Keith E. Shearwin

The role of individual Nedd4–2 (KIAA0439) WW domains in binding and regulating epithelial sodium channels

The amiloride‐sensitive epithelial sodium channel (ENaC) is essential for fluid and electrolyte homeostasis. ENaC consists of α, β, and γ subunits, each of which contains a PPxY motif that interacts with the WW domains of the ubiquitin‐protein ligases Nedd4 and Nedd4–2. Disruption of this interaction, as in Liddles syndrome in which mutations delete or alter the PPxY motif of either the β or the γ subunits, results in increased ENaC activity. We report here that Nedd4–2 has two major isoforms that show tissue‐specific expression; however, both isoforms can inhibit ENaC in Xenopus oocytes. Because there are four WW domains in Nedd4–2, we analyzed binding kinetics and affinity between individual WW domains and ENaC subunits. Using whole cell patch‐clamp techniques, we studied the role of individual WW domains in the regulation of ENaC in mammalian cells. We report here that unlike Nedd4, only two of the Nedd4–2 WW domains, WW3 and WW4, are required for both the binding to ENaC subunits and the regulation of Na+ feedback control of ENaC. Although both WW3 and WW4 individually can interact with all three ENaC subunits in vitro, both domains together are essential for in vivo function of Nedd4–2 in ENaC regulation. These data suggest that Nedd4–2 WW3 and WW4 interact with distinct, noninterchangeable sites in ENaC and that to prevent Na+ feedback control of ENaC it is necessary to occlude both sites.

Potent transcriptional interference by pausing of RNA polymerases over a downstream promoter

Elongating RNA polymerases (RNAPs) can interfere with transcription from downstream promoters by inhibiting DNA binding by RNAP and activators. However, combining quantitative measurement with mathematical modeling, we show that simple RNAP elongation cannot produce the strong asymmetric interference observed between a natural face-to-face promoter pair in bacteriophage lambda. Pausing of elongating polymerases over the RNAP-binding site of the downstream promoter is demonstrated in vivo and is shown by modeling to account for the increased interference. The model successfully predicts the effects on interference of treatments increasing or reducing pausing. Gene regulation by pausing-enhanced occlusion provides a general and potentially widespread mechanism by which even weak converging or tandem transcription, either coding or noncoding, can bring about strong in cis repression.

Transcriptional interference by RNA polymerase pausing and dislodgement of transcription factors.

Transcriptional interference is the in cis suppression of one transcriptional process by another. Mathematical modeling shows that promoter occlusion by elongating RNA polymerases cannot produce strong interference. Interference may instead be generated by (1) dislodgement of slow-to-assemble pre-initiation complexes and transcription factors and (2) prolonged occlusion by paused RNA polymerases.

The Tum Protein of Coliphage 186 Is an Antirepressor

The tum gene of coliphage 186, encoded on a LexA controlled operon, is essential for UV induction of a 186 prophage. Primer extension analysis is used to confirm that Tum is the sole phage function required for prophage induction and that it acts against the maintenance repressor, CI, to relieve repression of the lytic promoters, p R andp B, and thereby bring about lytic development.In vitro experiments with purified proteins demonstrate that Tum prevents CI binding to its operator sites. Tum does not compete with CI for binding sites on DNA, and unlike RecA mediated induction of lambda prophage, the action of Tum on CI is reversible. Mechanisms by which Tum may act against CI are discussed.

Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR

The basic helix–loop–helix (bHLH).PAS dimeric transcription factors have crucial roles in development, stress response, oxygen homeostasis and neurogenesis. Their target gene specificity depends in part on partner protein choices, where dimerization with common partner Aryl hydrocarbon receptor nuclear translocator (Arnt) is an essential step towards forming active, DNA binding complexes. Using a new bacterial two-hybrid system that selects for loss of protein interactions, we have identified 22 amino acids in the N-terminal PAS domain of Arnt that are involved in heterodimerization with aryl hydrocarbon receptor (AhR). Of these, Arnt E163 and Arnt S190 were selective for the AhR/Arnt interaction, since mutations at these positions had little effect on Arnt dimerization with other bHLH.PAS partners, while substitution of Arnt D217 affected the interaction with both AhR and hypoxia inducible factor-1α but not with single minded 1 and 2 or neuronal PAS4. Arnt uses the same face of the N-terminal PAS domain for homo- and heterodimerization and mutational analysis of AhR demonstrated that the equivalent region is used by AhR when dimerizing with Arnt. These interfaces differ from the PAS β-scaffold surfaces used for dimerization between the C-terminal PAS domains of hypoxia inducible factor-2α and Arnt, commonly used for PAS domain interactions.

Thermodynamic nonideality as a probe of reversible protein unfolding effected by variations in pH and temperature: Studies of ribonuclease

Thermodynamic nonideality arising from the space-filling effect of added sucrose is employed to confirm that the reversible unfolding of ribonuclease A effected by acid may be described as an equilibrium between native and unfolded states of the enzyme. However, the extent of the volume change is far too small for the larger isomer to be the fully expanded state, a result signifying that the acid-mediated unfolding of ribonuclease does not conform with the two-state equilibrium model of protein denaturation. Although the thermal denaturation of ribonuclease A is characterized by a larger increase in volume, quantitative reappraisal of published results on the effects of glycerol on this transition at pH 2.8 (Gekko, K., and Timasheff, S. N., 1981 Biochemistry 20, 4677-4686) leads to an estimated volume increase that is much smaller than that inferred from hydrodynamic studies--a disparity attributed to the dual actions of glycerol as a space-filling solute and as a ligand that binds preferentially to the thermally unfolded form of the enzyme. Even in this unfavorable circumstance the fact that glycerol exerts a net excluded volume effect at least confirms that the thermal unfolding of ribonuclease A is an equilibrium transition between two discrete states. The strengths and limitations of using thermodynamic nonideality as a probe of the two-state equilibrium model of protein denaturation are discussed in the light of these findings.

Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and λ repressors.

Significance Proteins bound to DNA often interact with proteins bound elsewhere on the same DNA to regulate gene expression. The intervening DNA tethers the proteins near each other, making their interaction efficient and specific, but the importance of this tethering effect is poorly understood at large DNA separations. We quantitated tethering inside bacterial cells, using two different proteins at separations up to 10,000 bp, to show that tethering is strong enough to drive efficient interactions over these distances. The same interactions were ∼10-fold weaker outside cells, implying that cellular factors enhance tethering. However, tethering was lost at a DNA separation of 500,000 bp inside bacteria, indicating special mechanisms inside eukaryotic cells to provide efficient and specific interactions over such distances. Efficient and specific interactions between proteins bound to the same DNA molecule can be dependent on the length of the DNA tether that connects them. Measurement of the strength of this DNA tethering effect has been largely confined to short separations between sites, and it is not clear how it contributes to long-range DNA looping interactions, such as occur over separations of tens to hundreds of kilobase pairs in vivo. Here, gene regulation experiments using the LacI and λ CI repressors, combined with mathematical modeling, were used to quantitate DNA tethering inside Escherichia coli cells over the 250- to 10,000-bp range. Although LacI and CI loop DNA in distinct ways, measurements of the tethering effect were very similar for both proteins. Tethering strength decreased with increasing separation, but even at 5- to 10-kb distances, was able to increase contact probability 10- to 20-fold and drive efficient looping. Tethering in vitro with the Lac repressor was measured for the same 600-to 3,200-bp DNAs using tethered particle motion, a single molecule technique, and was 5- to 45-fold weaker than in vivo over this range. Thus, the enhancement of looping seen previously in vivo at separations below 500 bp extends to large separations, underlining the need to understand how in vivo factors aid DNA looping. Our analysis also suggests how efficient and specific looping could be achieved over very long DNA separations, such as what occurs between enhancers and promoters in eukaryotic cells.

Functional Alignment of Regulatory Networks: A Study of Temperate Phages

The relationship between the design and functionality of molecular networks is now a key issue in biology. Comparison of regulatory networks performing similar tasks can provide insights into how network architecture is constrained by the functions it directs. Here, we discuss methods of network comparison based on network architecture and signaling logic. Introducing local and global signaling scores for the difference between two networks, we quantify similarities between evolutionarily closely and distantly related bacteriophages. Despite the large evolutionary separation between phage λ and 186, their networks are found to be similar when difference is measured in terms of global signaling. We finally discuss how network alignment can be used to pinpoint protein similarities viewed from the network perspective.

The dual role of Apl in prophage induction of coliphage 186

In the present study we show that the Apl protein of the temperate coliphage 186 combines, in one protein, the activities of the coliphage lambda proteins Cro and Xis. We have shown previously that Apl represses both the lysogenic promoter, pL, and the major lytic promoter, pR, and is required for excision of the prophage. Apl binds at two locations on the phage chromosome, i.e. between pR and pL and at the phage‐attachment site. Using an in vivo recombination assay, we now show that the role of ApI in excision is in the process itself and is not simply a consequence of repression of pR or pL. To study the repressive role of Apl at the switch promoters we isolated Apl‐resistant operator mutants and used them to demonstrate a requirement for Apl in the efficient derepression of the lysogenic promoter during prophage induction. We conclude that Apl is both an excisionase and transcriptional repressor.

Enhancer-like long-range transcriptional activation by λ CI-mediated DNA looping.

How distant enhancer elements regulate the assembly of a transcription complex at a promoter remains poorly understood. Here, we use long-range gene regulation by the bacteriophage λ CI protein as a powerful system to examine this process in vivo. A 2.3-kb DNA loop, formed by CI bridging its binding sites at OR and OL, is known already to enhance repression at the lysogenic promoter PRM, located at OR. Here, we show that CI looping also activates PRM by allowing the C-terminal domain of the α subunit of the RNA polymerase bound at PRM to contact a DNA site adjacent to the distal CI sites at OL. Our results establish OL as a multifaceted enhancer element, able to activate transcription from long distances independently of orientation and position. We develop a physicochemical model of our in vivo data and use it to show that the observed activation is consistent with a simple recruitment mechanism, where the α–C-terminal domain to DNA contact need only provide ∼2.7 kcal/mol of additional binding energy for RNA polymerase. Structural modeling of this complete enhancer–promoter complex reveals how the contact is achieved and regulated, and suggests that distal enhancer elements, once appropriately positioned at the promoter, can function in essentially the same way as proximal promoter elements.