Biomedical subjects

Anan Wang

Publications and source records attributed to Anan Wang.

4 recordsLinked to original sources

PubMed2005

A complementation method for functional analysis of mammalian genes.

Our progress in understanding mammalian gene function has lagged behind that of gene identification. New methods for mammalian gene functional analysis are needed to accelerate the process. In yeast, the powerful genetic shuffle system allows deletion of any chromosomal gene by homologous recombination and episomal expression of a mutant allele in the same cell. Here, we report a method for mammalian cells, which employs a helper-dependent adenoviral (HD-Ad) vector to synthesize small hairpin (sh) RNAs to knock-down the expression of an endogenous gene by targeting untranslated regions (UTRs). The vector simultaneously expresses an exogenous version of the same gene (wild-type or mutant allele) lacking the UTRs for functional analysis. We demonstrated the utility of the method by using PRPF3, which encodes the human RNA splicing factor Hprp3p. Recently, missense mutations in PRPF3 were found to cause autosomal-dominant Retinitis Pigmentosa, a form of genetic eye diseases affecting the retina. We knocked-down endogenous PRPF3 in multiple cell lines and rescued the phenotype (cell death) with exogenous PRPF3 cDNA, thereby creating a genetic complementation method. Because Ad vectors can efficiently transduce a wide variety of cell types, and many tissues in vivo, this method could have a wide application for gene function studies.

Adenoviridae↗

PubMed2005

Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference.

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-alpha, IL-1beta or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-kappaB, or on the protein levels of IkappaB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases.

Adenoviridae↗

PubMed2002

Central region of the human splicing factor Hprp3p interacts with Hprp4p.

Human splicing factors Hprp3p and Hprp4p are associated with the U4/U6 small nuclear ribonucleoprotein particle, which is essential for the assembly of an active spliceosome. Currently, little is known about the specific roles of these factors in splicing. In this study, we characterized the molecular interaction between Hprp3p and Hprp4p. Constructs were created for expression of Hprp3p or its mutants in bacterial or mammalian cells. We showed that antibodies against either Hprp3p or Hprp4p were able to pull-down the Hprp3p-Hprp4p complex formed in Escherichia coli lysates. By co-immunoprecipitation and isothermal titration calorimetry, we demonstrated that purified Hprp3p and its mutants containing the central region, but lacking either the N-terminal 194 amino acids or the C-terminal 240 amino acids, were able to interact with Hprp4p. Conversely, Hprp3p mutants containing only the N- or C-terminal region did not interact with Hprp4p. In addition, by co-immunoprecipitation, we showed that intact Hprp3p and its mutants containing the central region interacted with Hprp4p in HeLa cell nuclear extracts. Primer extension analysis illustrated that the central region of Hprp3p is required to maintain the association of Hprp3p-Hprp4p with U4/U6 small nuclear RNAs, suggesting that this Hprp3p/Hprp4p interaction allows the recruitment of Hprp4p, and perhaps other protein(s), to the U4/U6 small nuclear ribonucleoprotein particle.

Amino Acid Motifs↗

PubMed2002

Regulation of human Clara cell 10 kD protein expression by chicken ovalbumin upstream promoter transcription factors (COUP-TFs).

Clara cell 10 kD protein (CC10) is expressed specifically in a portion of nonciliated airway epithelial cells. The molecular mechanisms that determine its high specificity are not clear. Transcription factors implicated in the regulation of CC10 in rodents do not show the same level of cell specificity. We report here that a 3.3 kb human CC10 DNA fragment, containing the 5' flanking region and promoter, directs lacZ reporter expression in a small portion of Clara cells of the airway epithelia of transgenic mice, indicating the requirement of additional regulatory elements for expression. Addition of an intron containing a transcription enhancer from the human cytokeratin 18 gene greatly enhances the level of transgene expression and broadens epithelial specificity. To gain insight into the mechanisms underlying the cell specificity of human CC10 expression, we performed a promoter analysis of the CC10 gene and a yeast one-hybrid screening to identify factors that regulate the promoter. We have found that chicken ovalbumin upstream promoter transcription factors (COUP-TFs) interact with a proximal promoter region and confirmed the interaction by gel-shift assays. Cotransfection analyses with reporter constructs in cultured cells indicated that COUP-TFs inhibit human CC10 expression. These experiments suggest that COUP-TFs may play a pivotal role in cell specificity of the human CC10 gene by inhibiting its expression in nonpermissive cells.

Animals↗