Andrew G. Elefanty

The critical regulator of embryonic hematopoiesis, SCL, is vital in the adult for megakaryopoiesis, erythropoiesis, and lineage choice in CFU-S12

Gene targeting studies have shown that the transcription factor SCL is critically important for embryonic hematopoiesis, but the early lethality of SCL null mice has precluded the genetic analysis of its function in the adult. We have now generated a conditional knockout of SCL by using Cre/Lox technology and an IFN-inducible Cre transgenic mouse. Deletion of SCL in adult mice perturbed megakaryopoiesis and erythropoiesis with the loss of early progenitor cells in both lineages. This led to a blunted response to the hematopoietic stress induced by polyinosinic-polycytidylic acid, with a persistently low platelet count and hematocrit compared with controls. In contrast, progenitors of granulocyte and macrophage lineages were not affected, even in the setting of stress. Immature progenitor cells (day 12 colony-forming unit spleen) with multilineage capacity were still present in the SCL null bone marrow, but these progenitors had lost the capacity to generate erythroid and megakaryocyte cells, and colonies were composed of only myeloid cells. These results suggest that SCL is critical for megakaryopoiesis and erythropoiesis, but is dispensable for production of myeloid cells during adult hematopoiesis.

Cloning, expression analysis, and chromosomal localization of murine and human homologues of a Xenopus Mix gene

We report the cloning and chromosomal localization of murine and human Mix genes, members of a subclass of paired‐like homeobox genes of which the Xenopus laevis Mix.1 gene is the founding member. The murine Mix gene was mapped to the distal region of chromosome 1 and the human region to the syntenic region 1q41‐42. Northern analysis and RT‐PCR of murine adult and embryonic tissues demonstrated that Mix expression was restricted to the early embryo. Whole‐mount in situ hybridization revealed patchy but symmetrical Mix expression in visceral endoderm of embryonic day (E)5.5 embryos. In slightly older embryos, the expression was skewed to one side of the embryo and by E6.5, at the onset of gastrulation, expression was seen in the epiblast, visceral endoderm, nascent mesoderm, and the primitive streak. This expression pattern was maintained in mid‐ and late‐streak embryos. In early bud‐stage embryos, expression was strongest in the proximal two thirds of the streak, extending to the base of the allantois. By the headfold‐stage, expression was confined to the remnant of the primitive streak in the caudal region of the embryo and, after E8.0, in the caudal notochord and tail bud mesoderm. Mix transcripts were no longer detectable after embryonic day 9.5.

Transcription of the SCL gene in erythroid and CD34 positive primitive myeloid cells is controlled by a complex network of lineage-restricted chromatin-dependent and chromatin-independent regulatory elements.

The SCL gene (also known as TAL-1) encodes a basic helix – loop – helix transcription factor that is essential for the development of all haematopoietic lineages, and ectopic expression of which results in T cell leukaemia. SCL is expressed in normal pluripotent haematopoietic stem cells and its expression is maintained during differentiation along erythroid, mast and megakaryocytic lineages, but is extinguished following commitment to other cell types. The mechanisms responsible for this pattern of expression are poorly understood, but are likely to illuminate the molecular basis for stem cell development and lineage commitment. We have identified multiple lineage-restricted DNase I hypersensitive sites in a 45 kb region spanning the murine SCL locus. Committed erythroid cells and CD34 positive primitive myeloid cells exhibited both shared and unique DNase I hypersensitive sites whereas none were found in T cells. The function of each hypersensitive site was studied using both transient and stable reporter assays in erythroid, primitive myeloid and T cells. Multiple positive and negative regulatory elements were characterised and found to display lineage-specificity, promoter-specificity and/or chromatin-dependence. These results represent the first description of key components of a complex network of regulatory elements controlling SCL expression during haematopoiesis.

Apoptosis and beta-cell destruction in pancreatic islets of NOD mice with spontaneous and cyclophosphamide-accelerated diabetes

Summary Autoimmune-mediated destruction of pancreatic islet beta cells leads to insulin-dependent diabetes in non-obese diabetic (NOD) mice. Although both direct cytotoxic T cell- and indirect cytokine-, nitric oxide- or free radical-mediated mechanisms induce beta-cell apoptosis in vitro, beta-cell death in vivo in spontaneous autoimmune diabetes is not well-characterized. Furthermore, whether beta cells die gradually, or rapidly in the late pre-clinical stage, is a question of current interest. To investigate beta-cell death in vivo, we measured the frequency and intra-islet localisation of apoptosis, defined as DNA strand breaks by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) technique, during spontaneous and cyclophosphamide-accelerated diabetes in NOD mice. In spontaneous diabetes, the frequency of apoptosis in islets correlated with the progression of beta-cell destruction with age. Although apoptosis was detected at low frequency within the reduced insulin-positive islet area of pre-diabetic mice at 90 days of age, it was rarely co-localised to beta cells. After acceleration of beta-cell destruction with cyclophosphamide, the frequency of apoptosis reached maximum at 12 days, at which time 3.2 % of apoptotic cells were beta cells. Apoptosis was most frequent in the insulin-negative islet area comprised of mononuclear cell infiltrate and was localized to CD8+ T cells. The rarity of detectable apoptotic beta cells in spontaneous pre-diabetic mice with pronounced insulitis and reduced insulin-positive islet areas most likely reflects the rapid clearance of apoptotic beta cells. Our findings are more consistent with gradual destruction of non-renewable beta-cells in spontaneous diabetes, than with their rapid, accelerated destruction (as after cyclophosphamide) in the late pre-clinical stage. [Diabetologia (1998) 41: 1381–1388]

GATA transcription factors associate with a novel class of nuclear bodies in erythroblasts and megakaryocytes.

The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA‐1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA‐1 to consensus sites in the beta‐globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA‐1 foci seen by immunofluorescence. The GATA‐1 nuclear bodies differed from previously described nuclear structures and there was no co‐localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c‐Jun and TBP) or haemopoietic (NF‐E2) transcription factors. Interestingly, GATA‐2 and GATA‐3 proteins also localized to the same nuclear bodies in cell lines co‐expressing GATA‐1 and −2 or GATA‐1 and −3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein‐protein interaction with other components of the nuclear bodies via the GATA zinc finger domain.

Leishmania major proteophosphoglycan is expressed by amastigotes and has an immunomodulatory effect on macrophage function

Proteophosphoglycan (PPG) is a newly described mucin-like glycoprotein found on the surface of Leishmania major promastigotes and secreted in the culture supernatant. We show here that antigenically similar PPGs are present in several Leishmania species. PPG could also be detected on the surface of amastigotes and in small, parasite-free vesicles in infected macrophages. Because of the similarity of its carbohydrate chains to lipophosphoglycan, a parasite receptor for host macrophages, PPG was tested for binding to macrophages. PPG bound to macrophages and was internalized in a time-dependent manner. PPG inhibited the production of tumor necrosis factor-alpha and synergized with interferon-gamma to stimulate the production of nitric oxide by macrophages. PPG may contribute to the binding of Leishmania to host cells and may play a role in modulating the biology of the infected macrophage at the early stage of infection.

Distinct Mechanisms Direct SCL/tal-1 Expression in Erythroid Cells and CD34 Positive Primitive Myeloid Cells

The SCL/tal-1 gene (hereafter designated SCL) encodes a basic helix-loop-helix transcription factor which is pivotal for the normal development of all hematopoietic lineages and which is expressed in committed erythroid, mast, and megakaryocytic cells as well as in hematopoietic stem cells. The molecular basis for expression of SCL in stem cells and its subsequent modulation during lineage commitment is of fundamental importance for understanding how early “decisions” are made during hematopoiesis. We now compare the activity of SCL promoters 1a and 1b in erythroid cells and in CD34 positive primitive myeloid cells. SCL mRNA expression in CD34 positive myeloid cells did not require GATA-1. Promoter 1a activity was weak or absent in CD34 positive myeloid cells and appeared to correlate with the presence or absence of low levels of GATA-1. However, promoter 1b, which was silent in committed erythroid cells, was strongly active in transient assays using CD34 positive myeloid cells, and functioned in a GATA-independent manner. Interestingly, RNase protection assays demonstrated that endogenous promoter 1b was active in both erythroid and CD34 positive myeloid cells. These results demonstrate that fundamentally different mechanisms regulate the SCL promoter region in committed erythroid cells and in CD34 positive myeloid cells. Moreover these observations suggest that in erythroid, but not in CD34 positive myeloid cells, promoter 1b required integration in chromatin and/or additional sequences for its activity. Stable transfection experiments showed that both core promoters were silent following integration in erythroid or CD34 positive myeloid cells. Our data therefore indicate that additional regulatory elements were necessary for both SCL promoters to overcome chromatin-mediated repression.

The hemangioblast—an elusive cell captured in culture

The close temporal and spatial association between blood and endothelial cell development during embryogenesis was first documented almost 100 years ago. In recent years, gene expression studies have further strengthened this link. Now, using cultures of mouse embryonic stem cells, a common progenitor cell that gives rise to both blood cells and vascular endothelial cells, has been identified. The existence of the hemangioblast has been proved and experiments addressing its unique properties can begin.

Expression of BCR - ABL in M1 myeloid leukemia cells induces differentiation without arresting proliferation.

The mechanism leading to the expanding population of maturing myeloid cells which characterises chronic myeloid leukemia (CML) remains obscure. Because of its ability to mimic the proliferative and cell survival functions of hematopoietic growth factors, we hypothesized that the oncogene activated in CML, BCR – ABL, might also influence differentiation. To test this hypothesis, we examined the effects of expressing BCR – ABL on the myeloid differentiation of murine M1 leukemic cells, which cease dividing and differentiate into macrophages in the presence of the cytokines leukemia inhibitory factor (LIF) or interleukin (IL)-6. We found that BCR – ABL induced macrophage differentiation in M1 cells, accompanied by increased expression of macrophage cell surface markers and the acquisition of phagocytic ability. Interestingly, clones of M1 cells which expressed BCR – ABL remained in cell cycle and were refractory to the growth inhibition and apoptosis induced by IL-6 or LIF in parental M1 cells. These cells also expressed inappropriately high levels of c-MYC mRNA for their degree of differentiation, which may have been important in maintaining cellular proliferation. These data suggest that BCR – ABL can stimulate both differentiation and proliferation and that these characterisitics may contribute to the phenotype observed in CML.

Characterization of interactions between transcription factors and a regulatory region spanning nt -320 to -281 of the HIV-1 LTR in T-lymphoid and non-T-lymphoid cells.

HIV-1 gene expression is regulated by the interplay of transcription factors with multiple binding motifs present within the U3, R and U5 regions of the long terminal repeat (LTR). Here we report novel DNA binding complexes (termed 9a, 9b and 9c) between nuclear proteins from T-lymphoid and non-T-lymphoid cells and a region of the U3 LTR between nucleotides (nts) -320 to -281 in the HIV strain HXB2. Complex 9b bound a motif predicted to bind E-box or c-Myb proteins and a partially overlapping dyad symmetrical motif, and included basic helix-loop-helix proteins (E12, E47 or ITF-1) but surprisingly not c-Myb. Complex 9c, which bound to a pair of GATA sites, included GATA-3 and GATA-4 in Jurkat and MT-2 cells, respectively. We also demonstrate that the c-Myb/E-box and GATA sites form a bipartite motif required for the formation of complex 9a. Transient transfection experiments with T cells revealed that in the context of a minichromosome assembled full-length LTR, mutation of region -320 to -281 increased basal and PMA-stimulated LTR activity. These findings suggest that this region is an important component of the HIV-1 LTR required for response to different cellular transcription factors.