The leukemogenic AF4-MLL fusion protein causes P-TEFb kinase activation and altered epigenetic signatures

Institute of Pharmaceutical Biology/ZAFES, Goethe-University of Frankfurt, Biocenter, Frankfurt/Main, Germany.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K (Impact Factor: 10.43). 10/2010; 25(1):135-44. DOI: 10.1038/leu.2010.249
Source: PubMed


Expression of the AF4-MLL fusion protein in murine hematopoietic progenitor/stem cells results in the development of proB acute lymphoblastic leukemia. In this study, we affinity purified the AF4-MLL and AF4 protein complexes to elucidate their function. We observed that the AF4 complex consists of 11 binding partners and exhibits positive transcription elongation factor b (P-TEFb)-mediated activation of promoter-arrested RNA polymerase (pol) II in conjunction with several chromatin-modifying activities. In contrast, the AF4-MLL complex consists of at least 16 constituents including P-TEFb kinase, H3K4(me3) and H3K79(me3) histone methyltransferases (HMT), a protein arginine N-methyltransferase and a histone acetyltransferase. These findings suggest that the AF4-MLL protein disturbs the fine-tuned activation cycle of promoter-arrested RNA Pol II and causes altered histone methylation signatures. Thus, we propose that these two processes are key to trigger cellular reprogramming that leads to the onset of acute leukemia.

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Available from: Rolf Marschalek, Mar 25, 2014
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    • "The fusion proteins – deriving from the aforementioned MLL translocations – trigger very similar downstream events, namely the ability to bind and activate endogenous AF4/AF5 complexes (also named superelongation complexes), or the direct activation of RNA Polymerase II, as does the AF4-MLL fusion protein [4]. This causes an increase in mRNA levels and ectopic/extended histone methylation signatures (H3K4 and H3K79) [5] [6] [7]. Furthermore, these 4 MLL gene fusions account for more than 90% of ALL cases (infant, childhood and adult) and about 50% of AML cases (infant, childhood and adult). "
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    ABSTRACT: Our focus is the identification, characterization and functional analysis of different MLL fusions. In general, MLL fusion proteins are encoded by large cDNA cassettes that are difficult to transduce into haematopoietic stem cells. This is due to the size limitations of the packaging process of those vector-encoded RNAs into retro- or lentiviral particles. Here, we present our efforts in establishing a universal vector system to analyse different MLL fusions. The universal cloning system was embedded into the backbone of the Sleeping Beauty transposable element. This transposon has no size limitation and displays no integration preference, thereby avoiding the integration into active genes or their promoter regions. We utilised this novel system to test different MLL fusion alleles (MLL-NEBL, NEBL-MLL, MLL-LASP1, LASP1-MLL, MLL-MAML2, MAML2-MLL, MLL-SMAP1 and SMAP1-MLL) in appropriate cell lines. Stable cell lines were analysed for their growth behaviour, focus formation and colony formation capacity and ectopic Hoxa gene transcription. Our results show that only 1/4 tested direct MLL fusions, but 3/4 tested reciprocal MLL fusions exhibit oncogenic functions. From these pilot experiments, we conclude that a systematic analysis of more MLL fusions will result in a more differentiated picture about the oncogenic capacity of distinct MLL fusions.
    Cancer Letters 10/2014; 352(2). DOI:10.1016/j.canlet.2014.06.016 · 5.62 Impact Factor
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    • "The identification of these mutually exclusive CCAPs agrees precisely with traditional experimental approaches that identified these two distinct CDK9/CCNT1 complexes [26]. Additionally, the recently identified Super-elongation complex (SEC) complex (AFF4/AFF1/MLLT1/ELL2/MLLT3/; complex D [21,22,27]) was identified in our analysis, further validating this experimental approach to identify CCAPs of likely relevance to cellular and HIV-1 gene expression. "
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    ABSTRACT: Background HIV-1 Tat activates RNA Polymerase II (RNAP II) elongation of the integrated provirus by recruiting a protein kinase known as P-TEFb to TAR RNA at the 5′ end of nascent viral transcripts. The catalytic core of P-TEFb contains CDK9 and Cyclin T1 (CCNT1). A human endogenous complexome has recently been described – the set of multi-protein complexes in HeLa cell nuclei. We mined this complexome data set and identified 12 distinct multi-protein complexes that contain both CDK9 and CCNT1. We have termed these complexes CCAPs for CDK9/CCNT1-associated protein complexes. Nine CCAPs are novel, while three were previously identified as Core P-TEFb, the 7SK snRNP, and the Super-Elongation Complex. We have investigated the role of five newly identified CCAPs in Tat function and viral gene expression. Results We examined five CCAPs that contain: 1) PPP1R10/TOX3/WDR82; 2) TTF2; 3) TPR; 4) WRNIP1; 5) FBXO11/CUL1/SKP1. SiRNA depletions of protein subunits of the five CCAPs enhanced Tat activation of an integrated HIV-1 LTR-Luciferase reporter in TZM-bl cells. Using plasmid transfection assays in HeLa cells, we also found that siRNA depletions of TTF2, FBXO11, PPP1R10, WDR82, and TOX3 enhanced Tat activation of an HIV-1 LTR-luciferase reporter, but the depletions did not enhance expression of an NF-κB reporter plasmid with the exception of PPP1R10. We found no evidence that depletion of CCAPs perturbed the level of CDK9/CCNT1 in the 7SK snRNP. We also found that the combination of siRNA depletions of both TTF2 and FBXO11 sensitized a latent provirus in Jurkat cells to reactivation by sub-optimal amounts of αCD3/CD28 antibodies. Conclusions Our results identified five novel CDK9/CCNT1 complexes that are capable of negative regulation of HIV-1 Tat function and viral gene expression. Because siRNA depletions of CCAPs enhance Tat function, it is possible that these complexes reduce the level of CDK9 and CCNT1 available for Tat, similar to the negative regulation of Tat by the 7SK snRNP. Our results highlight the complexity in the biological functions of CDK9 and CCNT1.
    Retrovirology 10/2012; 9(1):90. DOI:10.1186/1742-4690-9-90 · 4.19 Impact Factor
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    • "Interestingly, even though the MLL N-terminal fusion protein MLL–AF9 does not contain the C-terminal catalytic methyltransferase domain, the wild-type allele is essential for leukaemogenesis [12]. Furthermore, reciprocal C-terminal MLL fusion proteins, such as AFF1–MLL, that retain H3K4me3 catalytic activity [13] are among the most potent leukaemogenic MLL fusions [14]. These studies suggest a role for the C-terminal catalytic SET domain in MLL-rearranged leukaemia. "
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    ABSTRACT: The WD40 protein, WDR5, is an essential component of the human trithorax-like family of SET1 methyltransferase complexes that carry out trimethylation of histone 3 lysine 4 (H3K4me3), play key roles in development, and are abnormally expressed in many cancers. Here we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide binding pocket with a KD of 450 nM, and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein-protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukemias or other cancers.
    Biochemical Journal 09/2012; 449(Pt 1). DOI:10.1042/BJ20121280 · 4.40 Impact Factor
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