TBP loading by AF4 through SL1 is the major rate-limiting step in MLL fusion-dependent transcription

Abstract

Gene rearrangement of the mixed lineage leukemia (MLL) gene causes leukemia by inducing the constitutive expression of a gene subset normally expressed only in the immature haematopoietic progenitor cells. MLL gene rearrangements often generate fusion products of MLL and a component of the AF4 family/ENL family/P-TEFb (AEP) complex. MLL-AEP fusion proteins have the potential of constitutively recruiting the P-TEFb elongation complex. Thus, it is hypothesized that relieving the promoter proximal pausing of RNA polymerase II is the rate-limiting step of MLL fusion-dependent transcription. AEP also has the potential to recruit the mediator complex via MED26. We recently showed that AEP activates transcription initiation by facilitating TBP loading to the TATA element through the SL1 complex. In the present study, we show that the key activity responsible for the oncogenic property of MLL-AEP fusion proteins is the TBP loading activity, and not the mediator recruitment or transcriptional elongation activities. Thus, we propose that TBP loading by AF4 through SL1 is the major rate-limiting step in MLL fusion-dependent transcription.

Keywords: AF4; MLL; SL1; TBP; leukemia; mediator; transcription.

Figure 1.

The pSER domain, but not the modules that recruit elongation factors, confer transforming ability to the MLL fusion proteins. (A) The structure of MLL fusion constructs. The properties of each construct are summarized on the right. MTM: the minimum structure of the PWWP domain of LEDGF and the minimum structure of the CXXC domain of MLL and its C-terminal basic region; black flag: FLAG epitope; red flag: the HA epitope. (B) Schematic representation of the modules required for MLL fusion-dependent transformation.

Figure 2.

The roles of 3 conserved motifs in the pSER domain. (A) Sequence alignments of the pSER domain. The pSER domain can be divided into 3 parts, each of which contains one conserved motif such as the DLXLS, SDE, and NKW motifs. (B) The structure of the FLAG-tagged GAL4-fusion constructs harboring various subdomains of the pSER domain. The properties of each construct are summarized on the right. (C) The domain responsible for association with MED26. Various FLAG-tagged GAL4 fusion constructs and HA-tagged MED26 construct were transfected into 293T-LUC cells and analyzed by IP-western blotting (WB). Each protein was visualized using antibodies for the indicated proteins/tags. (D) The domain responsible for recruitment of various factors and transactivation. Various FLAG-tagged GAL4 constructs and HA-tagged MED26 construct were transfected into 293T-LUC cells and analyzed by ChIP-qPCR for indicated proteins. The same set of FLAG-tagged GAL4 constructs and pRL-tk construct were transfected into 293T-LUC cells and analyzed for their transcriptional activation activity.

Figure 3.

The mediator complex co-localizes with MLL-AEP/SL1 complex in MLL leukemia cells. Localization of MLL, AF4, TAF1C, and MED15 at various loci in HB1119 cells. The genomic localization of each protein was determined by ChIP-qPCR. Precipitated DNA was analyzed using specific probes for the pre-TSS (−1.0 to −0.5 kb from the TSS), TSS (0 to +0.5 kb from the TSS), and post-TSS (+1.0 to +1.5 kb from the TSS) regions of the indicated genes. The ChIP signals are expressed as the percent input with error bars (SD of PCR triplicates). The data are partially redundant with those published previously.

Figure 4.

MED26 recruitment by MLL fusion proteins is dispensable for myeloid transformation. (A) Transforming ability of the MTM fusion constructs with various subdomains of the pSER domain. A schematic of the myeloid progenitor transformation assay is shown on top. Hoxa9 expression in the first round of colonies normalized to Gapdh expression is shown as the relative value of MTM-AF4-2C-bc (arbitrarily set at 100%) with error bars (SD of PCR triplicates). The number of colony-forming units (CFUs) of third and fourth rounds is shown with error bars (SD was calculated from data arising from more than 3 independent experiments). (B) Protein expression of various MTM fusion proteins in virus packaging cells. The whole cell extracts of the virus-packaging cells were analyzed by WB using anti-HA antibody.

Figure 5.

AEP activates gene expression by facilitating TBP loading, PIC formation, and transcriptional elongation. Working model of AEP-dependent transcriptional activation.

Figure 6.

TBP loading is the critical rate-limiting step activated by MLL-AEP fusion proteins. Working model of MLL-AEP-dependent transcriptional activation.