Definition of the Transcriptional Activation Domains of Three Human HOX Proteins Depends on the DNA-Binding Context

ABSTRACT Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

Download Full-text

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.

Download Full-text

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 ◽

1994 ◽

Vol 14 (7) ◽

pp. 4532-4545 ◽

Cited By ~ 39

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

Download Full-text

Reconstitution of transcriptional activation domains by reiteration of short peptide segments reveals the modular organization of a glutamine-rich activation domain

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6056-6067.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6056-6067

Author(s):

M Tanaka ◽

W Herr

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Binding Domain ◽

Dna Binding Domain ◽

Activation Domain ◽

Activation Domains ◽

Transcriptional Activation Domain ◽

Pou Domain

The POU domain activator Oct-2 contains an N-terminal glutamine-rich transcriptional activation domain. An 18-amino-acid segment (Q18III) from this region reconstituted a fully functional activation domain when tandemly reiterated and fused to either the Oct-2 or GAL4 DNA-binding domain. A minimal transcriptional activation domain likely requires three tandem Q18III segments, because one or two tandem Q18III segments displayed little activity, whereas three to five tandem segments were active and displayed increasing activity with increasing copy number. As with natural Oct-2 activation domains, in our assay a reiterated activation domain required a second homologous or heterologous activation domain to stimulate transcription effectively when fused to the Oct-2 POU domain. These results suggest that there are different levels of synergy within and among activation domains. Analysis of reiterated activation domains containing mutated Q18III segments revealed that leucines and glutamines, but not serines or threonines, are critical for activity in vivo. Curiously, several reiterated activation domains that were inactive in vivo were active in vitro, suggesting that there are significant functional differences in our in vivo and in vitro assays. Reiteration of a second 18-amino-acid segment from the Oct-2 glutamine-rich activation domain (Q18II) was also active, but its activity was DNA-binding domain specific, because it was active when fused to the GAL4 than to the Oct-2 DNA-binding domain. The ability of separate short peptide segments derived from a single transcriptional activation domain to activate transcription after tandem reiteration emphasizes the flexible and modular nature of a transcriptional activation domain.

Download Full-text

Direct activation of Sex-lethal transcription by the Drosophila runt protein

Development ◽

10.1242/dev.126.1.191 ◽

1999 ◽

Vol 126 (1) ◽

pp. 191-200 ◽

Cited By ~ 3

Author(s):

S.G. Kramer ◽

T.M. Jinks ◽

P. Schedl ◽

J.P. Gergen

Keyword(s):

Transcriptional Activation ◽

Target Genes ◽

Specific Binding ◽

Binding Activity ◽

Transcriptional Activation Domain ◽

Downstream Target ◽

Early Promoter ◽

Dna Binding Activity ◽

Sex Lethal

Runt functions as a transcriptional regulator in multiple developmental pathways in Drosophila melanogaster. Recent evidence indicates that Runt represses the transcription of several downstream target genes in the segmentation pathway. Here we demonstrate that runt also functions to activate transcription. The initial expression of the female-specific sex-determining gene Sex-lethal in the blastoderm embryo requires runt activity. Consistent with a role as a direct activator, Runt shows sequence-specific binding to multiple sites in the Sex-lethal early promoter. Using an in vivo transient assay, we demonstrate that Runt's DNA-binding activity is essential for Sex-lethal activation in vivo. These experiments further reveal that increasing the dosage of runt alone is sufficient for triggering the transcriptional activation of Sex-lethal in males. In addition, a Runt fusion protein, containing a heterologous transcriptional activation domain activates Sex-lethal expression, indicating that this regulation is direct and not via repression of other repressors. Moreover, we demonstrate that a small segment of the Sex-lethal early promoter that contains Runt-binding sites mediates Runt-dependent transcriptional activation in vivo.

Download Full-text

Runx proteins regulate Foxp3 expression

Journal of Experimental Medicine ◽

10.1084/jem.20090226 ◽

2009 ◽

Vol 206 (11) ◽

pp. 2329-2337 ◽

Cited By ~ 66

Author(s):

Ludovica Bruno ◽

Luca Mazzarella ◽

Maarten Hoogenkamp ◽

Arnulf Hertweck ◽

Bradley S. Cobb ◽

...

Keyword(s):

T Cells ◽

Dna Binding ◽

Cd4 T Cells ◽

Target Genes ◽

De Novo ◽

Foxp3 Expression ◽

Regulatory Elements ◽

Dominant Negative ◽

Downstream Target ◽

Runx Proteins

Runx proteins are essential for hematopoiesis and play an important role in T cell development by regulating key target genes, such as CD4 and CD8 as well as lymphokine genes, during the specialization of naive CD4 T cells into distinct T helper subsets. In regulatory T (T reg) cells, the signature transcription factor Foxp3 interacts with and modulates the function of several other DNA binding proteins, including Runx family members, at the protein level. We show that Runx proteins also regulate the initiation and the maintenance of Foxp3 gene expression in CD4 T cells. Full-length Runx promoted the de novo expression of Foxp3 during inducible T reg cell differentiation, whereas the isolated dominant-negative Runt DNA binding domain antagonized de novo Foxp3 expression. Foxp3 expression in natural T reg cells remained dependent on Runx proteins and correlated with the binding of Runx/core-binding factor β to regulatory elements within the Foxp3 locus. Our data show that Runx and Foxp3 are components of a feed-forward loop in which Runx proteins contribute to the expression of Foxp3 and cooperate with Foxp3 proteins to regulate the expression of downstream target genes.

Download Full-text

A heat shock-responsive domain of human HSF1 that regulates transcription activation domain function.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3354 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3354-3362 ◽

Cited By ~ 118

Author(s):

M Green ◽

T J Schuetz ◽

E K Sullivan ◽

R E Kingston

Keyword(s):

Heat Shock ◽

Dna Binding ◽

Transcriptional Activation ◽

Binding Domain ◽

Chimeric Proteins ◽

Dna Binding Domain ◽

Regulatory Domain ◽

Activation Domain ◽

Activation Domains ◽

Transcriptional Activation Domains

Human heat shock factor 1 (HSF1) stimulates transcription from heat shock protein genes following stress. We have used chimeric proteins containing the GAL4 DNA binding domain to identify the transcriptional activation domains of HSF1 and a separate domain that is capable of regulating activation domain function. This regulatory domain conferred heat shock inducibility to chimeric proteins containing the activation domains. The regulatory domain is located between the transcriptional activation domains and the DNA binding domain of HSF1 and is conserved between mammalian and chicken HSF1 but is not found in HSF2 or HSF3. The regulatory domain was found to be functionally homologous between chicken and human HSF1. This domain does not affect DNA binding by the chimeric proteins and does not contain any of the sequences previously postulated to regulate DNA binding of HSF1. Thus, we suggest that activation of HSF1 by stress in humans is controlled by two regulatory mechanisms that separately confer heat shock-induced DNA binding and transcriptional stimulation.

Download Full-text

Determination of the consensus DNA-binding sequence and a transcriptional activation domain for ESE-2

Biochemical Journal ◽

10.1042/bj20060375 ◽

2006 ◽

Vol 398 (3) ◽

pp. 497-507 ◽

Cited By ~ 19

Author(s):

Yeon Sook Choi ◽

Satrajit Sinha

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Transcriptional Activation ◽

Target Genes ◽

Regulatory Elements ◽

Activation Domain ◽

Transcriptional Activation Domain ◽

Ets Proteins ◽

Flanking Sequences ◽

Binding Sequence

The ESE (epithelium-specific Ets) subfamily of Ets transcription factors plays an important role in regulating gene expression in a variety of epithelial cell types. Although ESE proteins have been shown to bind to regulatory elements of some epithelial genes, the optimal DNA-binding sequence has not been experimentally ascertained for any member of the ESE subfamily of transcription factors. This has made the identification and validation of their targets difficult. We are studying ESE-2 (Elf5), which is highly expressed in epithelial cells of many tissues including skin keratinocytes. Here, we identify the preferred DNA-binding site of ESE-2 by performing CASTing (cyclic amplification and selection of targets) experiments. Our analysis shows that the optimal ESE-2 consensus motif consists of a GGA core and an AT-rich 5′- and 3′-flanking sequences. Mutational and competition experiments demonstrate that the flanking sequences that confer high DNA-binding affinity for ESE-2 show considerable differences from the known consensus DNA-binding sites of other Ets proteins, thus reinforcing the idea that the flanking sequences may impart recognition specificity for Ets proteins. In addition, we have identified a novel isoform of murine ESE-2, ESE-2L, that is generated by use of a hitherto unreported new exon and an alternate promoter. Interestingly, transient transfection assays with an optimal ESE-2 responsive reporter show that both ESE-2 and ESE-2L are weak transactivators. However, similar studies utilizing GAL4 chimaeras of ESE-2 demonstrate that while the DNA-binding ETS (E twenty-six) domain functions as a repressor, the PNT (pointed domain) of ESE-2 can act as a potent transcriptional activation domain. This novel transactivating property of PNT is also shared by ESE-3, another ESE family member. Identification of the ESE-2 consensus site and characterization of the transcriptional activation properties of ESE-2 shed new light on its potential as a regulator of target genes.

Download Full-text

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.

Download Full-text

Mutational analysis of the DNA binding, dimerization, and transcriptional activation domains of MEF2C.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2627 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2627-2636 ◽

Cited By ~ 130

Author(s):

J D Molkentin ◽

B L Black ◽

J F Martin ◽

E N Olson

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Mutational Analysis ◽

Gene Activation ◽

Positive Control ◽

Dominant Negative ◽

Activation Domains ◽

Muscle Gene Expression ◽

Transcriptional Activation Domains ◽

Muscle Gene

There are four members of the myocyte enhancer factor 2 (MEF2) family of transcription factors in vertebrates, MEF2A, -B, -C, and -D, which have homology within a MADS box at their amino termini and an adjacent motif known as the MEF2 domain. These factors activate muscle gene expression by binding as homo- and heterodimers to an A/T-rich DNA sequence in the control regions of muscle-specific genes. To understand the mechanisms of muscle gene activation of MEF2 factors, we generated a series of deletion and site-directed mutants of MEF2C. These mutants demonstrated that the MADS and MEF2 domains mediate DNA binding and dimerization, whereas the carboxyl terminus is required for transcriptional activation. Amino acids that are essential for MEF2 site-dependent transcription but which do not affect DNA binding were also identified in the MEF2 domain. This type of positive-control mutant demonstrates that the transcription activation domain of MEF2C, although separate from the MEF2 domain, is dependent on this domain for transcriptional activation through the MEF2 site. MEF2 mutants that are defective for DNA binding act as dominant negative mutants and can inhibit activation of MEF2-dependent genes by wild-type MEF2C.

Download Full-text

Deprivation of protein or amino acid induces C/EBPβ synthesis and binding to amino acid response elements, but its action is not an absolute requirement for enhanced transcription

Biochemical Journal ◽

10.1042/bj20071252 ◽

2008 ◽

Vol 410 (3) ◽

pp. 473-484 ◽

Cited By ~ 40

Author(s):

Michelle M. Thiaville ◽

Elizabeth E. Dudenhausen ◽

Can Zhong ◽

Yuan-Xiang Pan ◽

Michael S. Kilberg

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Transcriptional Activation ◽

Target Genes ◽

Activating Transcription Factor 3 ◽

Binding Studies ◽

Human Hepatoma Cells ◽

Amino Acid Response

A nutrient stress signalling pathway is triggered in response to protein or amino acid deprivation, namely the AAR (amino acid response), and previous studies have shown that C/EBPβ (CCAAT/enhancer-binding protein β) expression is up-regulated following activation of the AAR. DNA-binding studies, both in vitro and in vivo, have revealed increased C/EBPβ association with AARE (AAR element) sequences in AAR target genes, but its role is still unresolved. The present results show that in HepG2 human hepatoma cells, the total amount of C/EBPβ protein, both the activating [LAP* and LAP (liver-enriched activating protein)] and inhibitory [LIP (liver-enriched inhibitory)] isoforms, was increased in histidine-deprived cells. Immunoblotting of subcellular fractions and immunostaining revealed that most of the C/EBPβ was located in the nucleus. Consistent with these observations, amino acid limitation caused an increase in C/EBPβ DNA-binding activity in nuclear extracts and chromatin immunoprecipitation revealed an increase in C/EBPβ binding to the AARE region in vivo, but at a time when transcription from the target gene was declining. A constant fraction of the basal and increased C/EBPβ protein was phosphorylated on Thr235 and the phospho-C/EBPβ did bind to an AARE. Induction of AARE-enhanced transcription was slightly greater in C/EBPβ-deficient MEFs (mouse embryonic fibroblasts) or C/EBPβ siRNA (small interfering RNA)-treated HepG2 cells compared with the corresponding control cells. Transient expression of LAP*, LAP or LIP in C/EBPβ-deficient fibroblasts caused suppression of increased transcription from an AARE-driven reporter gene. Collectively, the results demonstrate that C/EBPβ is not required for transcriptional activation by the AAR pathway but, when present, acts in concert with ATF3 (activating transcription factor 3) to suppress transcription during the latter stages of the response.

Download Full-text