Human apul'inic/apyrimidinic endonuclease l/redox effector factor-1 (APEl/Ref·l) is a perfect paradigm of the functional complexity of a biological macromolecule. First, it plays a crucial role, by both redox-dependent and -independent mechanisms, as a transcriptional coactivator for different trnnscription factors, either ubiquitous (i.e., AP-1, Egr-1, NF-KB, p53, HIF) or tissue-specific (i.e., PEBP-2, Pax-Sand -8, TTF-1), in controlling different cellular processes such as apoptosis, proliferation, and differentiation. Second, it acts, as an apurinidapyrimidinic endonuclease, during the second step of the DNA base excision repair pathway, which is responsible for the repair of cellular alkylation and oxidative DNA damages. Thil'd, it controls the intracellular reactive oxygen species production by negatively regulating the activity of the Ras-related GTPase Rael. Despite these known functions of APEl/Ref-1, information is still scanty about the molecular mechanisms responsible for the coordinated control of its several activities. Some evidence suggests that the expression and subcellular localization of APEl/Ref-1 are finely tuned. APEl/Ref-1 is a ubiquitous protein, but its expression pattern differs according to the different cell types. APEl/Ref-1 subcellular localization is mainly nuclear, but cytop1asmic staining has also been reported, the latter being associated with mitochondria and/or presence within the endoplasmic reticulum. It is not by chance that both expression and sub cellular localization a1·e altered in several metabolic and proliferative disorde1·s, such as in tumors and aging. Moreover, a fundamental role played by different posttranslational modifications in modulating APEl/Ref·l functional activity is becoming eYident. In the present review, we tded to put togethe1· a growing body of information concerning APEl/Ref-l's different functions, shedding new light on present and future directions to understand fully this unique molecule. Antioxid. Redox Signal. 7,[367][368][369][370][371][372][373][374][375][376][377][378][379][380][381][382][383][384]

The cDNA for TTF‐1, a thyroid nuclear factor that binds to the promoter of thyroid specific genes, has been cloned. The protein encoded by the cDNA shows binding properties indistinguishable from those of TTF‐1 present in nuclear extracts of differentiated rat thyroid cells. The DNA binding domain of TTF‐1 is a novel mammalian homeodomain that shows considerable sequence homology to the Drosophila NK‐2 homeodomain. TTF‐1 mRNA and corresponding binding activity are detected in thyroid and lung. The chromosomal localization of the TTF‐1 gene has been determined in humans and mice and corresponds to chromosomes 14 and 12, respectively, demonstrating that the TTF‐1 gene is not located within previously described clusters of homeobox‐containing genes.

To explore the effect of fluctuating glucose on endothelial cells, human umbilical vein endothelial cells were incubated for 14 days in media containing different glucose concentrations: 5 mmol/l, 20 mmol/l, or a daily alternating 5 or 20 mmol/l glucose. Apoptosis was studied by different methods: viability assay, cell cycle analysis, DNA fragmentation, and morphological analysis. Furthermore, the levels of Bcl-2 and Bax, well known proteins involved in apoptosis, were evaluated. Stable high glucose induced apoptosis in human umbilical vein endothelial cells, a phenomenon accompanied by a significant decrease of Bcl-2 and a simultaneous increase of Bax expression. However, apoptosis was enhanced in human umbilical vein endothelial cells exposed to intermittent, rather than constant, high glucose concentration. In this condition, Bcl-2 was not detectable, whereas Bax expression was significantly enhanced. These findings suggest that variability in glycemic control could be more deleterious to endothelial cells than a constant high concentration of glucose.

APE1/Ref-1 protects cells from oxidative stress by acting as a central enzyme in base excision repair pathways of DNA lesions and through its independent activity as a redox transcriptional co-activator. Dysregulation of this protein has been associated to cancer development. At present, contrasting data have been published regarding the biological relevance of the two functions as well as the molecular mechanisms involved. Here, we combined both mRNA expression profiling and proteomic analysis to determine the molecular changes associated with APE1 loss-of-expression induced by siRNA technology. This approach identified a role of APE1 in cell growth, apoptosis, intracellular redox state, mitochondrial function and cytoskeletal structure. Thus, overall, our data show that APE1 acts as a hub in coordinating different and vital functions in mammalian cells, highlighting the molecular determinants of the multifunctional nature of APE1 protein.

The strictly regulated expression of most pleiotropic developmental control genes is critically dependent on the activity of long-range cis-regulatory elements. This was revealed by the identification of individuals with a genetic condition lacking coding-region mutations in the gene commonly associated with the disease but having a variety of nearby chromosomal abnormalities, collectively described as cis-ruption disease cases. The congenital eye malformation aniridia is caused by haploinsufficiency of the developmental regulator PAX6. We discovered a de novo point mutation in an ultraconserved cis-element located 150 kb downstream from PAX6 in an affected individual with intact coding region and chromosomal locus. The element SIMO acts as a strong enhancer in developing ocular structures. The mutation disrupts an autoregulatory PAX6 binding site, causing loss of enhancer activity, resulting in defective maintenance of PAX6 expression. These findings reveal a distinct regulatory mechanism for genetic disease by disruption of an autoregulatory feedback loop critical for maintenance of gene expression through development.

Gene duplication is a major driver of evolutionary divergence. In most vertebrates a single PAX6 gene encodes a transcription factor required for eye, brain, olfactory system, and pancreas development. In zebrafish, following a postulated whole-genome duplication event in an ancestral teleost, duplicates pax6a and pax6b jointly fulfill these roles. Mapping of the homozygously viable eye mutant sunrise identified a homeodomain missense change in pax6b, leading to loss of target binding. The mild phenotype emphasizes role-sharing between the co-orthologues. Meticulous mapping of isolated BACs identified perturbed synteny relationships around the duplicates. This highlights the functional conservation of pax6 downstream (3′) control sequences, which in most vertebrates reside within the introns of a ubiquitously expressed neighbour gene, ELP4, whose pax6a-linked exons have been lost in zebrafish. Reporter transgenic studies in both mouse and zebrafish, combined with analysis of vertebrate sequence conservation, reveal loss and retention of specific cis-regulatory elements, correlating strongly with the diverged expression of co-orthologues, and providing clear evidence for evolution by subfunctionalization.

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The thyroid transcription factor 1 (TTF-1) is a homeodomain-containing protein implicated in the activation of thyroid-specific gene expression. Here we report that TTF-1 is capable of activating transcription from thyroglobulin and, to a lesser extent, thyroperoxidase gene promoters in nonthyroid cells. Full transcriptional activation of the thyroglobulin promoter by TTF-1 requires the presence of at least two TTF-1 binding sites. TTF-1 activates transcription via two functionally redundant transcriptional activation domains that as suggested by competition experiments, could use a common intermediary factor.