EVI1 mediated stimulation of 3T3-L1 preadipocyte differentiation is CtBP dependent

Myelodysplasia syndrome 1 (MDS1) and Ecotropic viral integration site 1 (EVI1) complex (MECOM) locus encode multiple isoforms of the EVI1 protein that are essential for normal vertebrate development and when inappropriately expressed play a significant role in malignancy and in particular leukaemias. However, the function of individual EVI1 isoforms is not fully understood. Recently, EVI1 or PRDM3, which is structurally closely related to the brown adipose tissue determining factor PRDM16, was shown to be required for differentiation of adipocytes. In this study, we show that 3T3-L1 preadipocytes sustain expression of all Evi1 isoforms examined, including Mds1-Evi1, Evi1FL, Evi1Δ324, Evi1FL + 9 and Evi1Δ105 throughout the adipogenesis differentiation programme. We also show that differentiation markers are enhanced by enforced expression of either Evi1, Evi1FL + 9 or Evi1Δ105 isoforms. Interestingly 3T3-L1 differentiation markers are also moderately enhanced by enforced expression of Evi1Δ324, which lacks part of the N-ter-minal zinc finger domain (ZF1), demonstrating a biological activity for this particular isoform. Enforced expression of an Evi1 mutant lacking C-terminal binding protein (CtBP) co-repressor protein binding activity fails to stimulate 3T3-L1 differentiation markers and may have dominant negative activity, causing partial inhibition of this developmental programme. These studies show that multiple EVI1 isoforms are expressed in adipocytes and can stimulate adipogenic markers in a manner that is partially independent of the ZF1 DNA binding domain but fully dependent upon interaction with co-repressor CtBP proteins.


Introduction
Myelodysplasia syndrome 1 (MDS1) and Ecotropic virus integration site 1 (EVI1) complex (MECOM) locus gene transcripts include MDS1, EVI1 and a fusion of part of MDS1 with EVI1 [1] and their inappropriate expressions are associated with poor prognosis leukaemias and other malignancies [2] [3]. Those transcripts containing EVI1 encode transcription factors with multiple cys2his2 zinc finger DNA binding motifs [4] and are required for mammalian development [5]. EVI1 has been shown to contribute to a number of developmental programmes including maintenance of haemopoietic stem cells and various committed progenitor cells in haemopoiesis [6], neuroectodermal cell differentiation [7], nephrogenesis [8] and cardiac development [9]. EVI1 is also known as positive regulatory domain I-binding factor 1, retinoblastoma protein-binding zinc finger protein (PR) domain protein 3 (PRDM3) and the structurally similar PRDM16 is a key regulator of brown adipose tissue development [10]. Recent studies show that EVI1 also participates in adipogenesis [11] [12]. These studies show that EVI1 converts nonadipogenic cells to adipocytes and knockdown (KD) suppresses preadipocyte differentiation by impairing CCAAT/Enhancer-binding protein-beta (CEBPβ) assisted induction of peroxisome proliferator-activated receptor-gamma 2 (PPARγ 2 ).
There are multiple naturally occurring isoforms of EVI1 but it is not known which are expressed in preadipocytes and which might participate in adipogenesis as all are potentially affected in the knockdown (KD) study of Ishibashi et al., 2012. The isoforms include MDS1-EVI1, EVI1FL, EVI1Δ324 [13], EVI1RP+ and EVI1Δ105 (murine specific) [14]. MDS1-EVI1 comprises intergenic transcripts containing coding exons of both the MDS1 and the EVI1 genes and encodes an EVI1 protein with an N-terminal PR domain. EVI1FL is the original full length murine protein encoded by the cDNA first isolated from leukaemia cells [4]. EVI1RP+ is similar to EVI1FL but has an additional 9 amino acids inserted within the repressor domain (RP). EVI1Δ324 lacks 324 amino acids, including part of the first zinc finger domain up to, but excluding, RP and EVI1Δ105 has 105 C-terminal amino acids deleted. Various properties have been attributed to some of these isoforms and in some instances they have been shown to have opposing activities. For example, MDS1-EVI1 has been associated with tumor suppressing activity whereas EVI1FL is oncogenic. MDS1-EVI1 activates AGATA motif promoters whereas EVI1FL represses [1], EVI1FL inhibits 32Dcl3 cell response to granulocyte-colony stimulating factor (G-CSF) and transforming growth factor beta (TGFβ 1 ) whereas MDS1-EVI1 has no effect on G-CSF response and enhances TGFβ 1 signalling [15] and EVI1FL enhances proliferation of haemopoietic colonies from differentiating embryonal stem (ES) cells whereas MDS1-EVI1 represses these activities [16]. The MDS1-EVI1 isoform has a PR domain [17] which confers intrinsic histone H3 lysine 9 monomethyltransferase catalytic activity [18] which is absent from other EVI1 isoforms.  [19] and to date no biological activity has been assigned to this isoform.
In this study we investigate the profile of expression and biological activity of EVI1 isoforms in 3T3-L1 preadipocytes and throughout the adipocyte differentiation programme.

Western Blot Analysis
Protein extracts, SDS polyacrylamide gel electrophoresis and western blotting were performed as described previously [23] with either α-EVI1 (1806) or α-GAPDH

Enforced Expression of EVI1 in 3T3-L1 Preadipocytes
In order to investigate the effect of EVI1 expression on adipogenesis it was expressed in 3T3-L1 cells. Initially Plat-E cells were transiently transfected with the previously described Evi1FL encoding p50M5.6-neo retroviral vector [20]

EVI1 Enhances 3T3-L1 Adipocyte Differentiation
To investigate the impact of enforced EVI1FL expression on adipogenesis it was expressed in 3T3-L1 cells using the transient retroviral infection scheme and subsequent induction of adipocyte differentiation programme outlined in Figure   1

Naturally Occurring EVI1 Splice Variants, RP+, Δ105 and Δ324 Stimulate 3T3-L1 Adipocyte Differentiation
These data suggest enforced expression of Evi1 accelerates adipocyte differentiation of induced 3T3-L1 cells. Multiple, naturally occurring Evi1 splice variants exist in murine cells [14]. A schematic representation of the isoform shown to stimulate adipocyte differentiation ( Figure 2) is shown in Figure 3

Interaction with CtBP Proteins Is Required for EVI1 Mediated Stimulation of 3T3-L1 Adipogenesis
The results suggest that enforced expression of each naturally occurring Evi1 isoform tested can stimulate adipogenesis in induced 3T3-L1 cells. Evi1 interacts with CtBP proteins to mediate some biological activities and so we investigated if this interaction is required to stimulate adipocyte differentiation markers as well.

Discussion
In this study a transient  (Figure 1(b)) 3T3-L1 cells are unlikely to be confluent for 48 hrs prior to induction of differentiation as is normally the case [26] because of the need to optimize retroviral infection in dividing cells [27]. However, our results clearly show that differentiation is significantly enhanced by enforced expression of EVI1 under these conditions, based on the molecular markers examined. Following growth arrest, efficient induction of 3T3-L1 cell differentiation is accompanied by mitotic clonal expansion (MCE) [28]. Studies have shown that EVI1 stimulates cell proliferation [29] and this property may stimulate MCE, contributing to the enhanced expression of adipogenic markers observed here, which are consistent with previous observations [11].
All known naturally occurring EVI1 splice variants are expressed in preadipocytes and throughout differentiation of 3T3-L1 cells. The relative abundance of splice variants has not been determined in this study but others have shown that general EVI1 expression is low in proliferating preadipocytes, transiently peaks during chemical stimulation of differentiation then is low again for the remaining programme [11].
Enforced expression of splice variants EVI1FL, EVI1RP+, EVI1Δ105 and EVI1Δ324 are each capable of stimulating adipocyte differentiation based on relative increases in programme mediator (Cebpα, Pparγ 2 ) and marker (Fabp4, Ca3) gene expression ( Figure 2, Figure 5, Figure 6). It is interesting that EVI1Δ324 can stimulate adipogenic markers as this represents one of the few biological ac- The interaction of EVI1 with CtBP proteins has previously been shown to be essential for biological activities including cell transformation [25] and inhibition of TGFβ signaling [32]. This study shows EVI1 mediated stimulation of adipogenic markers is also CtBP binding dependent. Interestingly, EVI1ΔCtBP1 activity with regard to adipocyte differentiation. Other regulators of adipogenesis are also dependent upon CtBP complexes including Klf3 [33], Fog1 and Fog2 [34]. Both CtBP1 and CtBP2 are expressed throughout adipocyte differentiation ( Figure 6(c)) and their binding is required for both negative (Klf3, Fog1 and Fog2) and positive (EVI1) regulation. Furthermore, the EVI1 related protein PRDM16 also binds CtBP proteins to repress white fat specific genes and are displaced to promote brown adipose tissue development [35]. CtBP proteins bind NAD+ and NADH with higher affinity for the latter which promotes interaction with partner proteins [36]. CtBP proteins have been proposed to have a role in metabolic sensing [37]. High calorie intake is associated with increased levels of NADH.
Based on our study this would be predicted to promote association of EVI1 and CtBP and stimulate adipogenesis.
Obesity, the expansion of adipose tissue depots, is one underlying cause of major health conditions worldwide including both type 2 diabetes mellitus and cardiovascular disease, but the mechanisms involved are not fully understood.
Understanding the molecular mechanisms regulating adipogenesis might identify novel targets for therapeutic intervention. Regulation of the adipogenesis developmental programme is controlled by a complex network of transcription factors and EVI1 has only recently been identified to be involved in this process.
These studies show for the first time that multiple EVI1 isoforms are expressed in adipocytes and can stimulate adipogenic markers in a manner that is partially independent of the ZF1 DNA binding domain but fully dependent upon interaction with co-repressor CtBP proteins. Blocking EVI1/CtBP interaction may be a target for drug development controlling obesity.