Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 100 (14), 8342-7

MLL Repression Domain Interacts With Histone Deacetylases, the Polycomb Group Proteins HPC2 and BMI-1, and the Corepressor C-terminal-binding Protein

Affiliations

MLL Repression Domain Interacts With Histone Deacetylases, the Polycomb Group Proteins HPC2 and BMI-1, and the Corepressor C-terminal-binding Protein

Zhen-Biao Xia et al. Proc Natl Acad Sci U S A.

Abstract

The MLL (mixed-lineage leukemia) gene is involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia. We previously identified a transcriptional repression domain in MLL, which contains a region with homology to DNA methyltransferase. In chromosomal translocations, the MLL repression domain is retained in the leukemogenic fusion protein and is required for transforming activity of MLL fusion proteins. We explored the mechanism of action of the MLL repression domain. Histone deacetylase 1 interacts with the MLL repression domain, partially mediating its activity; binding of Cyp33 to the adjacent MLL-PHD domain potentiates this binding. Because the MLL repression domain activity was only partially relieved with the histone deacetylase inhibitor trichostatin A, we explored other protein interactions with this domain. Polycomb group proteins HPC2 and BMI-1 and the corepressor C-terminal-binding protein also bind the MLL repression domain. Expression of exogenous BMI-1 potentiates MLL repression domain activity. Functional antagonism between Mll and Bmi-1 has been shown genetically in murine knockout models for Mll and Bmi-1. Our new data suggest a model whereby recruitment of BMI-1 to the MLL protein may be able to modulate its function. Furthermore, repression mediated by histone deacetylases and that mediated by polycomb group proteins may act either independently or together for MLL function in vivo.

Figures

Fig. 1.
Fig. 1.
The MLL repression domain interacts with HDAC1. (A) Equivalent amounts of bacterially expressed GST alone, negative control GST-Egr1 repression domain, GST-MLL(1101–1400)(R/MT), MLL(1101–1250)(RD1), and MLL(1251–1400)(RD2), or positive control GST-Rb were used to pull down FLAG-HDAC proteins expressed by transient transfection in 293T cells and then were detected with anti-FLAG antibody. Bound HDAC1, input (5%), and markers are indicated. (B) FLAG-MLL(R/MT), (RD1), and (RD2) expressed in 293T cells and immunoprecipitated with anti-FLAG beads. Bound HDAC1 (Upper) was detected with anti-HDAC1 antibody. After stripping, the membrane was rehybridized with anti-FLAG antibody. (C) CAT reporter gene assay. TSA partially relieves MLL(RD2) repression in a reporter gene assay. MLL domains expressed as Gal4 (amino acids 1–147) fusion proteins, G4-MLL(R/ MT), G4-MLLRD1, G4-MLLRD2, or Gal4 alone were transiently cotransfected with the reporter gene, Gal45tkCAT (18), in HeLa cells. After 24 h, cells were split into two dishes, and one was treated with HDAC inhibitor TSA (100 ng/ml). The results are the mean (+SD) of at least three independent transfections. *, Statistically significant difference.
Fig. 2.
Fig. 2.
Full-length FLAG-MLL and FLAG-MLL(RD+PHD) interact with HDAC1 by coimmunoprecipitation. Equivalent amounts of full-length FLAG-MLL (A) or FLAG-MLL(RD+PHD) (B), plus HDAC1 and Cyp33 expressed by transient transfection in 293T cells, were incubated and immunoprecipitated with anti-FLAG beads. After electrophoresis and Western blot analysis, anti-HDAC1 antibody was used to detect HDAC1 bound to FLAG-MLL. Input (2.5%) and markers are indicated.
Fig. 3.
Fig. 3.
TSA inhibits the ability of Cyp33 to regulate HOX gene expression. Expression of HOXC8 and HOXC6 by semiquantitative RT-PCR in K562 cells transfected with expression vector for HA-Cyp33 (C33), for a truncated form of HA-Cyp33 encoding only the RRM domain and the spacer (ΔC33), or with the vector alone (C). HOXC8 expression is inhibited and HOXC6 expression is enhanced after expression of Cyp33, but not after expression of the truncated protein. These effects are suppressed by cyclosporin A, showing that they may be mediated by the cis/trans prolyl isomerase activity of Cyp33, and also by TSA, showing that they depend on the activity of HDACs.
Fig. 4.
Fig. 4.
The MLL(R/MT) and RD1 domains interact with CtBP and HPC2. (A) Gal4-CtBP protein expressed in 293T cells was pulled down with equivalent amounts of bacterially expressed GST alone, GST-MLL(R/MT), GST-MLL(RD1), or GST-MLL(RD2). Anti-Gal4 antibody was used to detect bound CtBP. Input (5%) is indicated. (B) Anti-FLAG IP. Transfected FLAG-tagged MLL(RD1), MLL(1101–1238), and MLL (1–672) (as a negative control) or FLAG vector were immunoprecipitated from 293T extracts with anti-FLAG beads. Anti-CtBP antibody was used to detect endogenous bound CtBP. (C) HPC2 interacts with MLL(R/MT) and MLL(RD1). Bacterially expressed GST-HPC2 was used to pull down FLAG-MLL(RD1) and FLAG-MLL(RD2) expressed by transient transfection in 293T cells and detected with anti-FLAG antibody. FLAG-CtBP and 293T extract were positive and negative controls, respectively. Input proteins (20%) are indicated. (D) GST-MLL pull-down assay. IVTT-expressed 35S-labeled full-length HPC2 (or HDAC1 as a positive control) was incubated with equivalent amounts of bacterially expressed GST alone, GST-MLL(RD1), or GST-MLL (R/MT). Bound proteins were detected by autoradiography. Input (2.5%) is indicated.
Fig. 5.
Fig. 5.
BMI-1 interacts with the MLL repression domain and enhances MLL(RD1) repression. (A) T7-tagged BMI-1 protein expressed in bacteria was pulled down with equivalent amounts of bacterially expressed GST alone, negative control GST-Egr1 repression domain, GST-MLL(R/MT), GST-MLL(RD1), GST-MLL(RD2), or positive control GST-HPC2. Anti-T7 antibody was used to detect bound BMI-1. Input (2.5%) is indicated. (B) FLAG-MLL(RD1), FLAG-MLL(RD2), or FLAG-MLL(1–672) (as a negative control) expressed by transient transfection in 293T cells that express endogenous BMI-1 protein were immunoprecipitated by using anti-FLAG beads. Proteins were detected by using anti-BMI-1 or anti-FLAG antibody. (C) CAT reporter gene assay. 293T cells were transiently cotransfected with Gal45tk CAT reporter plasmid plus Gal4 alone, Gal4-MLL (R/MT), GAL4-MLL(RD1), or Gal4-MLL(RD2) effectors, with or without additional PMT2SM-HA-BMI-1. Results shown are the mean (+SD) of at least three independent transfections. *, Statistically significant difference.
Fig. 6.
Fig. 6.
Model of repressor complexes associated with wild-type MLL vs. with an MLL fusion protein. The corepressor protein CtBP, the PcG proteins HPC2 and BMI-1, and HDAC1 (or HDAC2) bind to the MLL repression domain. Binding of HDAC1 is increased in the presence of the cyclophilin Cyp33. The coactivator CBP, which has acetyltransferase activity, can bind to the MLL activation domain. The MLL-SET domain has intrinsic histone H3, lysine 4 methyltransferase activity (HMTase H3K4). Corepressors and coactivators may both bind to wild-type MLL; the equilibrium of binding may be influenced by other factors that ultimately determine the function of MLL at a particular target gene locus. For leukemogenic MLL fusion proteins, the equilibrium of binding may be altered, resulting in the aberrant regulation of MLL target genes. Breakpoint cluster region (BCR) and MLL proteolytic cleavage site (arrow) are indicated. Numbering refers to MLL amino acids. The figure is not drawn to scale.

Similar articles

See all similar articles

Cited by 70 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback