A large targeted deletion of Hoxb1-Hoxb9 produces a series of single-segment anterior homeotic transformations.

Hox genes regulate axial regional specification during animal embryonic development and are grouped into four clusters. The mouse HoxB cluster contains 10 genes, Hoxb1 to Hoxb9 and Hoxb13, which are transcribed in the same direction. We have generated a mouse strain with a targeted 90-kb deletion within the HoxB cluster from Hoxb1 to Hoxb9. Surprisingly, heterozygous mice show no detectable abnormalities. Homozygous mutant embryos survive to term and exhibit an ordered series of one-segment anterior homeotic transformations along the cervical and thoracic vertebral column and defects in sternum morphogenesis. Neurofilament staining indicates abnormalities in the IXth cranial nerve. Notably, simultaneous deletion of Hoxb1 to Hoxb9 resulted in the sum of phenotypes of single HoxB gene mutants. Although a higher penetrance is observed, no synergistic or new phenotypes were observed, except for the loss of ventral curvature at the cervicothoracic boundary of the vertebral column. Although Hoxb13, the most 5' gene, is separated from the rest by 70 kb, it has been suggested to be expressed with temporal and spatial colinearity. Here, we show that the expression pattern of Hoxb13 is not affected by the targeted deletion of the other 9 genes. Thus, Hoxb13 expression seems to be independent of the deleted region, suggesting that its expression pattern could be achieved independent of the colinear pattern of the cluster or by a regulatory element located 5' of Hoxb9.

The human insulin gene contains multiple transcriptional elements that respond to glucocorticoids.

Glucocorticoids inhibit insulin expression in cultured pancreatic islet cells. In this study, we provide evidence that transcriptional downregulation of insulin gene expression by glucocorticoids is the result of synergistic interaction between various elements of the insulin promoter. Similar synergistic effects on insulin gene transcription were previously reported for other key insulin regulators, cyclic adenosine monophosphate (cAMP) and glucose. Transfection of CAT constructs containing different segments of the insulin promoter into the pancreatic cell line, HIT T-15 2.2.2, demonstrated that dexamethasone decreased CAT activity in all constructs tested. However, differences were found in the relative sensitivities of the various constructs. Glucocorticoid inhibition of expression from plasmids containing A elements may result from decreased expression of the pancreatic homeodomain protein STF-1. However, a different mechanism must be invoked for insulin promoter constructs lacking A sites, which nevertheless still demonstrated negative regulation. Glucocorticoid-induced inhibition of one of these regions (-882 to -342) was seen to require pancreas-specific factors, because inhibition was observed in HIT T-15 2.2.2 cells but not in the nonpancreatic COS-1 cells. We conclude, therefore, that the human insulin gene contains multiple transcriptional elements that respond to glucocorticoids, some of which require beta cell-specific factors.

Ha-ras oncogene-induced transcription of human papillomavirus type 18 E6 and E7 oncogenes.

Human papillomavirus (HPV) DNA sequences are found in most carcinomas originating from the uterine cervix. HPV E6 and E7 oncogenes have been shown to cooperate with ras oncogenes to fully transform human epithelial cells. We investigated the effect of the Ha-ras oncogene on the transcriptional activity of HPV-18 and found that it induced the transcriptional activity of the viral promoter, whereas the normal gene had only a minimal effect. However, transfection of the normal Ha-ras gene and simultaneous inhibition of protein phosphatase sensitive to okadaic acid (OA) resulted in a cooperative transactivation of the viral promoter. When cloned upstream of a minimal promoter, the AP-1 binding sites present in the viral promoter conferred transcriptional responsiveness to Ha-ras and OA. Furthermore, HeLa cell clones permanently expressing the Ha-ras oncogene or high levels of the normal gene exhibited a twofold to threefold increase in E6*E7/E1 and E6*E7 transcripts. We propose that both Ha-ras and a protein phosphatase sensitive to OA regulate HPV oncogene expression through modulation of AP-1 activity and suggest that increased levels of E6 and E7, resulting from activated viral transcription in the presence of ras oncogenes, may in part explain the observed cooperation between these viral and cellular oncogenes in the transformation of human cells.

Identification of the human insulin negative regulatory element as a negative glucocorticoid response element.

Insulin gene transcription in adults is restricted to pancreatic beta cells. Studies with both transgenic mice and islet cell lines have demonstrated that beta cell specific expression is conferred by the 5' flanking region of the insulin gene. Transfection analysis has shown that cell specific expression involved an interaction between both positive and negative promoter cis elements. An upstream region (between -258 and -279) of the human insulin promoter served as a site of negative regulation. Transfection analysis in the pancreatic cell line HIT T-15 M 2.2.2 revealed that a DNA fragment containing this region causes a 45% reduction in promoter activity when linked to the native insulin promoter and a 72% reduction when linked to a heterologous tk promoter. Electrophoretic mobility shift analysis of this negative regulatory region (NRE) reveals a complex pattern of binding, wherein two major and several minor complexes are observed. Competition experiments demonstrated that formation of the fastest mobility complex is completely inhibited with excess cold glucocorticoid responsive element (GRE) consensus oligonucleotide. Purified glucocorticoid receptor binding domain (T7X556) demonstrated binding to the NRE oligonucleotide. Functional studies showed that dexamethasone treatment of HIT T-15 M 2.2.2 cells containing an NRE-tk CAT plasmid decreased CAT gene expression by 48%. Analysis of the NRE revealed 73% homology with the negative GRE consensus sequence. These data show that the human insulin NRE is a negative glucocorticoid response element.

A single element mediates glucocorticoid hormone response of HPV18 with no functional interactions with AP1 or hbrm.

We determined that the human papillomavirus type 18 (HPV18) regulatory region contains one functional GRE sequence that interacts with the glucocorticoid receptor. This sequence conferred a moderate hormonal activation to the HPV18 P105 promoter. Two modulators of glucocorticoid hormone activity, AP1 and hbrm, both involved in P105 transcription, were found not to interfere with this hormonal activation.

Hepatocyte homologous beta 2-adrenergic desensitization is associated with a decrease in number of plasma membrane beta 2-adrenoceptors.

Preincubation of rat hepatocytes with isoproterenol induces homologous beta-adrenergic desensitization evidenced both in whole cells (cyclic AMP accumulation) and membranes (adenylyl cyclase activity). This desensitization is associated with and quantitatively similar to a loss of beta 2-adrenoceptors from the plasma membrane. Desensitization did not alter the affinities of isoproterenol for the [125I]iodocyanopindolol binding sites nor reduce the ability of guanine nucleotides to modulate agonist affinity, i.e., the receptors that remain in the surface of plasma membrane after desensitization (approximately 50%) retain their functional integrity. When membranes from isoproterenol-desensitized hepatocytes were treated with alkaline phosphatase, no attenuation of the desensitization was observed. Cholera toxin-catalyzed ADP-ribosylation was not decreased but rather slightly increased in membranes from desensitized cells as compared to the controls. Our data indicate that in hepatocytes, a loss of beta 2-adrenoceptors from the plasma membrane is closely associated to the homologous desensitization induced by isoproterenol.