Hox as well as Meis proteins are known to bind DNA as heterodimers with members of the Pbx family, and it is believed that such complexes mediate the in vivo functions of Hox and Meis. To begin exploring the role of hoxb1b and meis3 in vertebrate development, we isolated and characterized a zebrafish pbx cDNA which encodes a novel member of the pbx family, which we called pbx4. In situ analysis revealed that pbx4 RNA is maternally deposited and is detected throughout the zebrafish embryo during blastula stages. It becomes excluded from ventroanterior structures at late gastrula stages and is detected within the developing central nervous system during segmentation stages. pbx4 expression overlaps with that of hoxb1b and meis3, in the region of the presumptive caudal hindbrain during gastrula stages. In vitro binding experiments revealed that Pbx4/Meis3 and Pbx4/Hoxb1b, as well as a novel trimeric complex containing Pbx4, Meis3 and Hoxb1b form in vitro. Thus, protein complexes of different combinations of Pbx4, Meis3 and Hoxb1b form in vitro and importantly pbx4, meis3 and hoxb1b are coexpressed in a domain at the level of the presumptive caudal hindbrain during zebrafish gastrula stages. These findings raised the possibility that similar complexes may exist in vivo and may be involved in the specification of distinct developmental fates. To address this possibility we overexpressed meis3,pbx4 and hoxb1b in zebrafish embryos and we tested for the effect on endogenous gene expression, morphology and neuronal specification. Our results demonstrate that Hoxb1b/Pbx4/Meis3-containing complexes induce extensive expression of several hindbrain genes (hoxb1a, hoxb2, krox20 and valentino) anterior to their normal expression domains, and mediate the transformation of anterior (forebrain and midbrain) fates to posterior (hindbrain) ones, including the formation of excess ectopic Mauthner neurons. Ectopic expression of Hoxb1b/Pbx4/Meis3-containing complexes also leads to truncation of the embryonic axis anteriorly. In contrast, Hoxb1b/Pbx4 expression induces ectopic expression of only hoxb1a (primarily in r2), but does not mediate axial truncations, and Hoxb1b (or its mouse homolog, HoxA1) has been reported to induce an ectopic pair of Mauthner neurons in r2 (Alexandre et al., 1996). Thus, binding of Meis3 to Hoxb1b/Pbx4 generates Hoxb1b/Pbx4/Meis3-containing complexes that have qualitatively (e.g. induction of hoxb2 expression) and quantitatively (e.g. larger number of ectopic Mauthner neurons) different effects than Hoxb1b/Pbx4-containing complexes. These results suggest that Meis3/Pbx4/Hoxb1b-containing complexes may be responsible for specification of hindbrain fates in vivo. In addition to meis3, three other members of the meis/prep gene family are expressed during early embryogenesis in zebrafish. Analysis of gene expression patterns revealed both common as well as unique spatial and temporal expression patterns for each of these genes. This finding raises the question of whether all family members are functionally similar to meis3 or meis3 performs unique functions. To address this question we overexpressed meis1.1, meis2.2 and prep1 in zebrafish embryos and we asked whether they are able to induce hindbrain fates like meis3 does. Overexpression of any Meis protein, or Prep, along with Pbx4 and Hoxb1b resulted in embryos that were truncated anteriorly and exhibited massive ectopic hoxb1a and hoxb2 expression anterior to their normal expression domains. Furthermore, in vitro analysis demonstrated that they are all able to form dimers with Pbx4 in vitro. In addition, analysis of their subcellular localization defined Pbx4 interaction as a prerequisite for nuclear localization of all Meis and Prep proteins. Thus, at least in the overexpression assay there are no functional differences among meis/prep genes. These results raise the question of what is exactly the function of Meis/Prep proteins. Is binding to Pbx proteins and to DNA their only function, or do they have additional roles? To address this question we performed a deletional analysis of Meis3 protein and we tested the requirement of each domain in the overexpression assay. Our experiments revealed that the domain N-terminal to the Pbx-Interaction-Domain (PID) as well as the domain C-terminal to the Homeodomain are not required for the function of Meis3, at least in the overexpression assay. Furthermore, the homeodomain and the domain between the PID and the homeodomain are not required. From our previous analysis (Vlachakis et al., 2001) we know that the PID is required for the Meis3/Pbx4/Hoxb1b synergistic induction of hindbrain fates. Our deletion analysis extended this fmding showing that the PID is also sufficient to provide the Meis3 function in vivo, at least in our overexpression assay. Furthermore, a mutant PID that does not bind Pbx, when fused to the Pbx4 homeodomain induced hindbrain fates upon overexpression along with Hoxb1b. This finding suggests that the PID (motifs M1, M2 and the domain in between them, ID) besides binding to Pbx may also bind another protein that is required for the Meis3/Pbx4/Hoxb1b synergistic induction of hindbrain fates. Taking all our results together, we propose the following roles for Meis proteins in the transcriptional activation complexes. First, they are involved in the nuclear localization of Pbx4. Second, they bind to DNA as heterodimers with Pbx4 facilitating binding of Hoxb1b to Pbx4, which occurs only on DNA. In doing so, they provide the specificity for DNA binding since the Meis3/Pbx4 dimer first recognizes the "hox response element" and then the Hox protein is recruited. Third, they stabilize the binding of Hoxb1/Pbx4 complex on DNA. Fourth, they are responsible for recruiting additional factors to DNA, necessary for activation of target genes. The complicate and dynamic spatial and temporal expression patterns of the meis/prep genes, suggest that they are involved in many different processes during embryogenesis, as well as in the adult organism. We believe that one or more members of the Meis/Prep family execute some of the functions listed above at different times and places during development, although all member are probably capable of executing all these functions.

No description available.

Stem cell biology is an exciting field that will lead to significant advancements in science and medicine. We hypothesize that inducing the expression of stem cell genes, using the embryonic stem cell gene nanog, will reprogram cells and dedifferentiate human mesenchymal stem cells into pluripotent stem cells capable of neural differentiation. The aims of initial studies are as follows: Aim 1: Demonstrate that forced expression of the embryonic stem cell gene nanog induces changes in human mesenchymal stem cells to an embryonic stem cell-like phenotype. Aim 2: Demonstrate that induced expression of nanog up-regulates the expression of multiple embryonic stem cell markers and expands the differentiation potential of the stem cells. Aim 3: Demonstrate that these nanog-expressing stem cells have the ability to differentiate along neural lineages in vitro and in vivo, while mock-transfected cells have an extremely limited capacity for transdifferentiation. Alternatively, we hypothesize that embryonic stem cell genes can become activated in malignant gliomas and differentially regulate the subpopulation of cancer stem cells. This study examines the role of embryonic stem cell genes in transformed cells, particularly cancer stem cells. These studies explore has the following objectives: Aim 1: Isolate different sub-populations of cells from tumors and characterize cells with stem cell-like properties. Aim 2: Characterize the expression of embryonic stem cell markers in the sub-population of cancer stem cells. Aim 3: Examine the effects of histone deacetylase inhibitors at inhibiting the growth and reducing the expression of stem cell markers. Our research has demonstrated the potential of the embryonic transcription factor, nanog, at inducing dedifferentiation of human bone marrow mesenchymal stem cells and allowing their recommitment to a neural lineage. Specifically, we used viral and non-viral vectors to induce expression of NANOG, which produced an embryonic stem cell-like morphology in transduced cells. We characterized these cells using real-time PCR and immunohistochemical staining and find an up-regulation of genes responsible for pluripotency and self-renewal. Embryonic stem cell markers including Sox2, Oct4 and TERT were up-regulated following delivery of nanog. The role of nanog in the expression of these markers was further demonstrated in our induced-differentiation method where we transfected embryonic stem cell-like cells, that have been transduced with nanog flanked by two loxP sites, with a vector containing Cre-recominase. We tested the ability of these nanog-transfected cells to undergo neural differentiation in vitro using a neural co-culture system or in vivo following intracranial transplantation. Our next study characterized patient-derived glioblastoma cancer stem cells. We found that cells isolated from serum-free stem cell cultures were enriched for stem cell markers and were more proliferative than the bulk population of cells grown in convention serum-supplemented media. These cancer stem cells expressed embryonic stem cell markers NANOG and OCT4 whereas non-tumor-derived neural stem cells do not. Moreover, the expression of stem cell markers was correlated with enhanced proliferation and could serve as a measure of drug effectiveness. We tested two different histone deacetylase inhibitors, trichostatin A and valproic acid, and found that both inhibited proliferation and significantly reduced expression of stem cell markers in our cancer stem cell lines. These data demonstrate the potential use of stem cell genes as therapeutic markers and supports the hypothesis that cancer stem cells are a major contributor to brain tumor malignancy.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 61-63).

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 13, 2009). Includes bibliographical references.

Thesis (Ph.D. in Biophysics & Genetics, Program in Molecular Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 115-132). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 196-214.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008.

From the yeast genome completed in 1996 to the 12 Drosophilagenomes published earlier this year; little more than a decade has provided an incredible amount of genomic data. Yet even with this mountain of genetic information the regulatory networks that control gene expression remain relatively undefined. In part, this is due to the enormous amount of non-coding DNA, over 98% of the human genome, which needs to be made sense of. It is also due to the large number of transcription factors, potentially 2,000 such factors in the human genome, which may contribute to any given network directly or indirectly. Certainly, one of the central limitations has been the paucity of transcription factor (TF) specificity data that would aid in the prediction of regulatory targets throughout a genome. The general lack of specificity data has hindered the prediction of regulatory targets for individual TFs as well as groups of factors that function within a common regulatory pathway. A large collection of factor specificities would allow for the combinatorial prediction of regulatory targets that considers all factors actively expressed in a given cell, under a given condition. Herein we describe substantial improvements to a previous bacterial one-hybrid system with increased sensitivity and dynamic range that make it amenable for the high-throughput analysis of sequence-specific TFs. Currently we have characterized 108 (14.3%) of the predicted TFs in Drosophilathat fall into a broad range of DNA-binding domain families, demonstrating the feasibility of characterizing a large number of TFs using this technology. To fully exploit our large database of binding specificities, we have created a GBrowse-based search tool that allows an end-user to examine the overrepresentation of binding sites for any number of individual factors as well as combinations of these factors in up to six Drosophila genomes (veda.cs.uiuc.edu/cgi-bin/gbrowse/gbrowse/Dmel4). We have used this tool to demonstrate that a collection of factor specificities within a common pathway will successfully predict previously validated cis-regulatory modules within a genome. Furthermore, within our database we provide a complete catalog of DNA-binding specificities for all 84 homeodomains in Drosophila. This catalog enabled us to propose and test a detailed set of recognition rules for homeodomains and use this information to predict the specificities of the majority of homeodomains in the human genome.

Hox genes encode a conserved family of homeodomain containing transcription factors essential for metazoan development. The establishment of overlapping Hox expression domains specifies tissue identities along the anterior-posterior axis during early embryogenesis and is regulated by chromatin architecture and retinoic acid (RA). Here we present the role nucleosome positioning plays in hox activation during embryogenesis. Using four stages of early embryo development, we map nucleosome positions at 37 zebrafish hox promoters. We find nucleosome arrangement to be progressive, taking place over several stages independent of RA. This progressive change in nucleosome arrangement on invariant sequence suggests that trans-factors play an important role in organizing nucleosomes. To further test the role of trans-factors, we created hoxb1b and hoxb1a mutants to determine if the loss of either protein effected nucleosome positions at the promoter of a known target, hoxb1a. Characterization of these mutations identified hindbrain segmentation defects similar to targeted deletions of mouse orthologs Hoxa1 and Hoxb1 and zebrafish hoxb1b and hoxb1a morpholino (MO) loss-of-function experiments. However, we also identified differences in hindbrain segmentation as well as phenotypes in facial motor neuron migration and reticulospinal neuron formation not previously observed in the MO experiments. Finally, we find that nucleosomes at the hoxb1a promoter are positioned differently in hoxb1b-/- embryos compared to wild-type. Together, our data provides new insight into the roles of hoxb1b and hoxb1a in zebrafish hindbrain segmentation and reticulospinal neuron formation and indicates that nucleosome positioning at hox promoters is dynamic, depending on sequence specific factors such as Hox proteins.