S70 Poster Presentations/ Experimental Hematology 41 (2013) S23–S75

P1180 - MECHANISM OF DEREGULATION OF HEMATOPOIESIS BY

MUTANT FORMS OF RUNX1

Regha Kakkad1, Monika Lichtinger1, Valerie Kouskoff2, Georges Lacaud2, and

Constanze Bonifer1

1School of Cancer Sciences, University of Birmingham, Birmingham, UK; 2Paterson

Institute for Cancer Research, University of Manchester, Manchester, UK

During embryonic hematopoiesis, hematopoietic stem cells (HSCs) arise from endo-

thelial cells (ECs) lining the dorsal aorta. Some ECs change their endothelial fate and

gain hematopoietic potential and the ability to differentiate into blood cells. Endothe-

lial cells with hematopoietic potential are called hemogenic endothelium (HE) and

they give rise to intra-aortic clusters of CD45+ HSCs. This progression of HE to

HSCs is known as endothelial-to-hematopoietic transition (EHT) and absolutely

requires the transcription factor RUNX1. RUNX1 expression starts at low levels in

the ECs and is highly upregulated in HE and HSCs. The transition of HE to HSCs

depends on the levels of RUNX1, a reduction alters HSC development. Mutations

in RUNX1 lead to abnormal hematopoiesis and leukaemia. Here we studied two

mutant RUNX1 proteins, RUNX1/ETO (t8:21 translocation) and RUNX1-R203X

(point mutation), both associated with acute myeloid leukaemia, to understand how

these proteins interfere at different stages of hematopietic differentiation. Although

many studies have addressed the block in differentiation by RUNX1/ETO, not

much is known about how they affect initial stages of hematopoiesis. Towards this

end we generated ES cell lines with inducible expression of either RUNX1/ETO

or RUNX1-R203X in a RUNX1 wild-type background. The changes in hematopoi-

esis caused by these mutant proteins were studied by in vitro differentiation of these

ES cell lines into blood precursor cells. Our studies indicate that expression of

RUNX1/ETO severely inhibits the progression of ECs to HE and precursors. The

RUNX1-R203X mutant showed altered kinetics of hematopoietic differentiation

with rapid appearance of CD41+ precursor cells. Experiments are being carried

out to get mechanistic insights into how these mutants alter the endothelial and hema-

topoietic gene expression programs and the results will be presented.

P1181 - PROGRESSIVE DEREGULATION OF GENE EXPRESSION

DURING EARLY HAEMOPOIETIC LINEAGE COMMITMENT FROM

SP1-/- ES CELLS

Jane Gilmour1, Salam Assi2, Sjaak Philipsen3, and Constanze Bonifer1

1Institute of Biomedical Research, University of Birmingham, Birmingham, UK;2Faculty of Biological Sciences, University of Leeds, Leeds, UK; 3Dept of Cell

Biology, Erasmus MC, Rotterdam, Netherlands

Sp1 is a ubiquitously expressed transcription factor which regulates expression of

a variety of genes involved in diverse cellular processes including differentiation,

proliferation and chromatin remodelling. Sp1-/- mice die by E11 of multiple hetero-

geneous developmental defects, however Sp1-/- embryonic stem cells grow normally.

Currently the reason for this discrepancy is not understood. To gain insight into how

the lack of Sp1 influences one specific developmental pathway, we have used these

ES cells to study early haemopoietic development. Sp1 deficient ES cells show

a reduced capacity for macrophage production by embryoid bodies grown in methyl-

cellulose. Haemopoietic colony assays show that Sp1-/- myeloid precursors are

produced at a lower frequency and are functionally deficient as evidenced by lower

colony forming capacity. A more detailed analysis of the block in differentiation

using a blast culture differentiation system revealed that Sp1-/- cells can form haemo-

poietic progenitors from the haemogenic endothelium (HE), but further development

of these progenitors to macrophages is impaired. We next performed gene expression

microarrays using RNA isolated from purified cell populations from wild type and

Sp1 deficient cells representing the Flk+ve haemangioblast, HE1, HE2 and progen-

itor stages of haemopoietic development. Analysis of this data demonstrates

increasing deregulation of multiple genes essential for normal cellular function in

Sp1-/- cells as differentiation progresses, providing a molecular explanation for

why differentiation is impaired. Gene ontology analysis revealed that a wide range

of cellular differentiation and metabolic processes are affected by Sp1 deficiency,

consistent with a global role for Sp1 in the expression of house-keeping genes. These

expression studies were complemented by ChIP-seq analysis of Sp1 binding in wild

type cell populations. In summary, our data shows that while Sp1 is dispensable for

mES cell growth and self-renewal, it is essential to maintain a normal program of

haemopoietic differentiation.

P1182 - JAK/ STAT SIGNALLING DURING NORMAL AND

PATHOLOGICAL MYELOPOIESIS WITH FOCUS ON ERYTHROPOIESIS

AND MEGAKARYOPOIESIS

Winnie Lau1,2, Caia Dominicus1, Rebecca Hannah1,2, Amy Jones3,

Anthony Green1,2, and Berthold Gottgens1,2

1Cambridge Institute for Medical Research, University of Cambridge, Cambridge,

UK; 2Wellcome Trust & MRC Cambridge Stem Cell Institute, University of

Cambridge, Cambridge, UK; 3Human Development and Health, Faculty of Medicine,

University of Southampton, Southampton, UK

The JAK/ STAT signalling pathway plays a crucial role in normal haematopoiesis

transducing signals from critical cytokines such as erythropoietin (EPO) and throm-

bopoietin (TPO) from the cell surface to target gene promoters in the nucleus. Dys-

regulation of JAK/ STAT pathway can lead to an array of haematological

malignancies. The single acquired somatic mutation JAK2 V617F contributes to

a number of phenotypically diverse myeloproliferative neoplasms (MPNs) character-

ised by an overproduction of mature myeloid cells with a tendency to transform to

acute leukaemia. This includes 97 % polycythaemia vera (PV) and 57 % essential

thrombocythaemia (ET) cases where individuals present with increased erythropoi-

esis and megakaryopoiesis respectively (Baxter el al., 2005). We are currently char-

acterising the transcriptional programmes downstream of wild type JAK2 in

EPO-treated erythroblasts (O90% CD71+/CD235a+) and TPO-treated megakaryo-

cytes (O60 % CD41+). Using Chromatin Immunoprecipitation (ChIP) and

sequencing of native regulatory elements bound by ChIP-Seq grade STAT antibodies,

we aim to identify and present differential STAT binding sites and novel STAT-regu-

lated genes in these two primary human myeloid cell types. Microarray analysis of

RNA isolated from both cell types, before and after cytokine treatment, will aid

the differentiation of cytokine-dependent and independent target genes. By defining

the STAT target genes in normal erythroblasts and megakaryocytes we intend to

provide a wild type framework in which to compare subverted STAT signalling as

a consequence of mutant JAK2. We have cultured primary human JAK2 V617F posi-

tive erythroblasts and will assimilate and present ChIP-Seq data from these cells

alongside analyses from normal erythroblasts. Together these findings will create

a more comprehensive understanding of wild type JAK/ STAT signalling and may

provide new insights into MPN pathogenesis.

P1183 - A NOVEL EMBRYONIC STEM CELL BASED ASSAY PROVIDES

SINGLE CELL QUANTITATIVE ANALYSIS OF ENHANCER ACTIVITY

DURING HAEMATOPOIETIC SPECIFICATION AND ALLOWS

DISSECTION OF REGULATORY INPUTS

Adam Wilkinson1,2, Yi Han Cheng1,2, Debbie Goode1,2, Sarah Kinston1,2,

Aileen Smith1,2, and Berthold G€ottgens1,2

1Department of Haematology, University of Cambridge, Cambridge, UK; 2Wellcome

Trust & MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge,

UK

The transcription factor Scl/Tal1 is a critical regulator of haematopoiesis and its

precise spatiotemporal expression is necessary for proper developmental patterning

in the mouse embryo. Several transcriptional cis-regulatory elements have been iden-

tified that determine the haematopoietic expression of Scl including the +19, -4 and

+40 enhancers. Investigation of such regulatory elements allows determination of

upstream regulators and assembly of TF cascades and networks.Assays currently

used to assess putative haematopoietic enhancers include expression analysis of

randomly integrated enhancer-linked reporter genes in transient F0 transgenic

embryos and luciferase assays. However, transgenic embryos only provide qualitative

data while luciferase assays are limited to population level analysis in non-develop-

mental cell types. These assays therefore provide an incomplete picture of enhancer

activity. The Scl+19 enhancer has been well characterised by these methods and

others; it is sufficient to drive Scl expression during developmental haematopoiesis

and contains a highly conserved Ets/Ets/Gata motif necessary for its activity.

However, the cell type specificity and the quantitative effects of motif mutation in

the Scl+19 during haematopoietic specification cannot be determined by current

methods.We have developed a haematopoietic enhancer assay that utilises embry-

onic stem (ES) cell gene targeting for single copy integration of an enhancer-linked

YFP reporter gene. Embryoid body differentiation of targeted ES cell lines provides

an accurate model of haematopoietic specification and abrogates the need of trans-

genic mice. Flow cytometry of YFP and cell surface marker expression captures

quantitative single cell data of enhancer activity and specificity. Gene targeting

and analysis of mutated enhancers allows further dissection of their regulatory in-

puts.We will present use of this novel enhancer assay to define Scl+19 activity