Nuclear factor 1 interferes with Sp1 binding through a composite element on the rat poly(ADP-ribose) polymerase promoter to modulate its activity in vitro.

Poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the rapid and extensive poly(ADP-ribosyl)ation of nuclear proteins in response to DNA strand breaks, and its expression, although ubiquitous, is modulated from tissue to tissue and during cellular differentiation. PARP-1 gene promoters from human, rat, and mouse have been cloned, and they share a structure common to housekeeping genes, as they lack a functional TATA box and contain multiple GC boxes, which bind the transcriptional activator Sp1. We have previously shown that, although Sp1 is important for rat PARP1 (rPARP) promoter activity, its finely tuned modulation is likely dependent on other transcription factors that bind the rPARP proximal promoter in vitro. In this study, we identified one such factor as NF1-L, a rat liver isoform of the nuclear factor 1 family of transcription factors. The NF1-L site on the rPARP promoter overlaps one of the Sp1 binding sites previously identified, and we demonstrated that binding of both factors to this composite element is mutually exclusive. Furthermore, we provide evidence that NF1-L has no effect by itself on rPARP promoter activity, but rather down-regulates the Sp1 activity by interfering with its ability to bind the rPARP promoter in order to modulate transcription of the rPARP gene.

Improving sensitivity of the electrophoretic mobility shift assay by restricting tissue phosphatase activities.

Elucidating DNA-protein interactions at the molecular level is a prerequisite in understanding the way a transcriptionally active gene is regulated in various tissues. A number of techniques are presently available for this particular type of analyses, of which, the electrophoretic mobility shift assay (EMSA) is certainly the preferred one and likely the most sensitive and powerful. EMSA is widely used in combination with nuclear extracts obtained from cultured cells. However, its use with crude extracts prepared from whole animal tissues is still restricted. The restriction is partly due to numerous enzymatic activities, such as proteases and deacetylases, that strongly interfere with the EMSA's sensitivity. In addition to endogenous proteases, which can be inhibited by the addition of protease inhibitors to the buffers used, crude nuclear extracts obtained from whole animal tissues are often contaminated with considerable amounts of highly active endogenous phosphatases. Some of these enzymes substantially interfere with the ability of the EMSA to unravel specific DNA-protein interactions by removing the 5'-[32P] end-labeled phosphate of the DNA probe used for the binding assay. In this study, we evaluated whether we can restore the sensitivity of the EMSA by modifying the conditions under which the DNA-protein binding reaction is normally performed. Dephosphorylation by endogenous tissue phosphatases of the labeled probe used in our assays was drastically prevented by simply reducing both the temperature at which the binding reaction was normally performed and the time allowed for the DNA-protein interaction to occur.

A nuclear factor other than Sp1 binds the GC-rich promoter of the gene encoding rat poly(ADP-ribose) polymerase in vitro.

Poly(ADP-ribose) polymerase is a nuclear enzyme that has been shown to exert a key role in many important cellular functions, including DNA repair. Its activity was shown to vary substantially between tissues; the testis and the thymus expressed the highest levels of PARP whereas the liver and the kidney (as well as a few other tissues) expressed only low levels of PARP proteins in vivo. The GC-rich nature of its upstream gene promoter, along with the lack of TATA and CAAT boxes, a feature common to most housekeeping genes, is consistent with a major regulatory function played by the positive transcription factor Sp1 in rat PARP gene transcription. Sp1 was indeed recently shown to interact with five distinct GC or GT boxes present in the rat PARP promoter. However, the observation that PARP activity was lower in rat liver than in other tissues was shown not to be the result of reduced Sp1 activity in liver cells but rather suggests the interplay of nuclear proteins other than Sp1 that are required to restrict PARP expression in this organ and maybe in others (such as the kidney). In this study, we investigated this possibility further by defining whether other nuclear proteins might bind the PARP promoter to modulate its transcription in liver cells. As a result, we identified a nuclear factor distinct from Sp1 that binds the PARP promoter at a site overlapping the F2 Sp1 element previously identified. Our results suggest that this protein likely belongs to the CTF-NF1 family of transcription factors.

Transcriptional regulation of the rat poly(ADP-ribose) polymerase gene by Sp1.

Expression of the gene encoding poly(ADP-ribose) polymerase (PARP), although ubiquitous, nevertheless varies substantially between tissues. We have recently shown that Sp1 binds five distinct target sequences (US-1 and F1-F4) in the rat PARP (rPARP) gene promoter. Here we used deletion analyses and site-directed mutagenesis to address the regulatory function played by these Sp1 sites on the basal transcriptional activity directed by the rPARP promoter. Transfection experiments revealed that the most proximal Sp1 site is insufficient by itself to direct any promoter activity. In addition, a weak negative regulatory element was identified between positions -101 and -60. The rPARP promoter directed high levels of chloramphenicol acetyltransferase activity in Jurkat T-lymphoblastoid and Ltk- fibroblast cells but only moderate levels in pituitary GH4C1 and liver HTC cells, correlating with the amounts of PARP detected in these cells by western blot analysis. However, the reduced promoter efficiency in HTC and GH4C1 cells did not result from the lack of Sp1 activity in these cells but suggested that yet uncharacterized regulatory proteins might turn off PARP gene expression by binding negative regulatory elements from the rPARP promoter. Similarly, site-directed mutagenesis on the three most proximal Sp1 elements suggested the influence exerted by Sp1 on the rPARP promoter activity to vary substantially between cell types. It also provided evidence for a basal rPARP promoter activity driven through the recognition of unidentified cis-acting elements by transcription factors other than Sp1.

Guidelines for euthanasia of domestic animals by firearms.

All animals that are to be killed, whether for food, for humane reasons, or because they are homeless, must receive a quick and painless death. In some smaller communities, veterinary or humane society expertise may not be readily available to humanely kill stray and unwanted animals. An alternative that provides for a humane death for the animal is by shooting. The following guidelines are intended to assist persons who must perform this usually distasteful task; they contain recommended techniques that will help to ensure that any animals killed by shooting will die in a humane way.