Abstract
Myeloid translocation genes (MTGs) are transcriptional corepressors originally identified in acute myelogenous leukemia that have recently been linked to epithelial malignancy with non-synonymous mutations identified in both MTG8 and MTG16 in colon, breast, and lung carcinoma in addition to functioning as negative regulators of WNT and Notch signaling. A yeast two-hybrid approach was used to discover novel MTG binding partners. This screen identified the Zinc fingers, C2H2 and BTB domain containing (ZBTB) family members ZBTB4 and ZBTB38 as MTG16 interacting proteins. ZBTB4 is downregulated in breast cancer and modulates p53 responses. Because ZBTB33 (Kaiso), like MTG16, modulates Wnt signaling at the level of TCF4, and its deletion suppresses intestinal tumorigenesis in the ApcMin mouse, we determined that Kaiso also interacted with MTG16 to modulate transcription. The zinc finger domains of Kaiso as well as ZBTB4 and ZBTB38 bound MTG16 and the association with Kaiso was confirmed using co-immunoprecipitation. MTG family members were required to efficiently repress both a heterologous reporter construct containing Kaiso binding sites (4×KBS) and the known Kaiso target, Matrix metalloproteinase-7 (MMP-7/Matrilysin). Moreover, chromatin immunoprecipitation studies placed MTG16 in a complex occupying the Kaiso binding site on the MMP-7 promoter. The presence of MTG16 in this complex, and its contributions to transcriptional repression both required Kaiso binding to its binding site on DNA, establishing MTG16-Kaiso binding as functionally relevant in Kaiso-dependent transcriptional repression. Examination of a large multi-stage CRC expression array dataset revealed patterns of Kaiso, MTG16, and MMP-7 expression supporting the hypothesis that loss of either Kaiso or MTG16 can de-regulate a target promoter such as that of MMP-7. These findings provide new insights into the mechanisms of transcriptional control by ZBTB family members and broaden the scope of co-repressor functions for the MTG family, suggesting coordinate regulation of transcription by Kaiso/MTG complexes in cancer.
Highlights
The myeloid translocation gene proteins (MTGs) are transcriptional corepressors, lacking both enzymatic and DNA-binding activities, that act as scaffolding proteins upon which other transcriptional corepressors, histone deacetylases (HDACs), and transcription factors assemble [1,2]
The ZBTB4 and ZBTB38 clones that were identified consisted of the highly homologous zinc finger region shared by ZBTB family members (Fig. 1A, S1) and suggested that Kaiso would likely be a third target for MTG16 binding
Directed yeast two-hybrid validation experiments demonstrated that ZBTB4, ZBTB38, and Kaiso each interact with MTG16 via a highly conserved zinc-finger domain (Fig. 1B) which is required for ZBTB16 (PLZF) MTG16 interaction [19], but not with ZBTB27 (BCL6) which demonstrates MTG16 binding within the fourth zinc finger motif [20](Fig. S1)
Summary
The myeloid translocation gene proteins (MTGs) are transcriptional corepressors, lacking both enzymatic and DNA-binding activities, that act as scaffolding proteins upon which other transcriptional corepressors (mSin3a, N-CoR, SMRT), histone deacetylases (HDACs), and transcription factors assemble [1,2]. Their modular nature permits contributions to multiple promoterspecific repressor complexes, making them master regulators of gene expression. ZBTB4 modulates the cellular responses to p53 activation [16] and is downregulated in breast cancer [17] These data suggest ZBTB proteins, and Kaiso have multidimensional roles in cancer development
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