Timothy A. Blauwkamp

C-terminal-binding protein directly activates and represses Wnt transcriptional targets in Drosophila.

Regulation of Wnt transcriptional targets is thought to occur by a transcriptional switch. In the absence of Wnt signaling, sequence‐specific DNA‐binding proteins of the TCF family repress Wnt target genes. Upon Wnt stimulation, stabilized β‐catenin binds to TCFs, converting them into transcriptional activators. C‐terminal‐binding protein (CtBP) is a transcriptional corepressor that has been reported to inhibit Wnt signaling by binding to TCFs or by preventing β‐catenin from binding to TCF. Here, we show that CtBP is also required for the activation of some Wnt targets in Drosophila. CtBP is recruited to Wnt‐regulated enhancers in a Wnt‐dependent manner, where it augments Armadillo (the fly β‐catenin) transcriptional activation. We also found that CtBP is required for repression of a subset of Wnt targets in the absence of Wnt stimulation, but in a manner distinct from previously reported mechanisms. CtBP binds to Wnt‐regulated enhancers in a TCF‐independent manner and represses target genes in parallel with TCF. Our data indicate dual roles for CtBP as a gene‐specific activator and repressor of Wnt target gene transcription.

CBP/p300 are bimodal regulators of Wnt signaling.

Many Wnts influence cell behavior by a conserved signaling cascade that promotes the stabilization and nuclear accumulation of β‐catenin (β‐cat), which then associates with TCF family members to activate target genes. The histone acetyltransferase CREB binding protein (CBP) can bind to TCF and inhibit Wnt signaling in Drosophila. In contrast, studies in vertebrates indicate a positive role for CBP and the closely related protein p300 as β‐cat binding transcriptional co‐activators. We address this discrepancy by demonstrating that in addition to its negative role, CBP has an essential positive role in Wnt signaling in flies. CBP binds directly to the C‐terminus of Armadillo (Arm, the fly β‐cat) and is recruited to a Wnt‐regulated enhancer (WRE) in a Wnt‐ and Arm‐dependent manner. In a human colorectal cancer cell line, we show that CBP and p300 can inhibit Wnt signaling and demonstrate that human p300 can bind directly to TCF4 in vitro. Our results argue that CBP/p300 has an evolutionarily conserved role as a buffer regulating TCF‐β‐cat/Arm binding. Subsequent to this interaction, it also has an essential role in mediating the transactivation activity of β‐cat/Arm.

Activation of the glnA, glnK, and nac promoters as Escherichia coli undergoes the transition from nitrogen excess growth to nitrogen starvation

The nitrogen-regulated genes and operons of the Ntr regulon of Escherichia coli are activated by the enhancer-binding transcriptional activator NRI approximately P (NtrC approximately P). Here, we examined the activation of the glnA, glnK, and nac promoters as cells undergo the transition from growth on ammonia to nitrogen starvation and examined the amplification of NRI during this transition. The results indicate that the concentration of NRI is increased as cells become starved for ammonia, concurrent with the activation of Ntr genes that have less- efficient enhancers than does glnA. A diauxic growth pattern was obtained when E. coli was grown on a low concentration of ammonia in combination with arginine as a nitrogen source, consistent with the hypothesis that Ntr genes other than glnA become activated only upon amplification of the NRI concentration.

Physiological role of the GlnK signal transduction protein of Escherichia coli : survival of nitrogen starvation

Escherichia coli contains two PII‐like signal trans‐duction proteins, PII and GlnK, involved in nitrogen assimilation. We examined the roles of PII and GlnK in controlling expression of glnALG , glnK and nac during the transition from growth on ammonia to nitrogen starvation and vice versa. The PII protein exclusively controlled glnALG expression in cells adapted to growth on ammonia, but was unable to limit nac and glnK expression under conditions of nitrogen starvation. Conversely, GlnK was unable to limit glnALG expression in cells adapted to growth on ammonia, but was required to limit expression of the glnK and nac promoters during nitrogen starvation. In the absence of GlnK, very high expression of the glnK and nac promoters occurred in nitrogen‐starved cells, and the cells did not reduce glnK and nac expression when given ammonia. Thus, one specific role of GlnK is to regulate the expression of Ntr genes during nitrogen starvation. GlnK also had a dramatic effect on the ability of cells to survive nitrogen starvation and resume rapid growth when fed ammonia. After being nitrogen starved for as little as 10 h, cells lacking GlnK were unable to resume rapid growth when given ammonia. In contrast, wild‐type cells that were starved immediately resumed rapid growth when fed ammonia. Cells lacking GlnK also showed faster loss of viability during extended nitrogen starvation relative to wild‐type cells. This complex phenotype resulted partly from the requirement for GlnK to regulate nac expression; deletion of nac restored wild‐type growth rates after ammonia starvation and refeeding to cells lacking GlnK, but did not improve viability during nitrogen starvation. The specific roles of GlnK during nitrogen starvation were not the result of a distinct function of the protein, as expression of PII from the glnK promoter in cells lacking GlnK restored the wild‐type phenotypes.

Context-Dependent Functions of the PII and GlnK Signal Transduction Proteins in Escherichia coli

Two closely related signal transduction proteins, PII and GlnK, have distinct physiological roles in the regulation of nitrogen assimilation. Here, we examined the physiological roles of PII and GlnK when these proteins were expressed from various regulated or constitutive promoters. The results indicate that the distinct functions of PII and GlnK were correlated with the timing of expression and levels of accumulation of the two proteins. GlnK was functionally converted into PII when its expression was rendered constitutive and at the appropriate level, while PII was functionally converted into GlnK by engineering its expression from the nitrogen-regulated glnK promoter. Also, the physiological roles of both proteins were altered by engineering their expression from the nitrogen-regulated glnA promoter. We hypothesize that the use of two functionally identical PII-like proteins, which have distinct patterns of expression, may allow fine control of Ntr genes over a wide range of environmental conditions. In addition, we describe results suggesting that an additional, unknown mechanism may control the cellular level of GlnK.

Antagonism of PII signalling by the AmtB protein of Escherichia coli

Escherichia coli AmtB is a member of the MEP/Amt family of ammonia transporters found in archaea, eubacteria, fungi, plants and animals. In prokaryotes, AmtB homologues are co‐transcribed with a PII paralogue, GlnK, in response to nitrogen limitation. Here, we show that AmtB antagonizes PII signalling through NRII and that co‐expression of GlnK with AmtB overcomes this antagonism. In cells lacking GlnK, expression of AmtB during nitrogen starvation prevented deinduction of Ntr gene expression when a nitrogen source became available. The absence of AmtB in cells lacking GlnK allowed rapid reduction of Ntr gene expression during this transition, indicating that one function of GlnK is to prevent AmtB‐mediated antagonism of PII signalling after nitrogen starvation. Other roles of GlnK in controlling Ntr gene expression and maintaining viability during nitrogen starvation were unaffected by AmtB. Expression of AmtB from a constitutive promoter under nitrogen‐rich conditions induced full expression of glnALG and elevated expression of glnK in wild‐type and glnK cells; thus, the ability of AmtB to raise Ntr gene expression did not require a factor found only in nitrogen‐starved cells. Experiments with intact cells showed that AmtB acted downstream of a uridylyl transferase uridylyl‐removing enzyme (UTase/UR) and upstream of NRII, suggesting that the target was PII. AmtB also slowed the deuridylylation of PII∼UMP upon ammonia addition, showing that multiple PII interactions were affected by AmtB. Our data are consistent with a hypothesis that AmtB interacts with PII and GlnK, and that co‐transcription of glnK and amtB prevents titration of PII when AmtB is highly expressed.

Nac-mediated repression of the serA promoter of Escherichia coli

Escherichia coli and related bacteria contain two paralogous PII‐like proteins involved in nitrogen regulation, the glnB product, PII, and the glnK product, GlnK. Previous studies have shown that cells lacking both PII and GlnK have a severe growth defect on minimal media, resulting from elevated expression of the Ntr regulon. Here, we show that this growth defect is caused by activity of the nac product, Nac, a LysR‐type transcription factor that is part of the Ntr regulon. Cells with elevated Ntr expression that also contain a null mutation in nac displayed growth rates on minimal medium similar to the wild type. When expressed from high‐copy plasmids, Nac imparts a growth defect to wild‐type cells in an expression level‐dependent manner. Neither expression of Nac nor lack thereof significantly affected Ntr gene expression, suggesting that the activity of Nac at one or more promoters outside the Ntr regulon was responsible for its effects. The growth defect of cells lacking both PII and GlnK was also eliminated upon supplementation of minimal medium with serine or glycine for solid medium or with serine or glycine and glutamine for liquid medium. These observations suggest that high Nac expression results in a reduction in serine biosynthesis. β‐Galactosidase activity expressed from a Mu d1 insertion in serA was reduced approximately 10‐fold in cells with high Nac expression. We hypothesize that one role of Nac is to limit serine biosynthesis as part of a cellular mechanism to reduce metabolism in a co‐ordinated manner when cells become starved for nitrogen.

Spenito and Split ends act redundantly to promote Wingless signaling

Wingless (Wg)/Wnt signaling directs a variety of cellular processes during animal development by promoting the association of Armadillo/beta-catenin with TCFs on Wg-regulated enhancers (WREs). Split ends (Spen), a nuclear protein containing RNA recognition motifs (RRMs) and a SPOC domain, is required for optimal Wg signaling in several fly tissues. In this report, we demonstrate that Spenito (Nito), the only other fly protein containing RRMs and a SPOC domain, acts together with Spen to positively regulate Wg signaling. The partial defect in Wg signaling observed with spen RNAi was enhanced by simultaneous knockdown of nito while it was rescued by expression of nito in wing imaginal discs. In cell culture, depletion of both factors causes a greater defect in the activation of several Wg targets than RNAi of either spen or nito alone. These nuclear proteins are not required for Armadillo stabilization or the recruitment of TCF and Armadillo to a WRE. Loss of Wg target gene activation in cells depleted for spen and nito was not dependent on the transcriptional repressor Yan or Suppressor of Hairless, two previously identified targets of Spen. We propose that Spen and Nito act redundantly downstream of TCF/Armadillo to activate many Wg transcriptional targets.

Wnt/β‐catenin‐mediated transcriptional regulation

Many Wnts act by stabilizing β‐catenin and promoting its nuclear localization, where it can influence gene expression by associating with DNA‐binding proteins. The best understood of these transcription factors are members of the TCF/LEF1 (TCF) family, which recognize specific DNA sequences via a high mobility group (HMG) domain. There is strong support for a model where TCFs repress target gene expression in the absence of Wnt signaling, through recruitment of corepressors. Upon Wnt stimulation, high levels of nuclear β‐catenin bind to TCF and promote a switch to transcriptional activation. This is achieved by the recruitment of transcriptional coactivators and the subsequent modification of the chromatin surrounding Wnt regulated enhancers (WREs). While invertebrate TCFs can both repress and activate transcription of Wnt targets, genetic evidence in vertebrate systems suggests that these activities may have become separated among specific TCFs. In addition, recent findings demonstrate that β‐catenin/TCF complex can also directly repress some Wnt target genes and β‐catenin can also regulate transcription independently of TCFs, by associating with other DNA‐ binding proteins. Wnt‐signaling components previously thought to act only in the cytoplasm or as nuclear shuttles for β‐catenin have been found to associate directly with WREs. This added complexity to the already elaborate model of Wnt‐mediated transcriptional regulation may help account for the extraordinary diversity of transcriptional responses to the Wnt/β‐catenin pathway.

Wnt-Mediated Repression via Bipartite DNA Recognition by TCF in the Drosophila Hematopoietic System

The Wnt/β-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/β-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/β-catenin signaling and allosteric regulation of a transcription factor by DNA.