Cooperative gene activation by AF4 and DOT1L drives MLL-rearranged leukemia
Hiroshi Okuda, … , Akifumi Takaori-Kondo, Akihiko Yokoyama
Hiroshi Okuda, … , Akifumi Takaori-Kondo, Akihiko Yokoyama
Published April 10, 2017
Citation Information: J Clin Invest. 2017;127(5):1918-1931. https://doi.org/10.1172/JCI91406.
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Research Article Hematology Oncology

Cooperative gene activation by AF4 and DOT1L drives MLL-rearranged leukemia

Abstract

The eleven-nineteen leukemia (ENL) protein family, composed of ENL and AF9, is a common component of 3 transcriptional modulators: AF4–ENL–P-TEFb complex (AEP), DOT1L-AF10-ENL complex (referred to as the DOT1L complex) and polycomb-repressive complex 1 (PRC1). Each complex associates with chromatin via distinct mechanisms, conferring different transcriptional properties including activation, maintenance, and repression. The mixed-lineage leukemia (MLL) gene often fuses with ENL and AF10 family genes in leukemia. However, the functional interrelationship among those 3 complexes in leukemic transformation remains largely elusive. Here, we have shown that MLL-ENL and MLL-AF10 constitutively activate transcription by aberrantly inducing both AEP-dependent transcriptional activation and DOT1L-dependent transcriptional maintenance, mostly in the absence of PRC1, to fully transform hematopoietic progenitors. These results reveal a cooperative transcriptional activation mechanism of AEP and DOT1L and suggest a molecular rationale for the simultaneous inhibition of the MLL fusion–AF4 complex and DOT1L for more effective treatment of MLL-rearranged leukemia.

Authors

Hiroshi Okuda, Boban Stanojevic, Akinori Kanai, Takeshi Kawamura, Satoshi Takahashi, Hirotaka Matsui, Akifumi Takaori-Kondo, Akihiko Yokoyama

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Figure 1

Genomic localization of 3 ENL-containing complexes.

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Genomic localization of 3 ENL-containing complexes. (A) ENL-associating ...
(A) ENL-associating factors on nucleosomes. 293T cells transiently expressing Xpress-tagged ENL (xENL) or its AHD-lacking mutant (ΔAHD) were analyzed by nucfrIP copurification using an anti-Xpress antibody. The sample shown in the input lane is indicated by an asterisk. (B) Cofactor binding of MLL-ENL. 293T cells transiently expressing FLAG-tagged MLL-ENL were analyzed by fanChIP copurification using an anti-FLAG antibody. (C) CBX8-associating factors on nucleosomes. 293T cells transiently expressing Xpress-tagged ENL, with or without FLAG-tagged CBX8, were analyzed by nucfrIP copurification using an anti-FLAG antibody. (D) Histone H3K27 modification of the nucleosomes coprecipitated with ENL. Purified nucleosomes, as in A, were subjected to MS analysis. The ratio of each modification identified by MS on histone H3K27 is shown in pie charts. The n values shown indicate the number of peptides analyzed. ac, acetylated. (E) Genomic localization of ENL-containing complexes and various histone modifications in 293T cells. fanChIP or faxChIP (for H3K79me2), followed by deep sequencing, was performed on the chromatin of 293T cells for the indicated proteins and modifications. ChIP signals were visualized using the Integrative Genomics Viewer (The Broad Institute). The minimum value of the y axis is set at 0, while the maximum value for each sample is indicated. The RNA-seq profile is also shown for comparison. CGI, CpG island; FOXL2, forkhead box L2; MGME1, mitochondrial genome maintenance exonuclease 1. (F) Distribution patterns of various proteins and modifications at the ENL target loci. A set of genes (852 genes) whose ENL ChIP signal within the region 0 to +2 kb from the TSS was more than 2-fold of the input signal was defined as the representative ENL target set. The frequency of each protein and modification at the ENL target TSSs (black lines) or all TSSs (gray lines) is shown along with the input DNA (dashed lines). The y axis indicates the frequency of the ChIP-seq tag count (ppm) in 25-bp increments.