The inv(16) Fusion Protein Associates with Corepressors via a Smooth Muscle Myosin Heavy-Chain Domain

Abstract

Inversion (16) is one of the most frequent chromosomal translocations found in acute myeloid leukemia (AML), occurring in over 8% of AML cases. This translocation results in a protein product that fuses the first 165 amino acids of core binding factor ␤ to the coiled-coil region of a smooth muscle myosin heavy chain (CBF␤/SMMHC). CBF␤ interacts with AML1 to form a heterodimer that binds DNA; this interaction increases the affinity of AML1 for DNA. The CBF␤/SMMHC fusion protein cooperates with AML1 to repress the transcription of AML1-regulated genes. We show that CBF␤/SMMHC contains a repression domain in the C-terminal 163 amino acids of the SMMHC region that is required for inv(16)-mediated transcriptional repression. This minimal repression domain is sufficient for the association of CBF␤/SMMHC with the mSin3A corepressor. In addition, the inv(16) fusion protein specifically associates with histone deacetylase 8 (HDAC8). inv(16)-mediated repression is sensitive to HDAC inhibitors. We propose a model whereby the inv(16) fusion protein associates with AML1 to convert AML1 into a constitutive transcriptional repressor.Human acute leukemias often arise from chromosomal translocations targeting regulatory genes that affect cell growth, differentiation, and apoptosis (30). These translocations often fuse transcription factors to other proteins resulting in oncogenic chimeric proteins. inv (16) is found in approximately 8% of acute myeloid leukemia (AML) cases (35), making it one of the most frequent translocations in AML. inv(16) fuses the first 165 amino acids (aa) of core binding factor ␤ (CBF␤) to the C-terminal coiled-coil region of a smooth muscle myosin heavy chain (SMMHC [the gene name is MYH11]) (28). CBF␤ interacts with the AML1 (also known as RUNX1) transcription factor to increase the affinity of AML1 for DNA (15,40,47) and to stimulate the ability of AML1 to either activate or repress transcription (22,32). Both components of the CBF␤-AML1 transcription factor complex are disrupted by chromosomal translocations. The translocations that target AML1 include t(8;21) and t(12;21) that result in the leukemogenic fusion proteins AML1/ETO and TEL/AML1 (10, 16).The CBF␤-AML1 complex regulates genes encoding cytokines and their receptors, genes involved in differentiation such as T-cell receptors, neutrophil peptide 3, and myeloperoxidase as well as the p14 ARF tumor suppressor (4,12,14,27,36,39,43,46,52). Furthermore, CBF␤/SMMHC slows the cell cycle transition from G 1 to S in hematopoietic cells; thus, cell cycle regulatory genes such as cdk4 may also be targeted by this complex (5).Animal studies indicate that CBF␤ and AML1 are required for hematopoiesis. CBF␤ and AML-1 knockout mice share a phenotype; these mice lack fetal liver hematopoiesis and are embryonic lethal at embryonic day 12.5 (e12.5) to e13.5 (41,49). A similar phenotype is observed in mice expressing CBF␤/ SMMHC from a "knocked-in" CBFB-MYH11 gene (7), suggesting that inv(16) creates a dominant repressor of AML1-and CBF␤-regulated genes. Chimer...

RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription

“…RUNX1 residues 1-204 are also retained in the t(8;21) fusion protein, which again predicts that this fusion protein can recruit SUV39H1, although this association may negatively affect DNA binding (Chakraborty et al, 2003). Although the inv(16) contains no homology to RUNX1, it acts as a corepressor for RUNX1 (Lutterbach et al, 1999;Durst et al, 2003). Our initial analysis failed to detect an association between SUV39H1 and the inv(16) fusion protein.…”

“…In addition to the association between SUV39H1 and RUNX1, RUNX1 can bind to HDAC1, HDAC3 and HDAC9 and to a lesser degree HDAC2, HDAC5 and HDAC6 (Durst et al, 2003). Therefore, we tested whether HDAC1 or HDAC3 might also contact the motifs that are required for repression within RD2 by screening these factors against the collection of GAL4-RUNX1 fusion proteins.…”

“…To begin to dissect the function of the subdomains that contact HDACs and SUV39H1, we performed reporter assays using the p21 cyclin-dependent kinase inhibitor promoter linked to firefly luciferase, which contains multiple RUNX1-binding sites (Lutterbach et al, 1999;Durst et al, 2003). RUNX1 was able to repress this promoter up to five-fold in the absence of its co-factor CBFb, and the co-expression of CBFb stimulated RUNX1-mediated repression to nearly 10-fold (Figure 6d).…”

“…RUNX1 function is also impaired by the inv(16), which fuses the RUNX1 associating factor, core binding factor b (CBFb or polyoma enhancer binding protein 2 beta) to the smooth muscle myosin heavy-chain gene MYH11, in approximately 8% of acute myeloid leukemia (Liu et al, 1993). Like the t(8;21) and the t(12;21), the inv(16) also encodes a transcriptional repressor, but this factor must associate with RUNX1 to regulate transcription (Lutterbach et al, 1999;Durst et al, 2003).…”

“…Given the roles of RUNX family members in both establishment and maintenance of repression, as well as gene silencing during T-cell development, we hypothesized that RUNX1 and RUNX3 may associate with factors that regulate the 'histone code' (Durst et al, 2003). The histone code hypothesis suggests that deacetylation of key lysine residues in the conserved tails of histones is followed by methylation that allows the association of silencing factors such as heterochromatin protein-1 (HP1), which binds to methylated histone H3 (Firestein et al, 2000;Jacobs et al, 2001;Jenuwein and Allis, 2001;Lachner et al, 2001).…”