Functional dissection of the developmentally restricted BEN domain chromatin boundary factor Insensitive.

BACKGROUND: Boundaries in the Drosophila bithorax complex delimit autonomous regulatory domains that activate the parasegment (PS)-specific expression of homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains that control Abd-B expression in PS11 and PS12. This boundary is composed of multiple functionally redundant elements and has two key activities: it blocks crosstalk between iab-6 and iab-7 and facilitates boundary bypass. RESULTS: Here, we have used a structure-function approach to elucidate the biochemical properties and the in vivo activities of a conserved BEN domain protein, Insensitive, that is associated with Fab-7. Our biochemical studies indicate that in addition to the C-terminal BEN DNA-binding domain, Insv has two domains that mediate multimerization: one is a coiled-coil domain in the N-terminus, and the other is next to the BEN domain. These multimerization domains enable Insv to bind simultaneously to two canonical 8-bp recognition motifs, as well as to a ~ 100-bp non-canonical recognition sequence. They also mediate the assembly of higher-order multimers in the presence of DNA. Transgenic proteins lacking the N-terminal coiled-coil domain are compromised for boundary function in vivo. We also show that Insv interacts directly with CP190, a protein previously implicated in the boundary functions of several DNA-binding proteins, including Su(Hw) and dCTCF. While CP190 interaction is required for Insv binding to a subset of sites on polytene chromosomes, it has only a minor role in the boundary activity of Insv in the context of Fab-7. CONCLUSIONS: The subdivision of eukaryotic chromosomes into discrete topological domains depends upon the pairing of boundary elements. In flies, pairing interactions are specific and typically orientation dependent. They occur in cis between neighboring heterologous boundaries, and in trans between homologous boundaries. One potential mechanism for ensuring pairing-interaction specificity is the use of sequence-specific DNA-binding proteins that can bind simultaneously with two or more recognition sequences. Our studies indicate that Insv can assemble into a multivalent DNA-binding complex and that the N-terminal Insv multimerization domain is critical for boundary function.

The BEN Domain Protein Insensitive Binds to the Fab-7 Chromatin Boundary To Establish Proper Segmental Identity in Drosophila.

Boundaries (insulators) in the Drosophila bithorax complex (BX-C) delimit autonomous regulatory domains that orchestrate the parasegment (PS)-specific expression of the BX-C homeotic genes. The Fab-7 boundary separates the iab-6 and iab-7 regulatory domains, which control Abd-B expression in PS11 and PS12, respectively. This boundary is composed of multiple functionally redundant elements and has two key functions: it blocks cross talk between iab-6 and iab-7 and facilitates boundary bypass. Here, we show that two BEN domain protein complexes, Insensitive and Elba, bind to multiple sequences located in the Fab-7 nuclease hypersensitive regions. Two of these sequences are recognized by both Insv and Elba and correspond to a CCAATTGG palindrome. Elba also binds to a related CCAATAAG sequence, while Insv does not. However, the third Insv recognition sequences is ∼100 bp in length and contains the CCAATAAG sequence at one end. Both Insv and Elba are assembled into large complexes (∼420 and ∼265-290 kDa, respectively) in nuclear extracts. Using a sensitized genetic background, we show that the Insv protein is required for Fab-7 boundary function and that PS11 identity is not properly established in insv mutants. This is the first demonstration that a BEN domain protein is important for the functioning of an endogenous fly boundary.

Manufacturing costs of HPV vaccines for developing countries.

BACKGROUND: Nearly all of the 500,000 new cases of cervical cancer and 270,000 deaths occur in middle or lower income countries. Yet the two most prevalent HPV vaccines are unaffordable to most. Even prices to Gavi, the Vaccine Alliance, are unaffordable to graduating countries, once they lose Gavi subsidies. Merck and Glaxosmithkline (GSK) claim their prices to Gavi equal their manufacturing costs; but these costs remain undisclosed. We undertook this investigation to estimate those costs. METHODS: Searches in published and commercial literature for information about the manufacturing of these vaccines. Interviews with experts in vaccine manufacturing. FINDINGS: This detailed sensitivity analysis, based on the best available evidence, finds that after a first set of batches for affluent markets, manufacturing costs of Gardasil for developing countries range between $0.48 and $0.59 a dose, a fraction of its alleged costs of $4.50. Because volume of Cervarix is low, its per unit costs are much higher, though at comparable volumes, its costs would be similar. INTERPRETATION: Given the recovery of fixed and annual costs from sales in affluent markets, Merck's break-even price to Gavi could be $0.50-$0.60, not $4.50. These savings could support Gavi programs to strengthen delivery and increase coverage. Outside Gavi, prices to lower- and middle-income countries, with profit, could also be lowered and made available to millions more adolescents at risk. These estimates and their policy implications deserve further discussion.

The structure of Sec12 implicates potassium ion coordination in Sar1 activation.

Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a ß propeller fold. Conserved residues cluster around a loop we term the "K loop," which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange.