Roles of binding elements, FOXL2 domains, and interactions with cJUN and SMADs in regulation of FSHβ.

We previously identified FOXL2 as a critical component in FSHβ gene transcription. Here, we show that mice deficient in FOXL2 have lower levels of gonadotropin gene expression and fewer LH- and FSH-containing cells, but the same level of other pituitary hormones compared to wild-type littermates, highlighting a role of FOXL2 in the pituitary gonadotrope. Further, we investigate the function of FOXL2 in the gonadotrope cell and determine which domains of the FOXL2 protein are necessary for induction of FSHβ transcription. There is a stronger induction of FSHβ reporter transcription by truncated FOXL2 proteins, but no induction with the mutant lacking the forkhead domain. Specifically, FOXL2 plays a role in activin induction of FSHβ, functioning in concert with activin-induced SMAD proteins. Activin acts through multiple promoter elements to induce FSHβ expression, some of which bind FOXL2. Each of these FOXL2-binding sites is either juxtaposed or overlapping with a SMAD-binding element. We determined that FOXL2 and SMAD4 proteins form a higher order complex on the most proximal FOXL2 site. Surprisingly, two other sites important for activin induction bind neither SMADs nor FOXL2, suggesting additional factors at work. Furthermore, we show that FOXL2 plays a role in synergistic induction of FSHβ by GnRH and activin through interactions with the cJUN component of the AP1 complex that is necessary for GnRH responsiveness. Collectively, our results demonstrate the necessity of FOXL2 for proper FSH production in mice and implicate FOXL2 in integration of transcription factors at the level of the FSHβ promoter

TBP recruitment to the U1 snRNA gene promoter is disrupted by substituting a U6 proximal sequence element A (PSEA) for the U1 PSEA

In eukaryotes, small nuclear RNAs (snRNAs) are required for pre-mRNA splicing. Most snRNAs, such as U1, U2 ,U4 and U5, are synthesized by RNA polymerase II, but U6 snRNA is synthesized by RNA polymerase III. Transcription of snRNA genes by either RNA polymerase is dependent upon a proximal sequence element (PSE) centered approximately 50- 55 base pairs upstream of the start site. The PSE is recognized by the small nuclear RNA activating protein complex (SNAPc), a multi-subunit transcription factor. In Drosphila melanogaster, the PSE is more specifically termed the PSEA to distinguish it from a second conserved element termed the PSEB present in the promoter of the Pol II transcribed fly snRNA genes. Interestingly, the fly U1 and U6 PSEAs are not functionally interchangeable, even though both are recognized by the same protein, DmSNAPc. A five-nucleotide substitution that changed the U1 PSEA to a U6 PSEA was shown to inactivate the U1 promoter. In light of this knowledge I wished to investigate why the U6 PSEA cannot functionally substitute for the U1 PSEA. I sought to determine whether the U6 PSEA substitution disrupts a specific step in RNA polymerase II transcription pre- initiation complex assembly in vivo. To accomplish this, I used a chromatin immunoprecipitation (ChIP) assay. In chapter 1, I describe the preparation of reagents needed for the ChIP assays. I expressed TBP and two of the three subunits of DmSNAPc in bacteria. I then purified the proteins and used them for polyclonal antibody production. In chapter 2, I demonstrate that the antibodies can be used in ChIPs to detect DmSNAP43, DmSNAP50 and TBP bound to the endogenous U1 promoter in vivo. I then generated cell lines stably transfected with reporter constructs for the U1 wild type promoter or the U1 promoter that contained a U6 PSEA. Interestingly, my ChIPs indicated that DmSNAPc assembled on both types of promoters. On the other hand, TBP assembled only on the wild type promoter. These results are consistent with a model in which DmSNAPc assumes a conformation on the U6 PSEA that prevents the assembly of a Pol II transcription pre-initiation comple

Identification of SNAPc Subunit Domains That Interact with Specific Nucleotide Positions in the U1 and U6 Gene Promoters ▿

The small nuclear RNA (snRNA)-activating protein complex (SNAPc) is essential for transcription of genes coding for the snRNAs (U1, U2, etc.). In Drosophila melanogaster, the heterotrimeric DmSNAPc recognizes a 21-bp DNA sequence, the proximal sequence element A (PSEA), located approximately 40 to 60 bp upstream of the transcription start site. Upon binding the PSEA, DmSNAPc establishes RNA polymerase II preinitiation complexes on U1 to U5 promoters but RNA polymerase III preinitiation complexes on U6 promoters. Minor differences in nucleotide sequence of the U1 and U6 PSEAs determine RNA polymerase specificity; moreover, DmSNAPc adopts different conformations on these different PSEAs. We have proposed that such conformational differences in DmSNAPc play a key role in determining the different polymerase specificities of the U1 and U6 promoters. To better understand the structure of DmSNAPc-PSEA complexes, we have developed a novel protocol that combines site-specific protein-DNA photo-cross-linking with site-specific chemical cleavage of the protein. This protocol has allowed us to map regions within each of the three DmSNAPc subunits that contact specific nucleotide positions within the U1 and U6 PSEAs. These data help to establish the orientation of each DmSNAPc subunit on the DNA and have revealed cases in which different domains of the subunits differentially contact the U1 versus U6 PSEAs

Global economic burden of unmet surgical need for appendicitis

Background There is a substantial gap in provision of adequate surgical care in many low- and middle-income countries. This study aimed to identify the economic burden of unmet surgical need for the common condition of appendicitis. Methods Data on the incidence of appendicitis from 170 countries and two different approaches were used to estimate numbers of patients who do not receive surgery: as a fixed proportion of the total unmet surgical need per country (approach 1); and based on country income status (approach 2). Indirect costs with current levels of access and local quality, and those if quality were at the standards of high-income countries, were estimated. A human capital approach was applied, focusing on the economic burden resulting from premature death and absenteeism. Results Excess mortality was 4185 per 100 000 cases of appendicitis using approach 1 and 3448 per 100 000 using approach 2. The economic burden of continuing current levels of access and local quality was US $92 492 million using approach 1 and$73 141 million using approach 2. The economic burden of not providing surgical care to the standards of high-income countries was $95 004 million using approach 1 and$75 666 million using approach 2. The largest share of these costs resulted from premature death (97.7 per cent) and lack of access (97.0 per cent) in contrast to lack of quality. Conclusion For a comparatively non-complex emergency condition such as appendicitis, increasing access to care should be prioritized. Although improving quality of care should not be neglected, increasing provision of care at current standards could reduce societal costs substantially