ABSTRACT
Eleven-nineteen leukemia (ENL) is a chromatin reader present in complexes stimulating transcriptional elongation. It is fused to mixed-lineage leukemia (MLL) in leukemia, and missense mutations have been identified in Wilms tumor and acute myeloid leukemia. Here we demonstrate that ENL overcomes polycomb silencing through recruitment of PAF1 via the conserved YEATS domain, which recognizes acetylated histone H3. PAF1 was responsible for antirepressive activities of ENL in vitro, and it determined the transforming potential of MLL-ENL. MLL-ENL target loci showed supraphysiological PAF1 binding, hyperubiquitination of histone H2B and hypomodification with H2AUb, resulting in accelerated transcription rates. YEATS mutations induced a gain of function, transforming primary hematopoietic cells in vitro and in transplantation assays through aberrant transcription and H2B ubiquitination of Hoxa9 and Meis1 Mechanistically, H3 and PAF1 competed for ENL interaction, with activating mutations favoring PAF1 binding, whereas the MLL moiety provided a constitutive PAF1 tether allowing MLL fusions to circumvent H3 competition.
Subject(s)
Carrier Proteins/metabolism , Cell Transformation, Neoplastic , DNA-Binding Proteins/metabolism , Gene Silencing , Leukemia/genetics , Polycomb-Group Proteins/genetics , Transcription Factors/metabolism , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Gene Expression Regulation, Leukemic , HEK293 Cells , Histones/metabolism , Humans , Leukemia/metabolism , Leukemia/pathology , Mice , Mice, Inbred BALB C , Mutation , Protein Binding/genetics , Protein Domains/genetics , Protein Processing, Post-Translational , Transcription Factors/chemistry , Transcription Factors/geneticsABSTRACT
Hox homeobox transcription factors drive leukemogenesis efficiently only in the presence of Meis or Pbx proteins. Here we show that Pbx3 and Meis1 need to dimerize to support Hox-induced leukemia and we analyze the molecular details of this cooperation. In the absence of Pbx3, Meis1 was highly unstable. As shown by a deletion analysis Meis1 degradation was contingent on a motif coinciding with the Pbx-binding domain. Either deletion of this sequence or binding to Pbx3 prolonged the half-life of Meis1 by preventing its ubiquitination. Meis1 break-down could also be blocked by inhibition of the ubiquitin proteasome system, indicating tight post-transcriptional control. In addition, Meis1 and Pbx3 cooperated genetically as overexpression of Pbx3 induced endogenous Meis1 transcription. These functional interactions translated into in vivo activity. Blocking Meis1/Pbx3 dimerization abrogated the ability to enhance proliferation and colony-forming cell numbers in primary cells transformed by Hoxa9. Furthermore, expression of Meis1 target genes Flt3 and Trib2 was dependent on Pbx3/Meis1 dimerization. This correlated with the requirement of Meis1 to bind Pbx3 in order to form high affinity DNA/Hoxa9/Meis1/Pbx3 complexes in vitro. Finally, kinetics and severity of disease in transplantation assays indicated that Pbx3/Meis1 dimers are rate-limiting factors for Hoxa9-induced leukemia.