MEF2C is activated by multiple mechanisms in a subset of T-acute lymphoblastic leukemia cell lines
In T-cell acute lymphoblastic leukemia (T-ALL) the cardiac homeobox gene NKX2-5 (at 5q35) is variously deregulated by regulatory elements coordinating with BCL11B (at 14q32.2), or the T-cell receptor gene TRD (at 14q11.2), respectively. NKX2-5 is normally expressed in developing spleen and heart, regulating fundamental processes, including differentiation and survival. In this study we investigated whether NKX2-5 expression in T-ALL cell lines reactivates these embryonal pathways contributing to leukemogenesis. Among 18 known targets analyzed, we identified three genes regulated by NKX2-5 in T-ALL cells, including myocyte enhancer factor 2C (MEF2C). Knockdown and overexpression assays confirmed MEF2C activation by NKX2-5 at both the RNA and protein levels. Direct interactions between NKX2-5 and GATA3 as indicated by co-immunoprecipitation data may contribute to MEF2C regulation. In T-ALL cell lines LOUCY and RPMI-8402 MEF2C expression was correlated with a 5q14 deletion, encompassing noncoding proximal gene regions. Fusion constructs with green fluorescent protein permitted subcellular detection of MEF2C protein in nuclear speckles interpretable as repression complexes. MEF2C consistently inhibits expression of NR4A1/NUR77, which regulates apoptosis via BCL2 transformation. Taken together, our data identify distinct mechanisms underlying ectopic MEF2C expression in T-ALL, either as a downstream target of NKX2-5, or via chromosomal aberrations deleting proximal gene regions.
Homeobox genes code for transcription factors impacting basic cellular processes, such as differentiation, proliferation and apoptosis.1 If mutated or dysregulated many examples have been shown to promote oncogenesis in both solid and hematopoietic tumors.2, 3 With respect to evolutionary history, homeobox genes have been classified as clustered or nonclustered according to their genomic localization, and in subfamilies according to their homeobox sequences.4, 5 In T-cell acute lymphoblastic leukemia (T-ALL) genes belonging to both clustered (HOXA) and the subfamily of NK-like homeobox genes exhibit pathological involvement.6, 7, 8 The NK-like subfamily includes TLX1, TLX3 and NKX2-5, all ectopically activated in T-ALL cells via chromosomal rearrangements.7, 9, 10
In T-ALL expression of certain oncogenes including homeobox genes TLX1 and TLX3 define disease subtypes.11, 12 Data obtained by expression profiling link these T-ALL subtypes to developmental staging of T cells, indicating specific differentiation arrest.13 T-ALL cases expressing TLX3 have poorer prognoses than those expressing TLX1 instead,13, 14 suggesting differences in downstream regulation between these two closely related homeobox genes. With few exceptions, ectopic expression of NK-like homeobox genes is activated via chromosomal aberrations.15 For all three homeobox genes TLX1, TLX3 and NKX2-5 juxtaposition to TRD at 14q11 has been described. Additionally, TLX1 is activated in t(7;10)(q35;q24) via fusion with TRB, and both, TLX3 and NKX2-5 by t(5;14)(q35;32) involving BCL11B.7, 9, 10, 16, 17, 18, 19
TLX3 and NKX2-5 are located at 5q35 separated by approximately 2 Mb. In t(5;14)-positive cells orphan enhancer elements originated from far downstream regions of BCL11B at 14q32 are juxtaposed with either homeobox gene.7 These elements co-localize to DNAse I hypersensitive sites, contain acetylated histones, bind to the nuclear matrix and are enriched in HMGA1-binding sites juxtaposed by t(5;14) rearrangements with PU.1 sites located in the promoter regions of both TLX3 and NKX2-5.20 Treatment with oligonucleotides matching enhancer elements at 3'-BCL11B effected downregulation of NKX2-5 in T-ALL PEER cells where this gene is juxtaposed to BCL11B by t(5,14).20
In the present study this enhancer inhibition assay was used to effect NKX2-5 knockdown in T-ALL cells therein to evaluate candidate downstream targets of this gene previously identified in other tissues, notably developing spleen and heart.21 We report aberrant myocyte enhancer factor 2C (MEF2C) expression, which may implicate analogous activation mechanisms in T-ALL cell lines. In addition we show that a 5q14 deletion targets loss of putative regulatory regions associated with aberrant expression of the MEF2C transcription factor recently shown to act as a cooperating oncogene in mouse leukemia together with SOX4 in an insertional retrovirus assay.22 Our data highlight the MEF2C transcription factor as a new dysregulatory target in T-ALL subsets and provide a new example of a putative oncogenic microdeletion.
Cell lines and treatments
Detailed descriptions of cell lines used in this study and their culture methods are given in Drexler (2005) and are accessible at www.dsmz.de.23 Electroporation of PEER was performed as previously described.20 Transfection of HeLa with plasmid DNA and siRNA oligonucleotides was performed according to the manufacturers' instructions (Qiagen, Hilden, Germany). Trichostatin-A (TSA) and p38 inhibitor SB203580 were obtained from Sigma (München, Germany), and the BCL2 inhibitor YC137 from Calbiochem (Darmstadt, Germany).