Structural basis of CBP/p300 recruitment in leukemia induction by E2A-PBX1

Abstract E-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein–encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein–driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1–induced leukemia.

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Critical Role for a Single Leucine Residue in Leukemia Induction by E2A-PBX1

Molecular and Cellular Biology ◽

10.1128/mcb.02025-05 ◽

2006 ◽

Vol 26 (17) ◽

pp. 6442-6452 ◽

Cited By ~ 20

Author(s):

Richard Bayly ◽

Takayuki Murase ◽

Brandy D. Hyndman ◽

Rachel Savage ◽

Salima Nurmohamed ◽

...

Keyword(s):

Bone Marrow ◽

Transcriptional Activation ◽

Bone Marrow Cells ◽

Lymphoblastic Leukemia ◽

Critical Role ◽

Chromosomal Translocation ◽

Leucine Residue ◽

Helix Formation ◽

Lxxll Motif ◽

Kix Domain

ABSTRACT In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.

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Role for Homodimerization in Growth Deregulation by E2a Fusion Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.20.16.5789-5796.2000 ◽

2000 ◽

Vol 20 (16) ◽

pp. 5789-5796 ◽

Cited By ~ 10

Author(s):

Richard Bayly ◽

David P. LeBrun

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Transcriptional Activation ◽

Fusion Proteins ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Chromosomal Translocation ◽

Early Observation ◽

Focus Formation ◽

Transcriptional Activation Domain

ABSTRACT The oncogenic transcription factor E2a-Pbx1 is expressed in some cases of acute lymphoblastic leukemia as a result of chromosomal translocation 1;19. The early observation that E2a-Pbx1 incorporates transcriptional activation domains from E2a and a DNA-binding homeodomain from Pbx1 inspired a model in which E2a-Pbx1 promotes leukemogenic transformation of lymphoid progenitor cells through transcriptional induction of target genes defined by the Pbx1 portion of the molecule. However, the subsequent demonstration that the only known DNA-binding module on the molecule, the Pbx1 homeodomain, is dispensable for the induction of lymphoblastic lymphoma in transgenic mice called into question the contribution made by the Pbx1 portion. In this study, we have used a domain swap approach coupled with a fibroblast-based focus formation assay to evaluate further the requirement for PBX1-encoded peptide elements in growth deregulation by E2a-Pbx1. No impairment of focus formation was observed when the entire Pbx1 portion was replaced with DNA-binding/dimerization domains derived from yeast transcription factor GAL4 or GCN4. Furthermore, replacement of Pbx1 with tandem FKBP domains that mediate homodimerization in the presence of a synthetic ligand led to striking growth deregulation exclusively in the presence of the dimerizing agent. N-terminal elements encoded by E2A, including the AD1 transcriptional activation domain, were required for dimerization-induced focus formation. We conclude that transcriptional target genes defined by heterologous C-terminal DNA-binding modules are not required in growth deregulation by E2a fusion proteins. We speculate that interactions between N-terminal E2a elements and undefined proteins that could function as components of a transcriptional coactivator complex may be more important.

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Extension of the Notch intracellular domain ankyrin repeat stack by NRARP promotes feedback inhibition of Notch signaling

Science Signaling ◽

10.1126/scisignal.aay2369 ◽

2019 ◽

Vol 12 (606) ◽

pp. eaay2369 ◽

Cited By ~ 3

Author(s):

Sanchez M. Jarrett ◽

Tom C. M. Seegar ◽

Mark Andrews ◽

Guillaume Adelmant ◽

Jarrod A. Marto ◽

...

Keyword(s):

Notch Signaling ◽

Negative Feedback ◽

Transcriptional Activation ◽

Cultured Cells ◽

Feedback Inhibition ◽

Lymphoblastic Leukemia ◽

Ankyrin Repeat ◽

Structural Basis ◽

Intracellular Domain ◽

Notch Intracellular Domain

Canonical Notch signaling relies on regulated proteolysis of the receptor Notch to generate a nuclear effector that induces the transcription of Notch-responsive genes. In higher organisms, one Notch-responsive gene that is activated in many different cell types encodes the Notch-regulated ankyrin repeat protein (NRARP), which acts as a negative feedback regulator of Notch responses. Here, we showed that NRARP inhibited the growth of Notch-dependent T cell acute lymphoblastic leukemia (T-ALL) cell lines and bound directly to the core Notch transcriptional activation complex (NTC), requiring both the transcription factor RBPJ and the Notch intracellular domain (NICD), but not Mastermind-like proteins or DNA. The crystal structure of an NRARP-NICD1-RBPJ-DNA complex, determined to 3.75 Å resolution, revealed that the assembly of NRARP-NICD1-RBPJ complexes relied on simultaneous engagement of RBPJ and NICD1, with the three ankyrin repeats of NRARP extending the Notch1 ankyrin repeat stack. Mutations at the NRARP-NICD1 interface disrupted entry of the proteins into NTCs and abrogated feedback inhibition in Notch signaling assays in cultured cells. Forced expression of NRARP reduced the abundance of NICD in cells, suggesting that NRARP may promote the degradation of NICD. These studies establish the structural basis for NTC engagement by NRARP and provide insights into a critical negative feedback mechanism that regulates Notch signaling.

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Inv(11)(q21q23) Fuses MLL to the NOTCH Co-Activator Mastermind-Like 2 in Secondary T Cell Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v108.11.4284.4284 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4284-4284

Author(s):

Markus Metzler ◽

Jan Zuna ◽

Martin S. Staege ◽

Lana Harder ◽

Claus Meyer ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Transcriptional Activation ◽

Selection Process ◽

Expression Profiles ◽

Lymphoblastic Leukemia ◽

Fusion Partner ◽

Secondary Leukemia ◽

Transcriptional Activation Domain ◽

Cell Acute Lymphoblastic Leukemia

Abstract Development of a secondary leukemia after chemotherapeutic treatment for childhood acute leukemia is associated with rearrangements of the MLL gene on chromosome 11q23 and characteristically results in acute myeloid leukemia (AML). We identified the NOTCH co-activator Mastermind-like 2 as MLL fusion partner in two cases of pediatric secondary leukemia. The MLL-MAML2 fusion results from a cryptic inv(11)(q21q23) only detectable by interphase fluorescence in situ hybridization with an MLL split signal probe. With a latency of twenty months and five years after initial diagnosis for MLL negative AML and precursor B acute lymphoblastic leukemia (ALL), respectively, both patients developed a therapy-related T-cell acute lymphoblastic leukemia. MAML2 is the first MLL fusion partner involved in human Notch-signaling and was only recently identified as recurrent translocation fusion partner in a subset of salivary gland tumors. The genomic MLL breakpoint shows similar localization and sequence features described for etoposide induced treatment-related AML. MLL-MAML2 positive cells were detectable up to two years prior to clinical apparent secondary leukemia in one case. The discrepant dynamics of clone expansion quantified by either the genomic fusion sequence or Ig/TCR gene rearrangements as clone specific markers suggests a selection process within the inversion positive population and the need for additional mutation events to promote overt leukemic disease. Whole genome expression profiles demonstrated differential expression of both typical MLL and NOTCH downstream genes, which suggests a modulatory role of the MAML2 transcriptional activation domain in MLL leukemogenesis and lineage assignment induced by the MLL-MAML2 fusion protein. MSM21620813 WSF3401600.

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Inhibition of cellular differentiation by the SCL/tal oncoprotein: transcriptional repression by an Id-like mechanism

Blood ◽

10.1182/blood.v85.2.465.bloodjournal852465 ◽

1995 ◽

Vol 85 (2) ◽

pp. 465-471 ◽

Cited By ~ 2

Author(s):

AN Goldfarb ◽

K Lewandowska

Keyword(s):

Transcriptional Activation ◽

Transcriptional Repression ◽

Cellular Differentiation ◽

Interaction Analysis ◽

Lymphoblastic Leukemia ◽

Ectopic Expression ◽

Protein Product ◽

Monocytic Differentiation ◽

E Proteins ◽

Helix Loop Helix

In cases of T-cell acute lymphoblastic leukemia (T-ALL), the basic helix-loop-helix (bHLH) oncogene SCL/tal undergoes frequent rearrangements activating ectopic expression. Despite the compelling epidemiological association of SCL/tal expression with T-ALL, no specific transforming function has been attributable to the protein product. However, investigators have recently demonstrated that forced overexpression of SCL/tal can block monocytic differentiation of M1 murine myeloid leukemia cells. Thus, inappropriate expression of wild- type SCL/tal protein may in part account for the maturation arrest phenotype observed in T-ALL cells. In this study, ectopic expression of the SCL/tal gene blocked the differentiation of C2C12 muscle precursor cells. Characterization of the mechanism of differentiation blockade showed that the SCL/tal protein repressed transcriptional activation by the myogenic bHLH factor MyoD. Protein interaction analysis showed that SCL/tal and MyoD compete for common partners (E bHLH proteins) but do not directly bind one other. A model is thus proposed in which ectopic SCL/tal protein, by its ability to titrate out E proteins, prevents the formation of bHLH complexes that drive cellular differentiation: the “Id-like” mechanism.

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Helical conformation of the AD1 peptide in the AML1-ETO–E-protein complex

Spectroscopy An International Journal ◽

10.1155/2009/781240 ◽

2009 ◽

Vol 23 (3-4) ◽

pp. 123-129

Author(s):

Horea Porumb

Keyword(s):

Synthetic Peptides ◽

Structure Prediction ◽

Secondary Structure Prediction ◽

Transcription Activation ◽

Helical Conformation ◽

Wild Type ◽

E Proteins ◽

E Protein ◽

Structural Details ◽

Acute Myeloid

The AML1-ETO fusion protein is responsible for 15% of the acute myeloid leukaemias due to its interference with transcription activation by E-proteins. The eTAFH region of the AML1-ETO competes for the AD1 domain of E-protein, thereby preventing wild-type transcription activation. The structural details concerning the eTAFH–bound AD1 domain are not known. We have previously shown that the eTAFH–AD1 interaction is strong (Kd=28 nM, H. Porumb,Spectroscopy22 (2008), 251–260). The secondary structure prediction algorithms are undecided as to the conformation of bound AD1 peptide. Here we demonstrate by circular dichroism that the bound AD1 peptide is fully helical. This will facilitate modeling of the interaction and launches a challenge as to using synthetic peptides to out compete the eTAFH–E-protein interaction.

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Structural basis of coronavirus E protein interactions with human PALS1 PDZ domain

Communications Biology ◽

10.1038/s42003-021-02250-7 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Airah Javorsky ◽

Patrick O. Humbert ◽

Marc Kvansakul

Keyword(s):

Epithelial Cells ◽

Protein Interactions ◽

Pdz Domain ◽

Cell Junction ◽

Structural Basis ◽

E Proteins ◽

Related Sequence ◽

Binding Motifs ◽

E Protein ◽

Life Threatening

AbstractSARS-CoV-2 infection leads to coronavirus disease 2019 (COVID-19), which is associated with severe and life-threatening pneumonia and respiratory failure. However, the molecular basis of these symptoms remains unclear. SARS-CoV-1 E protein interferes with control of cell polarity and cell-cell junction integrity in human epithelial cells by binding to the PALS1 PDZ domain, a key component of the Crumbs polarity complex. We show that C-terminal PDZ binding motifs of SARS-CoV-1 and SARS-CoV-2 E proteins bind the PALS1 PDZ domain with 29.6 and 22.8 μM affinity, whereas the related sequence from MERS-CoV did not bind. We then determined crystal structures of PALS1 PDZ domain bound to both SARS-CoV-1 and SARS-CoV-2 E protein PDZ binding motifs. Our findings establish the structural basis for SARS-CoV-1/2 mediated subversion of Crumbs polarity signalling and serve as a platform for the development of small molecule inhibitors to suppress SARS-CoV-1/2 mediated disruption of polarity signalling in epithelial cells.

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Structural basis of phosphorylation kinetics in the transcriptional activation domain (TAD) of c-Jun and insight of two new phosphorylation sites Ser58 and Thr62

10.1101/2021.02.27.433150 ◽

2021 ◽

Author(s):

Viraj Singh ◽

Arnit Kumar ◽

Shikha Bharti ◽

Anuj Jatav

Keyword(s):

Transcription Factors ◽

Posttranslational Modifications ◽

Transcriptional Activation ◽

Structural Basis ◽

Cell Cycle Protein ◽

Activation Domain ◽

Transcriptional Activation Domain ◽

Multisite Phosphorylation ◽

Fast Kinetic ◽

First Time

Protein phosphorylation is one of the most important posttranslational modifications observed on biomolecules. Nearly one-third of the cell cycle protein undergoes phosphorylation at some stage of the lifespan. Multi-site phosphorylation is well known in biological systems, including those in transcription factors. Multisite phosphorylation on transcription factors brings about their activation and/or inactivation. c-Jun is one of such transcription factors, whose function is dependent upon the state of phosphorylation. N-terminal phosphorylation required for c-Jun activity, while C-terminal one suppresses its activity. c-Jun contains a transcriptional activation domain (TAD) at N-terminus. It is known that four residues viz., Ser63, Ser73, Thr91 and Thr93 get phosphorylated which is required for its functional dimerization. However, there is no evidence if there exists any phosphorylation kinetics in c-Jun. In this paper, for the first time, it has been demonstrated that there exist phosphorylation kinetics within TAD. NMR based analysis suggested that Ser63 follows the fast kinetic while, Thr91 slow and Ser73 and Thr93 fall in the intermediate category. The four sites follow the following trend in their kinetics Ser63 > Ser73 > Thr93 > Thr91. Similar phosphorylation kinetics was also observed inside the C3H 10T0.5 fibroblast. NMR-based experiments also suggested the phosphorylation of two additional sites at Ser58 and Thr62. However, a detailed significance of these phosphorylation kinetic, as well as newly identified sites, is yet to be discovered.

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Structural basis of a potent human monoclonal antibody against Zika virus targeting a quaternary epitope

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1815432116 ◽

2019 ◽

Vol 116 (5) ◽

pp. 1591-1596 ◽

Cited By ~ 26

Author(s):

Feng Long ◽

Michael Doyle ◽

Estefania Fernandez ◽

Andrew S. Miller ◽

Thomas Klose ◽

...

Keyword(s):

Monoclonal Antibody ◽

Zika Virus ◽

Neutralizing Antibodies ◽

Human Monoclonal Antibody ◽

Structural Basis ◽

E Proteins ◽

Fab Fragment ◽

Cryo Electron Microscopy ◽

E Protein ◽

Mouse Model Of Infection

Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus in the Flaviviridae family. In contrast to most other insect-transmitted flaviviruses, ZIKV also can be transmitted sexually and from mother to fetus in humans. During recent outbreaks, ZIKV infections have been linked to microcephaly, congenital disease, and Guillain-Barré syndrome. Neutralizing antibodies have potential as therapeutic agents. We report here a 4-Å-resolution cryo-electron microscopy structure of the ZIKV virion in complex with Fab fragments of the potently neutralizing human monoclonal antibody ZIKV-195. The footprint of the ZIKV-195 Fab fragment expands across two adjacent envelope (E) protein protomers. ZIKV neutralization by this antibody is presumably accomplished by cross-linking the E proteins, which likely prevents formation of E protein trimers required for fusion of the viral and cellular membranes. A single dose of ZIKV-195 administered 5 days after virus inoculation showed marked protection against lethality in a stringent mouse model of infection.

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Integrative network analysis reveals USP7 haploinsufficiency inhibits E-protein activity in pediatric T-lineage acute lymphoblastic leukemia (T-ALL)

Scientific Reports ◽

10.1038/s41598-021-84647-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Timothy I. Shaw ◽

Li Dong ◽

Liqing Tian ◽

Chenxi Qian ◽

Yu Liu ◽

...

Keyword(s):

Network Analysis ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Cancer Therapeutics ◽

Physical Interaction ◽

Loss Of Function ◽

Protein Activity ◽

E Proteins ◽

Down Regulation ◽

E Protein

AbstractUSP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development. In T-ALL cell lines, we showed the physical interaction of USP7 with E-proteins and TAL1 by mass spectrometry and ChIP-seq. Haploinsufficient but not complete CRISPR knock-out of USP7 showed accelerated cell growth and validated transcriptional down-regulation of E-protein targets. Our study unveiled the synergistic effect of USP7 haploinsufficiency with aberrant TAL1 activation on T-ALL, implicating USP7 as a haploinsufficient tumor suppressor in T-ALL. Our findings caution against a universal oncogene designation for USP7 while emphasizing the dosage-dependent consequences of USP7 inhibitors currently under development as potential cancer therapeutics.

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