Publications

In Caenorhabditis elegans, the predicted transcription factor SKN-1 is required for embryonic endodermal and mesodermal specification and for maintaining differentiated intestinal cells post-embryonically. The SKN-1 DNA-binding region is related to the Cap'n'Collar (CNC) family of basic leucine zipper proteins, but uniquely, SKN-1 binds DNA as a monomer. CNC proteins are absent in C. elegans, however; and their involvement in the endoderm and mesoderm suggests some functional parallels to SKN-1. Using a cell culture assay, we show that SKN-1 induces transcription and contains three potent activation domains. The functional core of one domain is a short motif, the DIDLID element, which is highly conserved in a subgroup of vertebrate CNC proteins. The DIDLID element is important for SKN-1-driven transcription, suggesting a likely significance in other CNC proteins. SKN-1 binds to and activates transcription through the p300/cAMP-responsive element-binding protein-binding protein (CBP) coactivator, supporting the genetic prediction that SKN-1 recruits the C. elegans p300/CBP ortholog, CBP-1. The DIDLID element appears to act independently of p300/CBP, however, suggesting a distinct conserved target. The evolutionarily preservation of the DIDLID transcriptional element supports the model that SKN-1 and some CNC proteins interact with analogous cofactors and may have preserved some similar functions despite having divergent DNA-binding domains.

The immediate early protein tristetraprolin (TTP) is required to prevent inappropriate production of the cytokine TNF-α, and is a member of a zinc finger protein family that is associated with RNA binding. TTP expression is induced by TNF-α, and evidence indicates that TTP can bind and destabilize the TNF-α mRNA. TTP and the closely related TIS11b and TIS11d proteins are evolutionarily conserved, however, and induced transiently in various cell types by numerous diverse stimuli, suggesting that they have additional functions. Supporting this idea, continuous expression of each TTP/TIS11 protein at physiological levels causes apoptotic cell death. By various criteria, this cell death appears analogous to apoptosis induced by certain oncoproteins. It is also dependent upon the zinc fingers, suggesting that it involves action on appropriate cellular targets. TTP but not TIS11b or TIS11d also sensitizes cells to induction of apoptosis by TNF-α. The data suggest that the TTP and TIS11 immediate early proteins have similar but distinct effects on growth or survival pathways, and that TTP might influence TNF-α regulation at multiple levels.

Basic helix-loop-helix (bHLH) proteins perform a wide variety of biological functions. Most bHLH proteins recognize the consensus DNA sequence CAN NTG (the E-box consensus sequence is underlined) but acquire further functional specificity by preferring distinct internal and flanking bases. In addition, induction of myogenesis by MyoD-related bHLH proteins depends on myogenic basic region (BR) and BR-HLH junction residues that are not essential for binding to a muscle-specific site, implying that their BRs may be involved in other critical interactions. We have investigated whether the myogenic residues influence DNA sequence recognition and how MyoD, Twist, and their E2A partner proteins prefer distinct CAN NTGsites. In MyoD, the myogenic BR residues establish specificity for particular CAN NTG sites indirectly, by influencing the conformation through which the BR helix binds DNA. An analysis of DNA binding by BR and junction mutants suggests that an appropriate BR-DNA conformation is necessary but not sufficient for myogenesis, supporting the model that additional interactions with this region are important. The sequence specificities of E2A and Twist proteins require the corresponding BR residues. In addition, mechanisms that position the BR allow E2A to prefer distinct half-sites as a heterodimer with MyoD or Twist, indicating that the E2A BR can be directed toward different targets by dimerization with different partners. Our findings indicate that E2A and its partner bHLH proteins bind to CAN NTG sites by adopting particular preferred BR-DNA conformations, from which they derive differences in sequence recognition that can be important for functional specificity.

In the early Caenorhabditis elegans embryo, maternally expressed PIE-1 protein is required in germ-line blastomeres to inhibit somatic differentiation, maintain an absence of mRNA transcription, and block phosphorylation of the RNA polymerase II large subunit (Pol II) carboxy-terminal domain (CTD). We have determined that PIE-1 can function as a transcriptional repressor in cell culture assays. By fusing PIE-1 sequences to the yeast GAL4 DNA-binding domain, we have identified a PIE-1 repression domain that appears to inhibit the transcriptional machinery directly. A sequence element that is required for this repressor activity is similar to the Pol II CTD heptapeptide repeat, suggesting that the PIE-1 repression domain might target a protein complex that can bind the CTD. An alteration of this sequence element that blocks repression also impairs the ability of a transgene to rescue a pie-1mutation, suggesting that this repressor activity may be important for PIE-1 function in vivo.

Tristetraprolin (TTP), the prototype of a class of CCCH zinc finger proteins, is a phosphoprotein that is rapidly and transiently induced by growth factors and serum in fibroblasts. Recent evidence suggests that a physiological function of TTP is to inhibit tumor necrosis factor α secretion from macrophages by binding to and destabilizing its mRNA (Carballo, E., Lai, W.S., Blackshear, P.J., 1998. Science, 281, 1001–1005). To investigate possible functions of CCCH proteins in early development of Xenopus, we isolated four XenopuscDNAs encoding members of this class. Based on 49% overall amino acid identity and 84% amino acid identity within the double zinc finger domain, one of the Xenopus proteins (XC3H-1) appears to be the homologue of TTP. By similar analyses, XC3H-2 and XC3H-3 are homologues of ERF-1 (cMG1, TIS11B) and ERF-2 (TIS11D). A fourth protein, XC3H-4, is a previously unidentified member of the CCCH class of vertebrate zinc finger proteins; it contains four Cx₈Cx₅Cx₃H repeats, two of which are YKTEL Cx₈Cx₅Cx₃H repeats that are closely related to sequences found in the other CCCH proteins. Whereas XC3H-1, XC3H-2, and XC3H-3 were widely expressed in adult tissues, XC3H-4 mRNA was not detected in any of the adult tissues studied except for the ovary. Its expression appeared to be limited to the ovary, oocyte, egg and the early embryonic stages leading up to the mid-blastula transition. Its mRNA was highly expressed in oocytes of all ages, and was enriched in the animal pole cytosol of mature oocytes. Maternal expression was also seen with the other three messages, suggesting the possibility that these proteins are involved in regulating mRNA stability in oocyte maturation and/or early embryogenesis.

The Caenorhabditis elegans SKN-1 protein binds DNA through a basic region like those of bZIP proteins and through a flexible amino-terminal arm segment similar to those with which numerous helix-turn-helix proteins bind to bases in the minor groove. A recent X-ray crystallographic structure suggests that the SKN-1 amino-terminal arm provides only nonspecific DNA binding. In this study, however, we demonstrate that this segment mediates recognition of an AT-rich element that is part of the preferred SKN-1 binding site and thereby significantly increases the sequence specificity with which SKN-1 binds DNA. Mutagenesis experiments show that multiple amino acid residues within the arm are involved in binding. These residues provide binding affinity through distinct but partially redundant interactions and enhance specificity by discriminating against alternate sites. The AT-rich element minor groove is important for binding of the arm, which appears to affect DNA conformation in this region. This conformational effect does not seem to involve DNA bending, however, because the arm does not appear to affect a modest DNA bend that is induced by SKN-1. The data illustrate an example of how a small, flexible protein segment can make an important contribution to DNA binding specificity through multiple interactions and mechanisms.

The SKN-1 transcription factor specifies early embryonic cell fates in Caenorhabditis elegans.SKN-1 binds DNA at high affinity as a monomer, by means of a basic region like those of basic-leucine zipper (bZIP) proteins, which bind DNA only as dimers. We have investigated how the SKN-1 DNA-binding domain (the Skn domain) promotes stable binding of a basic region monomer to DNA. A flexible arm at the Skn domain amino terminus binds in the minor groove, but a support segment adjacent to the carboxy-terminal basic region can independently stabilize basic region–DNA binding. Off DNA, the basic region and arm are unfolded and, surprisingly, the support segment forms a molten globule of four α-helices. On binding DNA, the Skn domain adopts a tertiary structure in which the basic region helix extends directly from a support segment α-helix, which is required for binding. The remainder of the support segment anchors this uninterrupted helix on DNA, but leaves the basic region exposed in the major groove. This is similar to how the bZIP basic region extends from the leucine zipper, indicating that positioning and cooperative stability provided by helix extension are conserved mechanisms that promote binding of basic regions to DNA.

Maternally expressed Skn-1 protein is required for the correct specification of certain blastomere fates in early Caenorhabditis elegans embryos. Skn-1 contains a basic region similar to those of basic leucine zipper (bZIP) proteins but, paradoxically, it lacks a leucine zipper dimerization segment. Random sequence selection methods were used to show that Skn-1 binds to specific DNA sequences as a monomer. The Skn-1 basic region lies at the carboxyl terminus of an 85-amino acid domain that binds preferentially to a bZIP half-site and also recognizes adjacent 5′ AT-rich sequences in the minor groove, apparently with an amino (NH₂)-terminal "arm" related to those of homeodomain proteins. The intervening residues appear to stabilize interactions of these two subdomains with DNA. The Skn-1 DNA binding domain thus represents an alternative strategy for promoting binding of a basic region segment recognition helix to its cognate half-site. The results point to an underlying modularity in subdomains within established DNA binding domains.