Chemopreventive function of retinoid X receptors in human squamous cell carcinoma of the skin

Retinoid therapy has been successful for the treatment of skin squamous cell carcinoma (SCC). A suppression of the predominant retinoid X receptor expressed in skin, retinoid X receptor α (RXRα), has been reported in skin SCC. These observations have led to the hypothesis that retinoid receptor loss contributes to the tumorigenic phenotype of epithelial cancers. To test this hypothesis, the RXRα gene was mapped in order to generate a targeting construct. Additionally the transcriptional regulation of the human RXRα a gene in keratinocytes was characterized after identifying the transcription initiation sites, the promoter, and enhancer regions of this gene. The structure is highly conserved between human and mouse. A nontumorigenic human skin-derived cell line called near diploid immortalized keratinocytes (NIKS) has the advantage of growing as organotypic raft cultures, under physiological conditions closely resembling in-vivo squamous stratification. We have exploited the raft culture technique to develop an in-vitro model for skin SCC progression that includes the NIKS cells, HaCaT cells, a premalignant cell line, and SRB 12-p9 cells, a tumorigenic SCC skin cell line. The differentiation, proliferation and nuclear receptor ligand response characteristics of this system were studied and significant and novel results were obtained. RXRs are obligate heterodimerization partners with many of the nuclear hormone receptors, including retinoic acid receptors (RARs), vitamin D3 receptors (VDR), thyroid hormone receptors (T3 R) and peroxisome proliferator activate receptors (PPARs), which are all known to be active in skin. Treatment of the three cell lines in raft culture with the RXR specific ligand BMS649, BMS961 (RARγ-specific), vitamin D3 (VDR ligand), thryoid hormone (T3R ligand) and clofibrate (PPARa ligand), and the combination of BMS649 with each of the 4 receptor partner ligands, resulted in distinct effects on differentiation, proliferation and apoptosis. The effects of activation of RXRs in each of the four-receptor pathways; in the context of skin SCC progression, with an emphasis on the VDR/RXR pathway, are discussed. These studies will lead to a better understanding of RXRα action in human skin and will help determine its role in SCC tumorigenesis, as well as its potential as a target for the prevention, treatment, and control of skin cancer. ^

Roles of Alix in regulating cell morphology

Mammalian Alix (ALG2-interacting protein X&barbelow;) is a conserved adaptor protein that is involved in endosomal trafficking, apoptosis and growth factor receptor turnover. Accumulating evidence also indicates that Alix plays roles in promoting/maintaining spread and aligned fibroblast morphology in monolayer culture. Since cell morphology is determined by the structure and dynamics of an integrin-mediated transmembrane protein network that links extracellular matrix to intracellular cytoskeleton, we hypothesized that Alix plays direct or indirect roles in regulating certain components or steps in this transmembrane protein network. To test this hypothesis, we first examined the subcellular localization of Alix and discovered that, as a predominantly cytoplasmic protein, Alix is also present on the substratum/cell surface and in the conditioned medium of fibroblast cultures. Further, precoating of culture surfaces with recombinant Alix promotes spreading and fibronectin assembly to NIH/3T3 cells, and siRNA-mediated Alix knockdown in W138 cells has the opposite effects. These findings indicate the extracellular functions of Alix in regulating cell spreading and extracellular matrix assembly. In a separate study, we analyzed Alix immunocomplexes from normal fibroblast W138 cells by mass spectrometry and identified actin as a major partner protein of Alix. Follow-up studies demonstrated that Alix preferentially binds filamentous actin (F-actin) in vitro and is required for maintaining normal F-actin content and proper actin cytoskeleton assembly in W138 cells. These findings establish direct and essential roles of Alix in regulating actin cytoskeleton. Finally, we investigated the effects of Alix knockdown on the activation and subcellular localization of FAK and Pyk2, the focal adhesion kinases required for cell spreading/migration by promoting turnover of integrin-mediated cell adhesions. We discovered that Alix knockdown inhibits FAK and Pyk2 localizations to focal adhesions or plasma membrane, in association with characteristics of reduced turnover of focal adhesions. These findings reveal a positive role of Alix in focal adhesion turnover. Based on these results, we conclude that Alix targets both intracellularly and extracellularly components to regulate extracellular matrix remodeling, actin cytoskeleton assembly and focal adhesion turnover. A combination of these three functions of Alix explains its crucial role in regulating spread and aligned fibroblast morphology. ^

Mouse retinal development: Role of cell adhesion and mechanism of gene regulation

Cell differentiation and pattern formation are fundamental processes in animal development that are under intense investigation. The mouse retina is a good model to study these processes because it has seven distinct cell types, and three well-laminated nuclear layers that form during embryonic and postnatal life. β-catenin functions as both the nuclear effector for the canonical Wnt pathway and a cell adhesion molecule, and is required for the development of various organs. To study the function of β-catenin in retinal development, I used a Cre-loxP system to conditionally ablate β-catenin in the developing retina. Deletion of β-catenin led to disrupted laminar structure but did not affect the differentiation of any of the seven cell types. Eliminating β-catenin did not reduce progenitor cell proliferation, although enhanced apoptosis was observed. Further analysis showed that disruption of cell adhesion was the major cause of the observed patterning defects. Overexpression of β-catenin during retinal development also disrupted the normal retinal lamination and caused a transdifferentiation of neurons into pigmented cells. The results indicate that β-catenin functions as a cell adhesion molecule but not as a Wnt pathway component during retinal neurogenesis, and is essential for lamination but not cell differentiation. The results further imply that retinal lamination and cell differentiation are genetically separable processes. ^ Sonic hedgehog (shh) is expressed in retinal ganglion cells under the control of transcription factor Pou4f2 during retinal development. Previous studies identified a phylogenetically conserved region in the first intron of shh containing a Pou4f2 binding site. Transgenic reporter mice in which reporter gene expression was driven by this region showed that this element can direct gene expression specifically in the retina, but expression was not limited to the ganglion cells. From these data I hypothesized that this element is required for shh expression in the retina but is not sufficient for specific ganglion cell expression. To further test this hypothesis, I created a conditional allele by flanking this region with two loxP sites. Lines carrying this allele will be crossed with retinal-specific Cre lines to remove this element in the retina. My hypothesis predicts that alteration in shh expression and subsequent retinal defects will occur in the retinas of these mice. ^

Structure-function analysis of human Integrator subunit-4

Structure-function analysis of human Integrator subunit 4 Anupama Sataluri Advisor: Eric. J. Wagner, Ph.D. Uridine-rich small nuclear RNAs (U snRNA) are RNA Polymerase-II (RNAPII) transcripts that are ubiquitously expressed and are known to be essential for gene expression. snRNAs play a key role in mRNA splicing and in histone mRNA expression. Inaccurate snRNA biosynthesis can lead to diseases related to defective splicing and histone mRNA expression. Although the 3′ end formation mechanism and processing machinery of other RNAPII transcripts such as mRNA has been well studied, the mechanism of snRNA 3′ end processing has remained a mystery until the recent discovery of the machinery that mediates this process. In 2005, a complex of 14 subunits (the Integrator complex) associated with RNA Polymerase-II was discovered. The 14subunits were annotated Integrator 1-14 based on their size. The subunits of this complex together were found to facilitate 3′ end processing of snRNA. Identification of the Integrator complex propelled research in the direction of understanding the events of snRNA 3’end processing. Recent studies from our lab confirmed that Integrator subunit (IntS) 9 and 11 together perform the endonucleolytic cleavage of the nascent snRNA 3′ end to generate mature snRNA. However, the role of other members of the Integrator complex remains elusive. Current research in our lab is focused on deciphering the role of each subunit within the Integrator complex This work specifically focuses on elucidating the role of human Integrator subunit 4 (IntS4) and understanding how it facilitates the overall function of the complex. IntS4 has structural similarity with a protein called “Symplekin”, which is part of the mRNA 3’end processing machinery. Symplekin has been thoroughly researched in recent years and structure-function correlation studies in the context of mRNA 3’end processing have reported a scaffold function for Symplekin due to the presence of HEAT repeat motifs in its N-terminus. Based upon the structural similarity between IntS4 and Symplekin, we hypothesized that Integrator subunit 4 may be behaving as a Symplekin-like scaffold molecule that facilitates the interaction between other members of the Integrator Complex. To answer this question, the two important goals of this study were to: 1) identify the region of IntS4, which is important for snRNA 3′ end processing and 2) determine binding partners of IntS4 which promote its function as a scaffold. IntS4 structurally consists of a highly conserved N-terminus with 8 HEAT repeats, followed by a nonconserved C- terminus. A series of siRNA resistant N and C-terminus deletion constructs as well as specific point mutants within its N-terminal HEAT repeats were generated for human IntS4 and, utilizing a snRNA transcriptional readthrough GFP-reporter assay, we tested their ability to rescue misprocessing. This assay revealed a possible scaffold like property of IntS4. To probe IntS4 for interaction partners, we performed co-immunoprecipitation on nuclear extracts of IntS4 expressing stable cell lines and identified IntS3 and IntS5 among other Integrator subunits to be binding partners which facilitate the scaffold like function of hIntS4. These findings have established a critical role for IntS4 in snRNA 3′ end processing, identified that both its N and C termini are essential for its function, and mapped putative interaction domains with other Integrator subunits.

The effects of DNA cytosine methylation on H -ras promoter structure and function

The effect of DNA cytosine methylation on H-ras promoter activity was assessed using a transient expression system employing the plasmid H-rasCAT (NaeI H-ras promoter linked to the chloramphenicol acetyltransferase (CAT) gene). This 551 bp promoter is 80% GC rich, enriched with 168 CpG dinucleotides, and contains six functional GC box elements which represent major DNA methylation target sites. Prokaryotic methyltransferases HhaI (CGm$\sp5$CG) and HpaII (Cm$\sp5$CGG) alone or in combination with a human placental methyltransferase (HP MTase) were used to introduce methyl groups at different CpG sites within the promoter. To test for functional promoter activity, the methylated plasmids were introduced into CV-1 cells and CAT activity assessed 48 h post-transfection. Methylation at specific HhaI and HpaII sites reduced CAT expression by 70%, whereas more extensive methylation at generalized CpG sites with HP MTase inactivated the promoter $>$95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in nonpromoter regions. We also observed that DNA cytosine methylation of a 360 bp promoter fragment by HP MTase induced a local change in DNA conformation. Using three independent methodologies (nitrocellulose filter binding assays, gel mobility shifts, and Southwestern blots), we determined that this change in promoter conformation affected the interaction of nuclear proteins with cis-regulatory sequences residing in the promoter region. The results provide evidence to suggest that DNA methylation may regulate gene expression by inducing changes in local promoter conformation which in turn alters the interactions between DNA and protein factors required for transcription. The results provide supportive evidence for the hypothesis of Cedar and Riggs, who postulated that DNA methylation may regulate gene expression by altering the binding affinities of proteins for DNA. ^