The PHD fingers of MLL block MLL fusion protein–mediated transformation

Abstract

IntroductionBalanced translocations involving mixed lineage leukemia (MLL) are common in human acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). 1 MLL positively regulates Hox and other target gene expression through SET domaindependent histone H3 lysine 4 methyltransferase activity. 2,3 Like MLL, MLL fusion proteins bind directly to Hox loci and up-regulate their expression. 2-5 Deregulated expression of Hox genes including Hoxa9 and the Hox cofactor Meis1 appears to be central for MLL fusion protein mediated transformation. [6][7][8] Translocations in MLL occur within an 8.3-kb breakpoint cluster region (BCR) resulting in fusion of approximately 1400 amino acids of N-terminal MLL to 1 of more than 60 fusion partners. 9 The BCR region has been extensively studied and contains several structural elements including Alu elements, topoisomerase II cleavage sites, and DNase I hypersensitive sites. These along with error-prone nonhomologous end joining have been proposed as mechanisms for MLL translocations. 10 It is also possible that there is a functional reason for the location of the breaks. For example, sequences immediately 5Ј to the BCR encode the CXXC domain, which must be retained intact for MLL-fusion protein transformation. 11,12 Notably, sequences 3Ј to the BCR, encoding the PHD fingers, are consistently deleted in leukemogenic fusion proteins. The function of PHD fingers in general is obscure. Some have been reported to mediate protein-protein interactions such as binding to methylated histones or other chromatin associated proteins. 13,14 For example, HCF-1 binds to the HBM region between the bromodomain and PHD#4 of MLL and modulates transcription through E2F interactions. [15][16][17] In addition, the nuclear cyclophilin Cyp33 has been reported to bind the PHD fingers leading to enhanced binding of HPC2 and BMI-1 to the CXXC domain. 18,19 The consistent deletion of the PHD fingers in balanced translocations, the reports of interactions of the PHD fingers with corepressors, 20 the disrupted MLL architecture in MLL partial tandem duplications in AML 21 and the finding that MLL partial deletion of exons 7 and 8, which encode the first PHD finger, is associated with T-ALL 22 all raise the possibility that the domains play a negative regulatory role in MLL function. Understanding the contribution of the PHD domain would provide insights into not only the location of breaks in the BCR but also the possible functions of the enigmatic PHD domains in normal and leukemogenic MLL function. Here we assess the role of the PHD fingers in transcriptional regulation and test the significance of inclusion of the PHD fingers, located downstream of the BCR, on MLL-fusion protein mediated transformation. MethodsAll animal studies were approved by the University of Michigan Committee on Use and Care of Animals and Unit for Laboratory Medicine. For complete materials and methods information, see Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article)....

The molecular biology of mixed lineage leukemia

“…68,69 In addition, it was mandatory that the breakpoint in MLL was upstream of the PHD fingers because artificial MLL fusions including this domain lost their transforming capacity. 70,71 This explains the strict conservation of the fusion breakpoints found in leukemic blasts. Further studies suggested that the leukemogenic potential of truncated MLL could be activated in at least four different ways (Figure 3).…”

Proteolytically cleaved MLL subunits are susceptible to distinct degradation pathways

“…In particular, the third PHD finger (PHD3) was shown to associate with di-or tri-methylated histone H3 lysine 4, which might be regulated by Cyp33 binding (Fair et al, 2001;Chang et al, 2010;Milne et al, 2010;Wang et al, 2010b). PHD3 is not present in the leukemic MLL fusion proteins and diminishes oncogenic ability if artificially included in an MLL fusion protein (Muntean et al, 2008;Chen et al, 2008). MLL N associates with menin and LEDGF, which are also crucial for linking MLL proteins with target chromatin (Yokoyama and Cleary, 2008).…”

Zebrafish mll Gene Is Essential for Hematopoiesis

“…The MLL gene encodes a 430-kDa protein containing three AT hook motifs, two speckled nuclear localization domains (SNL1 and SNL2), a CXXC zinc finger motif, multiple plant homology domains (PHDs), 2 and a Su/var, Enhancer or zeste, Trithorax (SET), domain. The PHD fingers contribute to protein-protein interactions, and their deletion, as a result of chromosomal translocation, is necessary for MLL fusion proteins to induce hematopoietic cell immortalization (9). The SET domain contributes to modulation of homeobox (HOX) gene expression by facilitating histone H3 Lys-4 methylation at the proximal promoter region (10).…”