MIP-1alpha: A Structure-Function Study

The observation that MIP-1alpha can inhibit the proliferation of transiently engrafting haemopoietic stem cells was first reported more than ten years ago. However, very little has since emerged about the molecular mechanism underlying stem cell inhibition. The work presented in this thesis therefore aimed at shedding some more light on the molecular mechanism and on how the structural properties of MIP-1alpha relate to its function as a stem cell inhibitor. Two properties of MIP-1alpha, which it shares with most other chemokines, were first of all considered, its ability to self-aggregate and its interaction with proteoglycans. It has already been demonstrated that the aggregation of murine MIP-1alpha is influenced by the pH and the ionic and hydrophobic strength of the buffer. In addition, three acidic residues in the carboxy terminal region of murine MIP-1alpha have been previously shown to be involved in self-association as their neutralisation generates aggregation-incompetent mutants. Since the aggregation of human MIP-1alpha has previously been found to be concentration-dependent, similar experiments were carried out for murine MIP-1alpha which established that its self-association is also controlled in a comparable way which allowed the isolation of differentially aggregated murine MIP-1alpha by progressive dilution. However, the forces that stabilise the oligomers appear to be stronger in human MIP-1alpha as compared to murine MIP-1alpha. In the recent determination of the crystal structure of murine MIP-1alpha, two calcium ions were found in association with the tetramer that were proposed to mediate the formation of higher order aggregates. However, experiments carried out as part of this thesis demonstrate that this is not the case since the removal of these ions by the addition of EDTA and EGTA has no influence on the aggregation process. Secondly, investigations were made into the way murine MIP-1alpha interacts with the glycosaminoglycan heparin. More specifically, experiments were aimed at establishing whether there is a link between the aggregation state of MIP-1alpha and its affinity for heparin since it is known for some chemokines, such as PF4, that the individual heparin binding sites in the monomers display positive cooperativity upon aggregation, resulting in a higher affinity of the tetramer for heparin. This may also have implications for a possible interaction of MIP-1alpha with proteoglycans in the bone marrow microenvironment. In a first approach, three aggregation-incompetent murine MIP-1alpha mutants, a monomer, dimer and a tetramer, were analysed for their binding to a heparin matrix. Surprisingly, the tetramer exhibited the lowest affinity for heparin, followed by the dimer and then the monomer with the highest affinity. One possible interpretation of this observation is that the heparin binding site becomes progressively occluded upon aggregation, thereby decreasing heparin binding in MIP-1alpha oligomers. Alternatively, the neutralisation of the negative charges during the generation of these aggregation mutants may have altered the strength with which they bind to heparin since glycosaminoglycan-protein interactions are predominantly determined by electrostatic forces. In order to resolve this question, differentially aggregated murine MlP-1? was prepared by progressive dilution (see above) and the different oligomers tested for their heparin binding affinity. No difference was observed in the strength with which the different aggregates bound to immobilised heparin which was also confirmed with stably cross-linked murine MIP-1alpha oligomers. This suggests, that the aggregation state of murine MIP-1alpha has no impact on its affinity for heparin which is instead controlled by its overall charge. Attention was then turned to the interaction of murine MIP-1alpha with the receptor on haemopoietic stem cells through which it mediates its inhibitory effect, as demonstrated in an in vitro assay, known as the CFU-A assay. It was first of all established that this inhibitory receptor is none of the four known murine MIP-1alpha receptors (CCR1, CCR3, CCR5 and D6) for the following reasons; (1) none of the other chemokines tested, which included other ligands for the four MIP-1alpha receptors as well as for all of the other known CC chemokine receptors, displayed any activity in the CFU-A assay, (2) the four known human MIP-1alpha variants, which show differential binding to murine CCR1, CCR5 and D6, have indistinguishable potencies as stem cell inhibitors, and (3) stem cells from CCR1-/-, CCR3-/-, CCR5-/- and D6-/- mice were still inhibited in their proliferation by MIP-1alpha, even when a chemokine analogue, which has the capacity to displace MIP-1alpha from all of its four receptors, was included in the assays. All of these data suggest that MIP-1alpha's inhibitory signal is conveyed by a novel, as yet uncharacterised receptor. (Abstract shortened by ProQuest.)

The Ly-6A (Sca-1) GFP transgene is expressed in all adult mouse hematopoietic stem cells

The Sca-1 cell surface glycoprotein is used routinely as a marker of adult hematopoietic stem cells (HSCs), allowing a >100-fold enrichment of these rare cells from the bone marrow of the adult mouse. The Sca-1 protein is encoded by the Ly-6A/E gene, a small 4-exon gene that is tightly controlled in its expression in HSCs and several hematopoietic cell types. For the ability to sort and localize HSCs directly from the mouse, we initiated a transgenic approach in which we created Ly-6A (Sca-1) green fluorescent protein (GFP) transgenic mice. We show here that a 14-kb Ly-6A expression cassette directs the transcription of the GFP marker gene in all functional repopulating HSCs in the adult bone marrow. A >100-fold enrichment of HSCs occurred by sorting for the GFP-expressing cells. Furthermore, as shown by fluorescence-activated cell sorting and histologic analysis of several hematopoietic tissues, the GFP transgene expression pattern generally corresponded to that of Sca-1. Thus, the Ly-6A GFP transgene facilitates the enrichment of HSCs and presents the likelihood of identifying HSCs in situ

Gata3 targets Runx1 in the embryonic haematopoietic stem cell niche.

Runx1 is an important haematopoietic transcription factor as stressed by its involvement in a number of haematological malignancies. Furthermore, it is a key regulator of the emergence of the first haematopoietic stem cells (HSCs) during development. The transcription factor Gata3 has also been linked to haematological disease and was shown to promote HSC production in the embryo by inducing the secretion of important niche factors. Both proteins are expressed in several different cell types within the aorta-gonads-mesonephros (AGM) region, in which the first HSCs are generated; however, a direct interaction between these two key transcription factors in the context of embryonic HSC production has not formally been demonstrated. In this current study, we have detected co-localisation of Runx1 and Gata3 in rare sub-aortic mesenchymal cells in the AGM. Furthermore, the expression of Runx1 is reduced in Gata3 -/- embryos, which also display a shift in HSC emergence. Using an AGM-derived cell line as a model for the stromal microenvironment in the AGM and performing ChIP-Seq and ChIP-on-chip experiments, we demonstrate that Runx1, together with other key niche factors, is a direct target gene of Gata3. In addition, we can pinpoint Gata3 binding to the Runx1 locus at specific enhancer elements which are active in the microenvironment. These results reveal a direct interaction between Gata3 and Runx1 in the niche that supports embryonic HSCs and highlight a dual role for Runx1 in driving the transdifferentiation of haemogenic endothelial cells into HSCs as well as in the stromal cells that support this process.This work was supported by an Intermediate Fellowship (K.O.) and a Junior Fellowship (S.R.F.) from the Kay Kendall Leukaemia Fund, a British Society for Haematology Early Stage Investigator Fellowship (K.O.) as well as funding from Bloodwise (N.K.W. and B.G.), MRC (N.K.W. and B.G.) and the Wellcome Trust (N.K.W. and B.G.). MdB is funded by a programme in the MRC Molecular Hematology Unit Core award (Grant number: MC_UU_12009/2). Core facilities are supported by Strategic Award WT100140, equipment grant 093026 and centre grant MR/K017047/1

Mll-AF4 Confers Enhanced Self-Renewal and Lymphoid Potential during a Restricted Window in Development.

MLL-AF4+ infant B cell acute lymphoblastic leukemia is characterized by an early onset and dismal survival. It initiates before birth, and very little is known about the early stages of the disease's development. Using a conditional Mll-AF4-expressing mouse model in which fusion expression is targeted to the earliest definitive hematopoietic cells generated in the mouse embryo, we demonstrate that Mll-AF4 imparts enhanced B lymphoid potential and increases repopulation and self-renewal capacity during a putative pre-leukemic state. This occurs between embryonic days 12 and 14 and manifests itself most strongly in the lymphoid-primed multipotent progenitor population, thus pointing to a window of opportunity and a potential cell of origin. However, this state alone is insufficient to generate disease, with the mice succumbing to B cell lymphomas only after a long latency. Future analysis of the molecular details of this pre-leukemic state will shed light on additional events required for progression to acute leukemia.Core facilities at the Cambridge Institute for Medical Research are supported by Strategic Award WT100140 and equipment grant 093026; core facilities at the Edinburgh MRC Centre for Regenerative Medicine are supported by centre grant MR/K017047/1. This work was funded by a Bloodwise Bennett Senior Fellowship (10015 to K.O.), a Wellcome Trust Clinical PhD Studentship (097454/z/11/z to N.A.B.) the Gabrielle’s Angel Foundation for Cancer Research (to K.O.), and the Kay Kendall Leukaemia Fund (to K.O.).This is the final version of the article. It first appeared from Cell Press/Elsevier at http://dx.doi.org/10.1016/j.celrep.2016.06.046