GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of histone H3, lysine 27.

GATA4 is expressed in the proximal 85% of small intestine where it promotes a proximal intestinal ('jejunal') identity while repressing a distal intestinal ('ileal') identity, but its molecular mechanisms are unclear. Here, we tested the hypothesis that GATA4 promotes a jejunal versus ileal identity in mouse intestine by directly activating and repressing specific subsets of absorptive enterocyte genes by modulating the acetylation of histone H3, lysine 27 (H3K27), a mark of active chromatin, at sites of GATA4 occupancy. Global analysis of mouse jejunal epithelium showed a statistically significant association of GATA4 occupancy with GATA4-regulated genes. Occupancy was equally distributed between down- and up-regulated targets, and occupancy sites showed a dichotomy of unique motif over-representation at down- versus up-regulated genes. H3K27ac enrichment at GATA4-binding loci that mapped to down-regulated genes (activation targets) was elevated, changed little upon conditional Gata4 deletion, and was similar to control ileum, whereas H3K27ac enrichment at GATA4-binding loci that mapped to up-regulated genes (repression targets) was depleted, increased upon conditional Gata4 deletion, and approached H3K27ac enrichment in wild-type control ileum. These data support the hypothesis that GATA4 both activates and represses intestinal genes, and show that GATA4 represses an ileal program of gene expression in the proximal small intestine by inhibiting the acetylation of H3K27.

Conditional ablation of Gata4 and Fog2 genes in mice reveals their distinct roles in mammalian sexual differentiation.

Assembly of functioning testis and ovary requires a GATA4-FOG2 transcriptional complex. To define the separate roles for GATA4 and FOG2 proteins in sexual development of the testis we have ablated the corresponding genes in somatic gonadal cells. We have established that GATA4 is required for testis differentiation, for the expression of Dmrt1 gene, and for testis cord morphogenesis. While Sf1Cre-mediated excision of Gata4 permitted normal expression of most genes associated with embryonic testis development, gonadal loss of Fog2 resulted in an early partial block in male pathway and sex reversal. We have also determined that testis sexual differentiation is sensitive to the timing of GATA4 loss during embryogenesis. Our results now demonstrate that these two genes also have non-overlapping essential functions in testis development.

Therapeutic neovascularization for peripheral arterial diseases: advances and perspectives.

Recently, with the better understanding of the mechanisms of neovascularization, many new therapeutic approaches to enhance neovascularization have emerged. Of these diverse emerging methods, use of growth factors and cells are the two major ones. This review will provide an update on the present understanding of the basic mechanisms of angiogenesis, vasculogenesis, and arteriogenesis, as a basis for designing future pro-neovascularization treatments. Several angiogenic factors including vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) have been implicated in augmenting the neovascularization process. However, single growth factors are not sufficient to generate functional vessels. In synergistic or complementary manner, these factors may be used in harmony to form long-term functional vessels. Cell therapy has the potential to supply stem/progenitor cells and multiple angiogenic factors to the region of ischemia. However, the efficacy of stem cells transplantation may be impaired by low survival rate, insufficient cell number and impaired function in aging and diseases. Combination of cells or cells primed with growth factor(s) or genetic modification may augment the therapeutic efficacy. This paper reviews critical literature in depth to elucidate the mechanism of therapeutic neovascularization, angiogenic factor therapy and cell transplantation. Based on past experience and actual knowledge, we propose future strategies for clinical application and discuss the problems and controversies that need to be addressed in order to fully exploit the potential of growth factors and/or cell transplantation with clinical relevance.

ICln is essential for cellular and early embryonic viability.

pICln is a 26-kDa protein that is ubiquitously expressed and highly conserved from Xenopus laevis to Homo sapiens. The physiological functions of pICln remain to be established. To address this question, we disrupted the ICln gene in embryonic stem cells. We found that murine embryos lacking ICln die early in gestation (between stages E3.5 and E7.5). Furthermore, we found that ICln is essential for embryonic stem cell viability. Previously, we showed that pICln interacts directly with a homolog of a yeast protein that binds a PAK-like kinase and participates in the regulation of cell morphology and cell cycling. pICln also forms a complex with several core spliceosomal proteins, and this interaction may play a role in the regulation of spliceosomal biogenesis. Collectively, these data strongly suggest that pICln participates in critical cellular pathways, including regulation of the cell cycle and RNA processing.

pICln inhibits snRNP biogenesis by binding core spliceosomal proteins.

The U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins (snRNPs) form essential components of spliceosomes, the machinery that removes introns from pre-mRNAs in eukaryotic cells. A critical initial step in the complex process of snRNP biogenesis is the assembly of a group of common core proteins (Sm proteins) on spliceosomal snRNA. In this study we show by multiple independent methods that the protein pICln associates with Sm proteins in vivo and in vitro. The binding of pICln to Sm proteins interferes with Sm protein assembly on spliceosomal snRNAs and inhibits import of snRNAs into the nucleus. Furthermore, pICln prevents the interaction of Sm proteins with the survival of motor neurons (SMN) protein, an interaction that has been shown to be critical for snRNP biogenesis. These findings lead us to propose a model in which pICln participates in the regulation of snRNP biogenesis, at least in part by interfering with Sm protein interaction with SMN protein.

Diagnosis and management of agenesis of the right lung and left pulmonary artery sling.

The diagnostic, developmental, and surgical aspects of left pulmonary artery sling associated with right lung agenesis in 3 patients are described. Cardiac catheterization may be avoided by the combined use of echocardiography and magnetic resonance imaging and/or computed tomography.

Dimerization of leucine zippers analyzed by random selection.

The leucine zipper is a coiled coil that mediates specific dimerization of bZIP DNA-binding domains. A hydrophobic spine involving the conserved leucines runs down the coiled-coil and is thought to stabilize the dimer. We used the method of random selection to further define the primary sequence requirements for homodimer formation and heterodimer formation with Fos. When positions on either side of the hydrophobic spine of GCN4 are diversified to include the corresponding residues of Jun, a large percentage of the resulting sequences form homodimers, and a large percentage form heterodimers with Fos. Basic residues were preferred, but not essential, at position e of zippers which heterodimerize with Fos. When random sequences containing 5 heptad repeat of leucines are subject to a selection for homodimer formation, a diverse set of sequences is isolated. Certain residues are preferred at each position in the heptad repeat, although no essential primary sequence determinants could be identified. No pair of residues not involving the conserved leucines could be identified which strongly promotes homodimerization. These results suggest that factors determining leucine zipper dimerization are complex, with numerous interactions contributing to the association.

Mutations in the bZIP domain of yeast GCN4 that alter DNA-binding specificity.

The bZIP class of eukaryotic transcriptional regulators utilize a distinct structural motif that consists of a leucine zipper that mediates dimerization and an adjacent basic region that directly contacts DNA. Although models of the protein-DNA complex have been proposed, the basis of DNA-binding specificity is essentially unknown. By genetically selecting for derivatives of yeast GCN4 that activate transcription from promoters containing mutant binding sites, we isolate an altered-specificity mutant in which the invariant asparagine in the basic region of bZIP proteins (Asn-235) has been changed to tryptophan. Wild-type GCN4 binds the optimal site (ATGACTCAT) with much higher affinity than the mutant site (TTGACTCAA), whereas the Trp-235 protein binds these sites with similar affinity. Moreover, the Trp-235, Ala-235, and Gln-235 derivatives differ from GCN4 in their strong discrimination against GTGACTCAC. These results suggest a direct interaction between Asn-235 and the +/- 4 position of the DNA target site and are discussed in terms of the scissors-grip and induced-fork models of bZIP proteins.

Uracil interference, a rapid and general method for defining protein-DNA interactions involving the 5-methyl group of thymines: the GCN4-DNA complex.

We describe a novel uracil interference method for examining protein contacts with the 5-methyl group of thymines. The protein of interest is incubated with target DNA containing randomly distributed deoxyuracil substitutions that is generated by carrying out the polymerase chain reaction in the presence of a mixture of TTP and dUTP. After separating DNA-protein complexes away from unbound DNA, the locations of deoxyuracil residues that either do or do not interfere with DNA-binding are determined by cleavage with uracil-N-glycosylase followed by piperidine. Using this uracil interference assay, we show that the methyl groups of the four core thymines, but not the two peripheral thymines, of the optimal binding site (ATG-ACTCAT) are important for high affinity binding of GCN4. Similar, but not identical, results are obtained using KMnO4 interference, another method used for studying protein-DNA interactions involving thymine residues. These observations strongly suggest that GCN4 directly contacts the 5-methyl groups of the four core thymines that lie in the major groove of the target DNA. Besides providing specific structural information about protein-DNA complexes, uracil interference should also be useful for identifying DNA-binding proteins and their target sites in eukaryotic promoter regions.

Highly conserved residues in the bZIP domain of yeast GCN4 are not essential for DNA binding.

Yeast GCN4 and the Jun oncoprotein are transcriptional activators that bind DNA via a bZIP domain consisting of a leucine zipper dimerization element and an adjacent basic region that directly contacts DNA. Two highly conserved alanines (Ala-238 and Ala-239 in GCN4) and an invariant asparagine (Asn-235) in the basic region have been proposed to play important roles in DNA sequence recognition by bZIP proteins. Surprisingly, these conserved residues can be functionally replaced in GCN4 and in a derivative containing the Jun basic region (Jun-GCN4). The ability of an amino acid to functionally substitute for Asn-235 does not correlate with its preference for assuming the N-cap position of an alpha helix. This finding argues against the proposal of the scissors grip model that the invariant asparagine forms an N cap that permits the basic region to bend sharply and wrap around the DNA. In contrast to a prediction of the induced fork model, the pattern of functional substitutions of the conserved alanines together with the results of uracil interference experiments suggests that Ala-238 and Ala-239 do not make base-specific DNA contacts. Finally, the Jun-GCN4 chimeric proteins appear much more active in vivo than expected from their DNA-binding properties in vitro. The mechanistic and evolutionary implications of these results are discussed.

The leucine zipper symmetrically positions the adjacent basic regions for specific DNA binding.

The bZIP structural motif present in several eukaryotic transcription factors is defined by the leucine zipper, a coiled-coil dimerization interface, and an adjacent basic region that directly interacts with DNA. To examine the functional importance of the highly conserved spacing between the leucine zipper and the basic region, we have analyzed the DNA-binding ability of yeast GCN4 proteins containing amino acid insertions between these two subdomains. Proteins containing a surprisingly wide variety of seven-amino acid insertions, but none containing two-, four-, or six-amino acid insertions, are functional. However, heterodimers between wild-type GCN4 and functional derivatives containing seven amino acid insertions are unable to bind DNA. These observations provide strong experimental support for several aspects of the scissors grip and induced fork models for DNA-binding by bZIP proteins. Specifically, they demonstrate that continuous alpha-helices symmetrically diverging from the leucine zipper correctly position the two basic regions for specific binding to abutting DNA half-sites. In addition, the results indicate that GCN4 homodimers are primarily responsible for transcriptional activation in yeast cells.

UvrABC incision of N-methylmitomycin A-DNA monoadducts and cross-links.

The Escherichia coli UvrABC endonuclease is a multisubunit enzyme that initiates the repair of a wide variety of DNA lesions in vivo by making dual incisions on a damaged strand at the eighth or ninth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified base. It has been hypothesized that UvrABC is able to recognize a broad spectrum of lesions because it does not recognize the lesion per se but rather gross helical distortions that the lesion induces in the DNA. Several lesions have recently been studied which are thermal stabilizing and are not believed to distort the DNA grossly, including the CC-1065-N-3-adenine and anthramycin-N-2-guanine adducts. We have studied the activity of UvrABC in vitro on another thermal stabilizing and nondistortive adduct, N-methylmitomycin A (NMA), a bifunctional DNA-alkylating agent that reacts with guanine on the side facing the minor groove, yielding either monoadducts or interstrand cross-links. NMA adducts increase the thermal stability of DNA, and theoretical calculations indicate that NMA adducts do not grossly distort the DNA helix. Our results show that UvrABC makes incisions at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to an NMA monoadduct, consistent with the incision pattern observed for the majority of other lesions that are also recognized by UvrABC. DNA containing a site-specific NMA cross-link was also recognized and incised by UvrABC. The rate of incision of NMA cross-linked DNA was about 200-fold higher in supercoiled molecules than in relaxed molecules, whereas the rate of incision of DNA containing NMA monoadducts was stimulated approximately 2-fold by supercoiling. The signal for UvrABC recognition and incision of damaged DNA is discussed in relation to the ability of UvrABC to incise NMA adducts as well as other nondistortive lesions.