The HOX Homeodomain Proteins Block CBP Histone Acetyltransferase Activity

ABSTRACT Despite the identification of PBC proteins as cofactors that provide DNA affinity and binding specificity for the HOX homeodomain proteins, HOX proteins do not demonstrate robust activity in transient-transcription assays and few authentic downstream targets have been identified for these putative transcription factors. During a search for additional cofactors, we established that each of the 14 HOX proteins tested, from 11 separate paralog groups, binds to CBP or p300. All six isolated homeodomain fragments tested bind to CBP, suggesting that the homeodomain is a common site of interaction. Surprisingly, CBP-p300 does not form DNA binding complexes with the HOX proteins but instead prevents their binding to DNA. The HOX proteins are not substrates for CBP histone acetyltransferase (HAT) but instead inhibit the activity of CBP in both in vitro and in vivo systems. These mutually inhibitory interactions are reflected by the inability of CBP to potentiate the low levels of gene activation induced by HOX proteins in a range of reporter assays. We propose two models for HOX protein function: (i) HOX proteins may function without CBP HAT to regulate transcription as cooperative DNA binding molecules with PBX, MEIS, or other cofactors, and (ii) the HOX proteins may inhibit CBP HAT activity and thus function as repressors of gene transcription.

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Trimeric Association of Hox and TALE Homeodomain Proteins Mediates Hoxb2 Hindbrain Enhancer Activity

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.5134 ◽

1999 ◽

Vol 19 (7) ◽

pp. 5134-5142 ◽

Cited By ~ 172

Author(s):

Yakop Jacobs ◽

Catherine A. Schnabel ◽

Michael L. Cleary

Keyword(s):

Dna Binding ◽

Homeodomain Proteins ◽

Site Mutation ◽

Enhancer Activity ◽

Hox Proteins ◽

Combinatorial Regulation ◽

Protein Functions ◽

Tale Homeodomain

ABSTRACT Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element.

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Novel pathway for thyroid hormone receptor action through interaction with jun and fos oncogene activities

Molecular and Cellular Biology ◽

10.1128/mcb.11.12.6016-6025.1991 ◽

1991 ◽

Vol 11 (12) ◽

pp. 6016-6025

Author(s):

X K Zhang ◽

K N Wills ◽

M Husmann ◽

T Hermann ◽

M Pfahl

Keyword(s):

Signal Transduction ◽

Dna Binding ◽

Thyroid Hormone Receptor ◽

Gene Activation ◽

Signal Transduction Pathway ◽

Large Family ◽

Transduction Pathway ◽

Regulatory Pathway

Many essential biological pathways, including cell growth, development, and metabolism, are regulated by thyroid hormones (THs). TH action is mediated by intracellular receptors that belong to a large family of ligand-dependent transcription factors, including the steroid hormone and retinoic acid receptors. So far it has been assumed that TH receptors (TRs) regulate gene transcription only through the classical protein-DNA interaction mechanism. Here we provide evidence for a regulatory pathway that allows cross-talk between TRs and the signal transduction pathway used by many growth factors, oncogenes, and tumor promoters. In transient transfection studies, we observed that the oncogenes c-jun and c-fos inhibit TR activities, while TRs inhibit induction of the c-fos promoter and repress AP-1 site-dependent gene activation. A truncated TR that lacks only 17 amino acids from the carboxy terminus can no longer antagonize AP-1 activity. The cross-regulation between TRs and the signal transduction pathway appears to be based on the ability of TRs to inhibit DNA binding of the transcription factor AP-1 in the presence of THs. The constituents of AP-1, c-Jun, and c-Fos, vice versa, can inhibit TR-induced gene activation in vivo, and c-Jun inhibits TR DNA binding in vitro. This novel regulatory pathway is likely to play a major role in growth control and differentiation by THs.

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The homeodomain region controls the phenotype of HOX-induced murine leukemia

Blood ◽

10.1182/blood-2011-10-384685 ◽

2012 ◽

Vol 120 (19) ◽

pp. 4018-4027 ◽

Cited By ~ 13

Author(s):

Constanze Breitinger ◽

Emanuel Maethner ◽

Maria-Paz Garcia-Cuellar ◽

Robert K. Slany

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Expression Patterns ◽

Hox Proteins ◽

Hematopoietic Development ◽

N Terminus ◽

Fast Acting ◽

Murine Leukemia

Abstract HOX proteins are widely involved in hematopoietic development. These transcription factors combine a conserved DNA-binding homeobox with a divergent N-terminus that mediates interaction with variable cofactors. The resulting combinatorial diversity is thought to be responsible for mammalian HOX specificity. Contrasting this proposed mechanism for normal HOX function, here we demonstrate that, in the context of hematopoietic immortalization and leukemogenesis, individual HOX properties are governed almost exclusively by the homeodomain. Swap experiments between HOXA1 and HOXA9, 2 members of nonrelated paralog groups, revealed that gene expression patterns of HOX transformed cells in vitro are determined by the nature of the homeodomain. Similar results were seen in vivo during HOX-mediated leukemogenesis. An exchange of the homeodomains was sufficient to convert the slow, low-penetrance phenotype of HOXA1-induced leukemia to the aggressive fast-acting disease elicited by HOXA9 and vice versa. Mutation and deletion studies identified several subregions within the DNA binding domain responsible for paralog specificity. Previously defined binding sites for PBX cofactors within the exchangeable, nonhomeobox segment were dispensable for in vitro oncogenic HOX activity but affected in vivo disease development. The transcriptional activator domain shared by HOXA1 and HOXA9 at the very N-terminus proved essential for all transformation.

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Both Pbx1 and E2A-Pbx1 bind the DNA motif ATCAATCAA cooperatively with the products of multiple murine Hox genes, some of which are themselves oncogenes.

Molecular and Cellular Biology ◽

10.1128/mcb.15.7.3786 ◽

1995 ◽

Vol 15 (7) ◽

pp. 3786-3795 ◽

Cited By ~ 89

Author(s):

Q Lu ◽

P S Knoepfler ◽

J Scheele ◽

D D Wright ◽

M P Kamps

Keyword(s):

Dna Binding ◽

Target Genes ◽

Hox Genes ◽

Physical Interaction ◽

Homeodomain Protein ◽

Structural Development ◽

Cell Leukemia ◽

Homeodomain Proteins ◽

Hox Proteins

E2A-PBX1 is the oncogene produced at the t(1;19) chromosomal breakpoint of pediatric pre-B-cell leukemia. Expression of E2A-Pbx1 induces fibroblast transformation and myeloid and T-cell leukemia in mice and arrests differentiation of granulocyte macrophage colony-stimulating factor-dependent myeloblasts in cultured marrow. Recently, the Drosophila melanogaster protein Exd, which is highly related to Pbx1, was shown to bind DNA cooperatively with the Drosophila homeodomain proteins Ubx and Abd-A. Here, we demonstrate that the normal Pbx1 homeodomain protein, as well as its oncogenic derivative, E2A-Pbx1, binds the DNA sequence ATCAATCAA cooperatively with the murine Hox-A5, Hox-B7, Hox-B8, and Hox-C8 homeodomain proteins, which are themselves known oncoproteins, as well as with the Hox-D4 homeodomain protein. Cooperative binding to ATCAATCAA required the homeodomain-dependent DNA-binding activities of both Pbx1 and the Hox partner. In cotransfection assays, Hox-B8 suppressed transactivation by E2A-Pbx1. These results suggest that (i) Pbx1 may participate in the normal regulation of Hox target gene transcription in vivo and therein contribute to aspects of anterior-posterior patterning and structural development in vertebrates, (ii) that E2A-Pbx1 could abrogate normal differentiation by altering the transcriptional regulation of Hox target genes in conjunction with Hox proteins, and (iii) that the oncogenic mechanism of certain Hox proteins may require their physical interaction with Pbx1 as a cooperating, DNA-binding partner.

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Meis proteins are major in vivo DNA binding partners for wild-type but not chimeric Pbx proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5679 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5679-5687 ◽

Cited By ~ 148

Author(s):

C P Chang ◽

Y Jacobs ◽

T Nakamura ◽

N A Jenkins ◽

N G Copeland ◽

...

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Cultured Cells ◽

Binding Activity ◽

Wild Type ◽

Hox Proteins ◽

Amino Terminal ◽

Binding Partners

The Pbx1 and Meis1 proto-oncogenes code for divergent homeodomain proteins that are targets for oncogenic mutations in human and murine leukemias, respectively, and implicated by genetic analyses to functionally collaborate with Hox proteins during embryonic development and/or oncogenesis. Although Pbx proteins have been shown to dimerize with Hox proteins and modulate their DNA binding properties in vitro, the biochemical compositions of endogenous Pbx-containing complexes have not been determined. In the present study, we demonstrate that Pbx and Meis proteins form abundant complexes that comprise a major Pbx-containing DNA binding activity in nuclear extracts of cultured cells and mouse embryos. Pbx1 and Meis1 dimerize in solution and cooperatively bind bipartite DNA sequences consisting of directly adjacent Pbx and Meis half sites. Pbx1-Meis1 heterodimers display distinctive DNA binding specificities and cross-bind to a subset of Pbx-Hox sites, including those previously implicated as response elements for the execution of Pbx-dependent Hox programs in vivo. Chimeric oncoprotein E2a-Pbx1 is unable to bind DNA with Meis1, due to the deletion of amino-terminal Pbx1 sequences following fusion with E2a. We conclude that Meis proteins are preferred in vivo DNA binding partners for wild-type Pbx1, a relationship that is circumvented by its oncogenic counterpart E2a-Pbx1.

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Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3633 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3633-3641 ◽

Cited By ~ 39

Author(s):

T Chakraborty ◽

T J Brennan ◽

L Li ◽

D Edmondson ◽

E N Olson

Keyword(s):

Dna Binding ◽

Gene Activation ◽

Binding Activity ◽

Specific Gene ◽

Gene Products ◽

Basic Domain ◽

High Affinity ◽

Dna Binding Activity

Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.

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Chromatin Accessibility Plays a Key Role in Selective Targeting of Hox Proteins

10.1101/473850 ◽

2018 ◽

Author(s):

Damiano Porcelli ◽

Bettina Fischer ◽

Steven Russell ◽

Robert White

Keyword(s):

Binding Specificity ◽

Chromatin Accessibility ◽

Binding Selectivity ◽

Hox Proteins ◽

Systematic Analysis ◽

Binding Regions ◽

Hox Protein ◽

Accessible Chromatin

AbstractHox protein transcription factors specify segmental diversity along the anterior-posterior body axis in metazoans. Understanding the basis of Hox function has long faced the problem that, while the different members of the Hox family show clear functional specificity in vivo, they all show very similar binding specificity in vitro. Based on in vitro studies, cofactors may increase Hox binding selectivity however a satisfactory understanding of in vivo Hox target selectivity is still lacking.We have carried out a systematic analysis of the in vivo genomic binding profiles of all eight Drosophila Hox proteins using transient transfection in Kc167 cells to examine Hox protein targeting. We find that Hox proteins show considerable binding selectivity in vivo in the absence of the canonical Hox cofactors Extradenticle and Homothorax. Hox binding selectivity is strongly associated with chromatin accessibility; binding sites in less accessible chromatin show the highest selectivity and the different Hox proteins exhibit different propensities to bind less accessible chromatin. High Hox binding selectivity is also associated with high affinity binding regions, leading to a model where Hox proteins derive binding selectivity through an affinity-based competition with nucleosomes. Provision of the Extradenticle/Homothorax cofactors generally leads to an increase in the number of Hox binding regions and promotes the binding to regions in less accessible chromatin, however the provision of these cofactors has little effect on the overall selectivity of Hox targeting.These studies indicate that chromatin accessibility plays a key role in Hox selectivity and we propose that relative chromatin accessibility provides a basis for subtle differences in binding specificity and affinity to generate significantly different sets of genomic targets for different Hox proteins. We suggest that this mechanism may also be relevant to other transcription factor families.

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A micro-evolutionary change in target binding sites as key determinant of Ultrabithorax function in Drosophila.

10.1101/2021.08.17.456507 ◽

2021 ◽

Author(s):

Soumen Khan ◽

Saurabh J. Pradhan ◽

Guillaume Giraud ◽

Françoise Bleicher ◽

Rachel Paul ◽

...

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Morphological Changes ◽

Binding Motif ◽

Core Sequence ◽

Dna Binding Sites ◽

Target Binding ◽

Hox Protein

All Hox proteins are known to recognize, in vitro, similar DNA-binding sites containing a TAAT core sequence. This poor DNA-binding specificity is in sharp contrast with their specific functions in vivo. Here we report a new binding motif with TAAAT core sequence to which the Hox protein Ultrabithorax (Ubx) binds with higher affinity and specificity. Using transgenic and luciferase assays, we show that this new motif is critical for Ubx-mediated regulation of a target gene in Drosophila melanogaster. Interestingly, this new motif with TAAAT core sequences is not associated with the targets of Ubx in the honeybee, Apis mellifera, wherein hindwings are nearly identical to the forewings. We show that introduction of TAAAT motif in the place of TAAT motif is sufficient to bring an enhancer of a wing-promoting gene of A. mellifera under the regulation of Ubx. Our results, thus, suggest that binding motifs with a TAAAT core sequence may help identify functionally relevant direct targets of Ubx in D. melanogaster and the emergence of these binding sites may be crucial for Hox-mediated morphological changes during insect evolution.

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Hox proteins have different affinities for a consensus DNA site that correlate with the positions of their genes on the hox cluster

Molecular and Cellular Biology ◽

10.1128/mcb.14.7.4532-4545.1994 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4532-4545

Author(s):

I Pellerin ◽

C Schnabel ◽

K M Catron ◽

C Abate

Keyword(s):

Dna Binding ◽

Target Genes ◽

Hox Genes ◽

Regulatory Elements ◽

Binding Properties ◽

Hox Proteins ◽

Hox Cluster ◽

Dna Binding Properties

The hox genes, members of a family of essential developmental regulators, have the intriguing property that their expression in the developing murine embryo is colinear with their chromosomal organization. Members of the hox gene family share a conserved DNA binding domain, termed the homeodomain, which mediates interactions of Hox proteins with DNA regulatory elements in the transcriptional control regions of target genes. In this study, we characterized the DNA binding properties of five representative members of the Hox family: HoxA5, HoxB4, HoxA7, HoxC8, and HoxB1. To facilitate a comparative analysis of their DNA binding properties, we produced the homeodomain regions of these Hox proteins in Escherichia coli and obtained highly purified polypeptides. We showed that these Hox proteins interact in vitro with a common consensus DNA site that contains the motif (C/G)TAATTG. We further showed that the Hox proteins recognize the consensus DNA site in vivo, as determined by their ability to activate transcription through this site in transient transfection assays. Although they interact optimally with the consensus DNA site, the Hox proteins exhibit subtle, but distinct, preferences for DNA sites that contain variations of the nucleotides within the consensus motif. In addition to their modest differences in DNA binding specificities, the Hox proteins also vary in their relative affinities for DNA. Intriguingly, their relative affinities correlate with the positions of their respective genes on the hox cluster. These findings suggest that subtle differences in DNA binding specificity combined with differences in DNA binding affinity constitute features of the "Hox code" that contribute to the selective functions of Hox proteins during murine embryogenesis.

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