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Functional analysis of truncated forms of ETV6

2015; Wiley; Volume: 171; Issue: 4 Linguagem: Inglês

10.1111/bjh.13428

1365-2141

Parisa Rasighaemi, Clifford Liongue, Sara M.N. Onnebo, Alister C. Ward,

Chronic Myeloid Leukemia Treatments

The ETV6 (TEL) transcription factor has been shown to play a wide role in haematopoiesis, influencing the development of multiple lineages, while chromosomal translocations involving fusions of the ETV6 gene occur frequently in haematological malignancies (Rasighaemi et al, 2014). Recurrent mutations of ETV6 have been reported in cases of acute myeloid leukaemia (AML) (Barjesteh Van Waalwijk Van Doorn-Khosravani et al, 2005; Silva et al, 2008), childhood B cell acute lymphoblastic leukaemia (B-ALL) (Zhang et al, 2011) and early immature adult T-cell ALL (T-ALL) (Van Vlierberghe et al, 2011), along with alternative splicing of ETV6 in myelodysplastic syndrome (MDS) (Sasaki et al, 2004). These lead to expression of alternate forms of ETV6 lacking either the N-terminal PNT domain, involved in protein–protein interactions, or the C-terminal ETS DNA-binding domain, unable to repress transcription but capable of ablating the repressional activity of full-length ETV6 in vitro (Barjesteh Van Waalwijk Van Doorn-Khosravani et al, 2005; Van Vlierberghe et al, 2011). However, the in vivo role of these ETV6 truncations has not been characterized. The zebrafish represents an established model for the study of haematopoiesis and its perturbation, which has previously been used to investigate the role of ETV6 (Rasighaemi et al, 2015), and the consequences of enforced expression of ETV6-JAK2 (Onnebo et al, 2012). This study has utilized this model to analyse the in vivo function of truncated ETV6 proteins. Sequences encoding Flag-tagged versions of full-length zebrafish Etv6 (Etv6-FL) and truncated Etv6 forms corresponding to amino acids 1–324, thereby deleting the ETS domain (Etv6-ΔETS), and amino acids 325–444, thereby deleting the PNT domain (Etv6-ΔPNT) were generated (Fig 1A). Consistent with their mammalian counterparts, the truncated forms lacked the repressive properties of full-length Etv6, but interfered with the repression mediated by full-length Etv6 (Fig 1B), confirming that truncated versions of zebrafish Etv6 act in a similar dominant-negative manner in vitro to equivalent mammalian ETV6 proteins (Sasaki et al, 2004; Barjesteh Van Waalwijk Van Doorn-Khosravani et al, 2005). Some evidence of degradation of co-expressed full-length and truncated Etv6 protein was observed (Fig 1C–D), consistent with the absence of ETV6 protein observed in leukaemic blasts heterozygous for truncating ETV6 mutations (Patel et al, 2003; Sasaki et al, 2004; Barjesteh Van Waalwijk Van Doorn-Khosravani et al, 2005). To investigate the function of truncated isoforms of ETV6 in vivo, zebrafish embryos were microinjected with in vitro transcribed mRNA encoding Etv6-FL, Etv6-ΔETS or Etv6-ΔPNT (Fig 1E), which was stable to at least 5 d post-fertilization (dpf) (data not shown). We have previously shown that ablation of zebrafish Etv6 affects embryonic haematopoiesis (Rasighaemi et al, 2015). Therefore, embryos injected with truncated Etv6 isoforms were examined for similar haematological perturbations using specific blood lineage markers to directly assess their in vivo effects. During primitive haematopoiesis, embryos injected with Etv6-ΔETS and Etv6-ΔPNT showed increased expression of the early haematopoietic marker tal1 in the rostral lateral plate mesoderm (LPM) compared to control embryos at 20 h post fertilization (hpf), while those injected with Etv6-FL showed decreased tal1 expression (Fig 1F–I, N). In contrast, the caudal LPM tal1+ population was significantly decreased in Etv6-ΔETS- and Etv6-ΔPNT-injected embryos but increased in those injected with Etv6-FL (Fig 1J–M, O). Expression of the early erythroid marker gata1a at the same time point was also decreased in Etv6-ΔETS- and Etv6-ΔPNT-injected embryos but increased in Etv6-FL embryos relative to controls (Fig 1P–S, V). Co-injection of Etv6-FL with either Etv6-ΔETS or Etv6-ΔPNT resulted in decreased gata1a expression compared to controls (Fig 1T–V). Definitive haematopoiesis was analysed at 5 dpf. Embryos injected with either Etv6-ΔETS or Etv6-ΔPNT showed an overall decrease in the expression of the HSC marker myb and haemoglobin (Hb) staining, but an increased number of cells expressing the leucocyte marker lyz. In contrast, Etv6-FL embryos showed increased myb expression and Hb staining but reduced lyz+ cells relative to controls (Fig 2A–L, U). Analysis of the lymphoid marker ikzf1 at 5 dpf revealed increased expression in Etv6-ΔETS- and Etv6-ΔPNT-injected embryos but decreased expression in Etv6-FL-injected embryos compared to controls (Fig 2O–R, V). Co-injection of Etv6-FL with either Etv6-ΔETS or Etv6-ΔPNT resulted in increased lyz and ikzf1 expression but decreased Hb staining compared to controls (Fig 2M–N, S–T, U–V). The disruption of both primitive and definitive haematopoiesis observed following expression of both truncated ETV6 forms closely resembled that seen following Etv6 ablation (Rasighaemi et al, 2015). Furthermore, these effects were reversed in embryos injected with full-length Etv6, which suggests that levels of Etv6 represent a crucial determinant of haematopoietic cell development. Importantly, co-injection of Etv6-ΔPNT or Etv6-ΔETS not only ablated the effects of Etv6-FL expression, but elicited a similar phenotype to when they were injected alone, demonstrating they act in a dominant manner. This is the first study showing a dominant-negative effect of truncated forms of ETV6 on normal ETV6 function in vivo. This suggests that in cases with truncating ETV6 mutations in AML (Barjesteh Van Waalwijk Van Doorn-Khosravani et al, 2005; Silva et al, 2008), B-ALL (Zhang et al, 2011) and T-ALL (Van Vlierberghe et al, 2011) or alternate splicing in MDS (Sasaki et al, 2004), it is likely that wild-type ETV6 function is low or indeed absent. Such a conclusion is consistent with expression data showing a similar enrichment in genes in immature adult T-ALL carrying truncating ETV6 mutations as in a T-ALL cell line in which ETV6 was knocked down, including up-regulation of CD33, HOXA13, PTEN and PRDM16 (Van Vlierberghe et al, 2011). Collectively, this adds to our understanding of the contribution of ETV6 truncations to leukaemia aetiology at the molecular level. The authors acknowledge support from the Deakin University International Research Scholarship scheme (PR, SMNO) and Alfred Deakin Postdoctoral Research Fellow scheme (CL), and access to the resources of FishWorks: Collaborative Infrastructure for Zebrafish Research, an Australian Research Council LIEF initiative (ACW). The authors would like to thank the Deakin University Upper Animal House staff for their assistance with zebrafish husbandry. PR performed the bulk of the experimental work, analysed the data and assisted in manuscript preparation, while CL and SMNO provided additional input. ACW conceived the project and contributed to data analysis and preparation of the manuscript and takes overall responsibility for the manuscript. None of the authors have any competing interests to declare.