blood vessel formation is essential for organ development during embryogenesis and is associated with several physiological and pathological processes, such as wound healing and tumor development. The VE-statin/egfl7 gene is specifically expressed in endothelial cells during embryonic development and in the adult. We studied here the regulatory mechanisms that control this tissue-specific expression. RT-qPCR analyses showed that the specificity of expression of VE-statin/egfl7 in endothelial cells is not shared with its closest neighbor genes notch1 and agpat2 on the mouse chromosome 2. Chromatin-immunoprecipitation analysis of histone modifications at the VE-statin/egfl7 locus showed that the chromatin is specifically opened in endothelial cells, but not in fibroblasts at the transcription start sites. A 13 kb genomic fragment of promoter was cloned and analyzed by gene reporter assays which showed that two conserved regions are important for the specific expression of VE-statin/egfl7 in endothelial cells; a −8409/−7563 enhancer and the −252/+38 region encompassing the exon-1b transcription start site. The latter contains essential GATA and ETS-binding sites, as assessed by linker-scanning analysis and site-directed mutagenesis. An analysis of expression of the ETS and GATA transcription factors showed that Erg, Fli-1 and GATA-2 are the most highly expressed factors in endothelial cells. Erg and GATA-2 directly control the expression of the endogenous VE-statin/egfl7 while Fli-1 probably exerts an indirect control, as assessed by RNA interference and chromatin immunoprecipitation. This first detailed analysis of the mechanisms that govern the expression of the VE-statin/egfl7 gene in endothelial cells pinpoints the specific importance of ETS and GATA factors in the specific regulation of genes in this cell lineage

A Conformation-Sensitive Monoclonal Antibody against the A2 Domain of von Willebrand Factor Reduces Its Proteolysis by ADAMTS13

The size of von Willebrand factor (VWF), controlled by ADAMTS13-dependent proteolysis, is associated with its hemostatic activity. Many factors regulate ADAMTS13-dependent VWF proteolysis through their interaction with VWF. These include coagulation factor VIII, platelet glycoprotein 1bα, and heparin sulfate, which accelerate the cleavage of VWF. Conversely, thrombospondin-1 decreases the rate of VWF proteolysis by ADAMTS13 by competing with ADAMTS13 for the A3 domain of VWF. To investigate whether murine monoclonal antibodies (mAbs) against human VWF affect the susceptibility of VWF to proteolysis by ADAMTS13 in vitro, eight mAbs to different domains of human VWF were used to evaluate the effects on VWF cleavage by ADAMTS13 under fluid shear stress and static/denaturing conditions. Additionally, the epitope of anti-VWF mAb (SZ34) was mapped using recombinant proteins in combination with enzyme-linked immunosorbent assay and Western blot analysis. The results indicate that mAb SZ34 inhibited proteolytic cleavage of VWF by ADAMTS13 in a concentration-dependent manner under fluid shear stress, but not under static/denaturing conditions. The binding epitope of SZ34 mAb is located between A1555 and G1595 in the central A2 domain of VWF. These data show that an anti-VWF mAb against the VWF-A2 domain (A1555-G1595) reduces the proteolytic cleavage of VWF by ADAMTS13 under shear stress, suggesting the role of this region in interaction with ADAMTS13

Genome-Wide Analysis of Transcriptional Reprogramming in Mouse Models of Acute Myeloid Leukaemia

Acute leukaemias are commonly caused by mutations that corrupt the transcriptional circuitry of haematopoietic stem/progenitor cells. However, the mechanisms underlying large-scale transcriptional reprogramming remain largely unknown. Here we investigated transcriptional reprogramming at genome-scale in mouse retroviral transplant models of acute myeloid leukaemia (AML) using both gene-expression profiling and ChIP-sequencing. We identified several thousand candidate regulatory regions with altered levels of histone acetylation that were characterised by differential distribution of consensus motifs for key haematopoietic transcription factors including Gata2, Gfi1 and Sfpi1/Pu.1. In particular, downregulation of Gata2 expression was mirrored by abundant GATA motifs in regions of reduced histone acetylation suggesting an important role in leukaemogenic transcriptional reprogramming. Forced re-expression of Gata2 was not compatible with sustained growth of leukaemic cells thus suggesting a previously unrecognised role for Gata2 in downregulation during the development of AML. Additionally, large scale human AML datasets revealed significantly higher expression of GATA2 in CD34+ cells from healthy controls compared with AML blast cells. The integrated genome-scale analysis applied in this study represents a valuable and widely applicable approach to study the transcriptional control of both normal and aberrant haematopoiesis and to identify critical factors responsible for transcriptional reprogramming in human cancer