The transcriptional co-activator lens epithelium-derived growth factor/p75 (LEDGF/p75) plays an important role in the biology of the cell and in several human diseases, including MLL-rearranged acute leukemia, autoimmunity, and HIV-1 infection. In both health and disease, LEDGF/p75 functions as a chromatin tether that interacts with proteins such as MLL1 and HIV-1 integrase via its integrase-binding domain (IBD) and with chromatin through its N-terminal PWWP domain. Recently, dimerization of LEDGF/p75 was shown, mediated by a network of electrostatic contacts between amino acids from the IBD and the C-terminal α6-helix. Here, we investigated the functional impact of LEDGF/p75 variants on the dimerization using biochemical and cellular interaction assays. The data demonstrate that the C-terminal α6-helix folds back in cis on the IBD of monomeric LEDGF/p75. We discovered that the presence of DNA stimulates LEDGF/p75 dimerization. LEDGF/p75 dimerization enhances binding to MLL1 but not to HIV-1 integrase, a finding that was observed in vitro and validated in cell culture. Whereas HIV-1 replication was not dependent on LEDGF/p75 dimerization, colony formation of MLLr-dependent human leukemic THP-1 cells was. In conclusion, our data indicate that intricate changes in the quaternary structure of LEDGF/p75 modulate its tethering function.

• Keywords
• DNA-binding protein, DNA-induced protein binding, LEDGF/p75, chromatin structure, protein dynamic, protein–DNA interaction, protein–protein interaction,
• MeSH
• Chromatin * MeSH
• Dimerization MeSH
• DNA metabolism   MeSH
• Humans MeSH
• Intercellular Signaling Peptides and Proteins * metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin * MeSH
• DNA MeSH
• lens epithelium-derived growth factor MeSH Browser
• Intercellular Signaling Peptides and Proteins * MeSH

Interaction scaffolds that selectively recognize disordered protein strongly shape protein interactomes. An important scaffold of this type that contributes to transcription is the TFIIS N-terminal domain (TND). The TND is a five-helical bundle that has no known enzymatic activity, but instead selectively reads intrinsically disordered sequences of other proteins. Here, we review the structural and functional properties of TNDs and their cognate disordered ligands known as TND-interacting motifs (TIMs). TNDs or TIMs are found in prominent members of the transcription machinery, including TFIIS, super elongation complex, SWI/SNF, Mediator, IWS1, SPT6, PP1-PNUTS phosphatase, elongin, H3K36me3 readers, the transcription factor MYC, and others. We also review how the TND interactome contributes to the regulation of transcription. Because the TND is the most significantly enriched fold among transcription elongation regulators, TND- and TIM-driven interactions have widespread roles in the regulation of many transcriptional processes.

• Keywords
• intrinsically disordered proteins, molecular scaffolds, structural biology, transcription,
• MeSH
• Elongin metabolism   MeSH
• Gene Expression Regulation MeSH
• Transcriptional Elongation Factors * chemistry   genetics   metabolism   MeSH
• Transcription Factors * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Elongin MeSH
• transcription factor S-II MeSH Browser
• Transcriptional Elongation Factors * MeSH
• Transcription Factors * MeSH

During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.

• MeSH
• Adaptor Proteins, Signal Transducing chemistry   metabolism   MeSH
• DNA-Binding Proteins chemistry   metabolism   MeSH
• Transcription Elongation, Genetic * MeSH
• Gene Expression MeSH
• Protein Interaction Domains and Motifs genetics   MeSH
• Humans MeSH
• Protein Interaction Maps MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Cell Line, Tumor MeSH
• Protein Domains MeSH
• RNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• RNA Polymerase II chemistry   metabolism   MeSH
• Transcriptional Elongation Factors chemistry   metabolism   MeSH
• Transcription Factors chemistry   genetics   metabolism   MeSH
• Protein Binding MeSH
• Intrinsically Disordered Proteins chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• DNA-Binding Proteins MeSH
• Iws1 protein, human MeSH Browser
• PPP1R10 protein, human MeSH Browser
• RNA-Binding Proteins MeSH
• PSIP1 protein, human MeSH Browser
• RNA Polymerase II MeSH
• transcription factor S-II MeSH Browser
• Transcriptional Elongation Factors MeSH
• Transcription Factors MeSH
• Intrinsically Disordered Proteins MeSH

HDGF-related protein 2 (HRP-2) is a member of the Hepatoma-Derived Growth Factor-related protein family that harbors the structured PWWP and Integrase Binding Domain, known to associate with methylated histone tails or cellular and viral proteins, respectively. Interestingly, HRP-2 is a paralog of Lens Epithelium Derived Growth Factor p75 (LEDGF/p75), which is essential for MLL-rearranged (MLL-r) leukemia but dispensable for hematopoiesis. Sequel to these findings, we investigated the role of HRP-2 in hematopoiesis and MLL-r leukemia. Protein interactions were investigated by co-immunoprecipitation and validated using recombinant proteins in NMR. A systemic knockout mouse model was used to study normal hematopoiesis and MLL-ENL transformation upon the different HRP-2 genotypes. The role of HRP-2 in MLL-r and other leukemic, human cell lines was evaluated by lentiviral-mediated miRNA targeting HRP-2. We demonstrate that MLL and HRP-2 interact through a conserved interface, although this interaction proved less dependent on menin than the MLL-LEDGF/p75 interaction. The systemic HRP-2 knockout mice only revealed an increase in neutrophils in the peripheral blood, whereas the depletion of HRP-2 in leukemic cell lines and transformed primary murine cells resulted in reduced colony formation independently of MLL-rearrangements. In contrast, primary murine HRP-2 knockout cells were efficiently transformed by the MLL-ENL fusion, indicating that HRP-2, unlike LEDGF/p75, is dispensable for the transformation of MLL-ENL leukemogenesis but important for leukemic cell survival.

• Keywords
• animal model, cell culture, cell proliferation, hematopoietic stem cell, leukemia, molecular cell biology, nuclear magnetic resonance (NMR), protein complex, protein-protein interaction,
• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Histone-Lysine N-Methyltransferase genetics   metabolism   MeSH
• Carcinogenesis genetics   metabolism   pathology   MeSH
• Leukemia genetics   metabolism   pathology   MeSH
• Humans MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Cell Cycle Proteins genetics   metabolism   MeSH
• Myeloid-Lymphoid Leukemia Protein genetics   metabolism   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Cell Survival MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• Hdgfl2 protein, mouse MeSH Browser
• Histone-Lysine N-Methyltransferase MeSH
• KMT2A protein, human MeSH Browser
• Kmt2a protein, mouse MeSH Browser
• Cell Cycle Proteins MeSH
• Myeloid-Lymphoid Leukemia Protein MeSH
• PSIP1 protein, human MeSH Browser
• Transcription Factors MeSH

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

• Keywords
• LEDGF/p75, disordered proteins, leukemia, phosphorylation, protein–protein interactions,
• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Amino Acid Motifs MeSH
• Phosphorylation genetics   MeSH
• Histone-Lysine N-Methyltransferase genetics   metabolism   MeSH
• HIV Integrase genetics   metabolism   MeSH
• HIV enzymology   genetics   MeSH
• Humans MeSH
• Mediator Complex Subunit 1 genetics   metabolism   MeSH
• Cell Line, Tumor MeSH
• Myeloid-Lymphoid Leukemia Protein genetics   metabolism   MeSH
• Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• Histone-Lysine N-Methyltransferase MeSH
• HIV Integrase MeSH
• KMT2A protein, human MeSH Browser
• MED1 protein, human MeSH Browser
• Mediator Complex Subunit 1 MeSH
• Myeloid-Lymphoid Leukemia Protein MeSH
• PSIP1 protein, human MeSH Browser
• Transcription Factors MeSH

Lens epithelium-derived growth factor (LEDGF/p75) is an epigenetic reader and attractive therapeutic target involved in HIV integration and the development of mixed lineage leukaemia (MLL1) fusion-driven leukaemia. Besides HIV integrase and the MLL1-menin complex, LEDGF/p75 interacts with various cellular proteins via its integrase binding domain (IBD). Here we present structural characterization of IBD interactions with transcriptional repressor JPO2 and domesticated transposase PogZ, and show that the PogZ interaction is nearly identical to the interaction of LEDGF/p75 with MLL1. The interaction with the IBD is maintained by an intrinsically disordered IBD-binding motif (IBM) common to all known cellular partners of LEDGF/p75. In addition, based on IBM conservation, we identify and validate IWS1 as a novel LEDGF/p75 interaction partner. Our results also reveal how HIV integrase efficiently displaces cellular binding partners from LEDGF/p75. Finally, the similar binding modes of LEDGF/p75 interaction partners represent a new challenge for the development of selective interaction inhibitors.

• MeSH
• Dimerization MeSH
• Escherichia coli MeSH
• Histone-Lysine N-Methyltransferase metabolism   MeSH
• HIV Integrase metabolism   MeSH
• Consensus Sequence MeSH
• Lentivirus enzymology   MeSH
• Humans MeSH
• Intercellular Signaling Peptides and Proteins metabolism   MeSH
• Molecular Sequence Data MeSH
• RNA-Binding Proteins MeSH
• Proteins metabolism   MeSH
• Myeloid-Lymphoid Leukemia Protein metabolism   MeSH
• Repressor Proteins metabolism   MeSH
• Amino Acid Sequence MeSH
• Protein Structure, Tertiary MeSH
• Transcription Factors MeSH
• Transposases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CDCA7L protein, human MeSH Browser
• Histone-Lysine N-Methyltransferase MeSH
• HIV Integrase MeSH
• Iws1 protein, human MeSH Browser
• KMT2A protein, human MeSH Browser
• lens epithelium-derived growth factor MeSH Browser
• Intercellular Signaling Peptides and Proteins MeSH
• POGZ protein, human MeSH Browser
• RNA-Binding Proteins MeSH
• Proteins MeSH
• Myeloid-Lymphoid Leukemia Protein MeSH
• Repressor Proteins MeSH
• Transcription Factors MeSH
• Transposases MeSH