Étude sur la reconnaissance de l'ubiquitine par les domaines de transactivation acides des activateurs de transcription

Les domaines de transactivation (TAD) acides sont présents dans plusieurs protéines oncogéniques, virales et dans des facteurs de différenciation de cellules souches. Ces domaines acides contrôlent la transcription à travers une myriade d’interactions avec divers partenaires ce qui provoque l’activation de la transcription ou leur propre élimination. Cependant, dans la dernière décennie, de plus en plus de recherches ont démontré que les TAD possédaient un sous-domaine activation/dégradation (DAD) responsable pour une fonction d'activation de la transcription dépendante de la dégradation de la protéine. Un tel phénomène peut être accompli par plusieurs moyens tels que des modifications post-traductionnelles, l’association à des cofacteurs ou la formation d’un réseau d’interaction complexe en chaînes. Or, aucune preuve concrète n’a pu clairement démontrer le fonctionnement de la dépendance paradoxale entre ces deux fonctions sur un activateur de transcription. Le DAD, a été observé dans plusieurs facteurs de transcription incluant la protéine suppresseur de tumeur p53 et le facteur de différenciation érythrocyte EKLF. Un aspect particulier des DAD est que la composition de leur séquence d’acide aminé est fortement similaire à celle des domaines de liaison à l’ubiquitine (UBD) qui jouent un rôle clé dans le contrôle de la transcription à travers leur interaction non-covalente avec l’ubiquitine. Ainsi, dans ce mémoire, nous avons étudié la possibilité que les TAD acides soient capables d’agir comme UBD pour réguler leur fonction paradoxale à travers des interactions non-covalentes avec l’ubiquitine. L’analyse est faite en utilisant la résonnance magnétique nucléaire (RMN) ainsi qu’avec des essais fonctionnels de dégradation. En somme, cette étude amène une plus grande compréhension des protéines impliquées dans le contrôle des TAD et caractérise le tout premier exemple de TAD capable d’interagir avec l’ubiquitine.

Identification of novel and cell type enriched cofactors of the transcription activation domain of RelA (p65 NF-kappaB).

RelA (NF-kappaB) is a transcription factor inducible by distinct stimuli in many different cell types. To find new cell type specific cofactors of NF-kappaB dependent transcription, we isolated RelA transcription activation domain binding proteins from the nuclear extracts of three different cell types. Analysis by electrophoresis and liquid chromatography tandem mass spectrometry identified several novel putative molecular partners. Some were strongly enriched in the complex formed from the nuclear extracts of specific cell types.

Multicentric carpotarsal osteolysis is caused by mutations clustering in the amino-terminal transcriptional activation domain of MAFB.

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated simplex cases with MCTO were also heterozygous for previously unreported mutations within this same region, as were affected members of two families with autosomal-dominant MCTO. MAFB encodes a transcription factor that negatively regulates RANKL-induced osteoclastogenesis and is essential for normal renal development. Identification of this gene paves the way for development of novel therapeutic approaches for this crippling disease and provides insight into normal bone and kidney development.

Multicentric Carpotarsal Osteolysis Is Caused by Mutations Clustering in the Amino-Terminal Transcriptional Activation Domain of MAFB

Multicentric carpotarsal osteolysis (MCTO) is a rare skeletal dysplasia characterized by aggressive osteolysis, particularly affecting the carpal and tarsal bones, and is frequently associated with progressive renal failure. Using exome capture and next-generation sequencing in five unrelated simplex cases of MCTO, we identified previously unreported missense mutations clustering within a 51 base pair region of the single exon of MAFB, validated by Sanger sequencing. A further six unrelated simplex cases with MCTO were also heterozygous for previously unreported mutations within this same region, as were affected members of two families with autosomal-dominant MCTO. MAFB encodes a transcription factor that negatively regulates RANKL-induced osteoclastogenesis and is essential for normal renal development. Identification of this gene paves the way for development of novel therapeutic approaches for this crippling disease and provides insight into normal bone and kidney development.

The activation domain of the enhancer binding protein p45NF-E2 interacts with TAFII130 and mediates long-range activation of the α- and β-globin gene loci in an erythroid cell line

We have used the interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the human β-globin locus control region and the globin gene promoters as a paradigm to examine the mechanisms governing promoter/enhancer interactions in this locus. We have demonstrated that enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2, p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation domain in the NH2-terminal 80 amino acids of the protein. In contrast to the wild-type protein, expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails to reactivate expression of the endogenous β- or α-globin loci in this cell line. Protein-protein interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the transcription initiation complex, TATA binding protein associated factor TAFII130. These findings suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to the individual promoters.

N-terminal transcriptional activation domain of LZIP comprises two LxxLL motifs and the Host Cell Factor-1 binding motif

Host Cell Factor-1 (HCF-1, C1) was first identified as a cellular target for the herpes simplex virus transcriptional activator VP16. Association between HCF and VP16 leads to the assembly of a multiprotein enhancer complex that stimulates viral immediate-early gene transcription. HCF-1 is expressed in all cells and is required for progression through G1 phase of the cell cycle. In addition to VP16, HCF-1 associates with a cellular bZIP protein known as LZIP (or Luman). Both LZIP and VP16 contain a four-amino acid HCF-binding motif, recognized by the N-terminal β-propeller domain of HCF-1. Herein, we show that the N-terminal 92 amino acids of LZIP contain a potent transcriptional activation domain composed of three elements: the HCF-binding motif and two LxxLL motifs. LxxLL motifs are found in a number of transcriptional coactivators and mediate protein–protein interactions, notably recognition of the nuclear hormone receptors. LZIP is an example of a sequence-specific DNA-binding protein that uses LxxLL motifs within its activation domain to stimulate transcription. The LxxLL motifs are not required for association with the HCF-1 β-propeller and instead interact with other regions in HCF-1 or recruit additional cofactors.

A novel tetracycline-dependent transactivator with E2F4 transcriptional activation domain

A tetracycline-controlled gene expression system provides a powerful tool to dissect the functions of gene products. However, it often appears difficult to establish cell lines or transgenic animals stably expressing tetracycline-dependent transactivators, possibly as a result of toxicity of the transactivator domains used. In order to overcome this problem, we developed a novel tetracycline-dependent transactivator that works efficiently in mammalian cells. This transactivator is a fusion of the tet reverse repressor mutant and the transcriptional activating domain of human E2F4, which is ubiquitously expressed in vivo. We demonstrate here that this tetracycline-regulated gene expression system provides a two log transcriptional activation in mammalian cells as assessed by northern blot and luciferase analyses. Combining this system with green fluorescent protein reporter systems or microarray gene expression profiling will facilitate the study of gene function.

O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability

The posttranslational modification of eukaryotic intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is essential for cell viability, yet its precise functional roles are largely unknown. O-GlcNAc transferase utilizes UDP-GlcNAc, the end product of hexosamine biosynthesis, to catalyze this modification. The availability of UDP-GlcNAc correlates with glycosylation levels of intracellular proteins as well as with transcriptional levels of some genes. Meanwhile, transcription factors and RNA polymerase II can be modified by O-GlcNAc. A linkage between transcription factor O-GlcNAcylation and transcriptional regulation therefore has been postulated. Here, we show that O-GlcNAcylation of a chimeric transcriptional activator containing the second activation domain of Sp1 decreases its transcriptional activity both in an in vitro transcription system and in living cells, which is in concert with our observation that O-GlcNAcylation of Sp1 activation domain blocks its in vitro and in vivo interactions with other Sp1 molecules and TATA-binding protein-associated factor II 110. Furthermore, overexpression of O-GlcNAc transferase specifically inhibits transcriptional activation by native Sp1 in cells. Thus, our studies provide direct evidence that O-GlcNAcylation of transcription factors is involved in transcriptional regulation.

Activation domain-mediated enhancement of activator binding to chromatin in mammalian cells.

DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.

Selective constraints on the activation domain of transcription factor Pit-1.

The POU transcription factor Pit-1 activates members of the prolactin/growth hormone gene family in specific endocrine cell types of the pituitary gland. Although Pit-1 is structurally conserved among vertebrate species, evolutionary changes in the pattern of Pit-1 RNA splicing have led to a notable "contraction" of the transactivation domain in the mammalian lineage, relative to Pit-1 in salmonid fish. By site-directed mutagenesis we demonstrate that two splice insertions in salmon Pit-1, called beta (29 aa) and gamma (33 aa), are critical for cooperative activation of the salmon prolactin gene. Paradoxically, Pit-1-dependent activation of the prolactin gene in rat is enhanced in the absence of the homologous beta-insert sequence. This apparent divergence in the mechanism of activation of prolactin genes by Pit-1 is target gene specific, as activation of rat and salmon growth hormone genes by Pit-1 splice variants is entirely conserved. Our data suggest that efficient activation of the prolactin gene in the vertebrate pituitary has significantly constrained the pattern of splicing within the Pit-1 transactivation domain. Rapid evolutionary divergence of prolactin gene function may have demanded changes in Pit-1/protein interactions to accommodate new patterns of transcriptional control by developmental or physiological factors.

The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain.

Mutations in the whn gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/ winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene revealed the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. As the whnrnu mutant protein lacks this domain, our results indicate that a transactivation function is essential for the activity of the whn transcription factor.

The embryonic transcription factor stage specific activator protein contains a potent bipartite activation domain that interacts with several RNA polymerase II basal transcription factors.

Stage specific activator protein (SSAP) is a member of a newly discovered class of transcription factors that contain motifs more commonly found in RNA-binding proteins. Previously, we have shown that SSAP specifically binds to its recognition sequence in both the double strand and the single strand form and that this DNA-binding activity is localized to the N-terminal RNA recognition motif domain. Three copies of this recognition sequence constitute an enhancer element that is directly responsible for directing the transcriptional activation of the sea urchin late histone H1 gene at the midblastula stage of embryogenesis. Here we show that the remainder of the SSAP polypeptide constitutes an extremely potent bipartite transcription activation domain that can function in a variety of mammalian cell lines. This activity is as much as 3 to 5 times stronger than VP16 at activating transcription and requires a large stretch of amino acids that contain glutamine-glycine rich and serine-threonine-basic amino acid rich regions. We present evidence that SSAP's activation domain shares targets that are also necessary for activation by E1a and VP16. Finally, SSAP's activation domain is found to participate in specific interactions in vitro with the basal transcription factors TATA-binding protein, TFIIB, TFIIF74, and dTAF(II) 110.

Modulation of promoter occupancy by cooperative DNA binding and activation-domain function is a major determinant of transcriptional regulation by activators in vivo.

Binding of transcriptional activators to a promoter is a prerequisite process in transcriptional activation. It is well established that the efficiency of activator binding to a promoter is determined by the affinity of direct interactions between the DNA-binding domain of an activator and its specific target sequences. However, I describe here that activator binding to a promoter is augmented in vivo by the effects of two other determinants that have not been generally appreciated: (i) the number of activator binding sites present in a promoter and (ii) the potency of activation domains of activators. Multiple sites within a promoter can cooperatively recruit cognate factors regardless of whether they contain an effective activation domain. This cooperativity can result in the synergistic activation of transcription. The second effect is the enhancement of activator binding to a promoter by the presence of activation domains. In this case, activation domains are not simply tethered to the promoter by the DNA-binding domain but instead assist the DNA-binding domain being tethered onto the promoter. This effect of activation domains on DNA binding is instrumental in determining how potent activators can induce steep transcriptional increases at low concentrations.

E2F1 and E1A(12S) have a homologous activation domain regulated by RB and CBP.

The E2F1 transcription factor has a well-characterized activation domain at its C terminus and the E1A protein has a recently defined activation domain at its N terminus. Here we show that these activation domains are highly related in sequence. The sequence homology reflects, at least partly, the conservation of common binding sites for the RB and CBP/p300 proteins, which are preserved in the same relative order along E2F1 and E1A. Furthermore, the interaction of RB and CBP with these two activation domains results in the same functional consequences: RB represses both activation domains, whereas CBP stimulates them. We conclude that the activation domains of E1A(12s) and E2F1 belong to a novel functional class, characterized by specific protein binding sites. The implication of this conservation with respect to E1A-induced stimulation of E2F activity is discussed.

The activation domain of GAL4 protein mediates cooperative promoter binding with general transcription factors in vivo.

Most proteins that activate RNA polymerase II-mediated transcription in eukaryotic cells contain sequence-specific DNA-binding domains and "activation" regions. The latter bind general transcription factors and/or coactivators and are required for high-level transcription. Their function in vivo is unknown. Since several activation domains bind the TATA-binding protein (TBP), TBP-associated factors, or other general factors in vitro, one role of the activation domain may be to facilitate promoter occupancy by supporting cooperative binding of the activator and general transcription factors. Using the GAL4 system of yeast, we have tested this model in vivo. It is demonstrated that the presence of a TATA box (the TBP binding site) facilitates binding of GAL4 protein to low- and moderate-affinity sites and that the activation domain modulates these effects. These results support the cooperative binding model for activation domain function in vivo.