1994 — 1996 |
Tucker, Philip W. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Nucleic Acid Studies of Antiarsonate Hybridomas @ University of Texas Austin
The original goals of this project concerned the expression of anti-arsonate hybridomas. In particular, we hoped to understand something of the germline contribution to the anti-arsonate immune response. These goals have been largely met. During the past five years, this grant also funded work concerning transcription initiation through the VH-promoter-associated octamer motif. The VH genes of the arsonate system as well as other murine VH and VL genes were used to gain insight into the factors that regulated immunoglobulin gene expression. The present application is wholly directed toward this latter issue and is divided into three aims: First, we seek to continue our studies on the "conventional" ubiquitous octamer binding protein, OTF1, which promotes B-cell stage-specific transcription in lieu of OTF2. Aims 2 and 3 focus on two types of "unconventional" octamer-binding proteins (i.e., lack the prototypic POU domain of OTF1, OTF2 and related factors). One of these, P3, represents a new class of DNA-binding proteins and shares striking homology with a Drosophila protein involved in visual processing. The other is the well-known Ku antigen, a DNA-associated nuclear protein recognized by sera from patients with certain autoimmune diseases. We propose studies of both a molecular biological as well as a biochemical nature that should provide insight into the mechanisms of promoter region function in the murine immune and nervous systems.
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1994 — 1996 |
Tucker, Philip W. [⬀] |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
H Chain Gene Organization in Transformed B-Lymphocytes @ University of Texas Austin |
0.904 |
1995 — 1998 |
Tucker, Philip W. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulated Expression of T Cell Gamma and Delta Chains @ University of Texas Austin
In years 1-4 of this grant we studied both structure-function and gene regulation of gamma delta TcR. Our specific focus was the V gamma 3/V gamma 1 receptor expressed in the first fetal wave and on the majority of adult dendritic epidermal T cells. This renewal addresses the hypothesis that selective gamma delta expression derives from a developmental program for ordered V(D)J recombination mediated by factors that affect transcription from specific V gamma/V delta promoter- associated sequences. Experiments to test this model are grouped into two aims. First, we hope to better understand the rules of enhancer-dependent and independent V gamma 3/V delta 1 transcription. Next, we hope to use this information to develop direct links between germline transcription and V(D)J recombination. We feel that putative V-associated silencer sequences are central to the timing of both events. We have attempted to design hypothesis-driven experiments in cultured cells and transgenic mice that segregate the roles played by transacting factors, transcription per se, and chromatin state.
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1997 — 2001 |
Tucker, Philip W. [⬀] |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Heavy Chain Gene Organization in Transformed B Lymphocyt @ University of Texas Austin
The goal of this grant and its three predecessors is to understand molecular events related to Ig heavy chain gene expression. Alternative 3' end selection is the rate-limiting step for the biosynthesis of mu and delta mRNAs and there membrane (m) and secreted (s) forms. In the last period, we showed that the geometry of the Cmu-Cdelta transcription unit and the spacing strength, and downstream sequences associated with mu s and mu m poly(A) sites were major contributors. Consequent to this regulation and critical to subsequent transmembrane signaling, expression of the mIg antigen receptor is achieved. Through mutagenesis of antigen-specific transfectants we have defined differential roles for the mIg carboxyl-terminus in this process. Aim 1 seeks to continue studies on mu s/mu m regulation and on the function of mIg as an antigen receptor in immediate, late and differentiative events. Aim 2 intends to characterize two categories of B cell-specific proteins, potentially regulatory for the membrane-secretory developmental shift. These are a VH-promoter binding factor induced by lymphokines or LPS, and a family of putative DNA-binding proteins assessed only at the secretory B cell stage. We recently discovered that the JH-Cmu intronic enhancer (Emu) contains an origin of DNA replication preferentially active in B cells. Aim 3 will further define the sequences involved and how Emu may coordinate transcription and replication timing.
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1997 — 2000 |
Tucker, Philip W. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mar Mediated Genetic Switch For Igh Enhancer Control @ University of Texas Austin
DESCRIPTION (Adapted from the Investigator's Abstract): Nuclear matrix associating regions (MARs) have been implicated in transcriptional control of several genes, including the immunoglobulin heavy chain (IgH). The MARs flanking the IgH intronic enhancer (Em) are the targets of positive and negative regulators identified by this investigator. Both factors partition to the nuclear matrix in cell-type, developmental stage-restricted fashions: The positive regulator in mature/Ig-secreting B cells and the negative regulator in non-B cells. The differential occupancy of these two factors is hypothesized to contribute to enhancer control, perhaps by regulating nuclear matrix attachment and/or chromatin structure. In this model the MARs function as a genetic switch, contributing to Em activation in B cells and Em repression in non-B cells. Through a series of biochemical and genetic approaches the investigator proposes to further characterize these factors and their MAR interactions, to study the mechanism of the proposed switch, and to understand the consequences upon B cell lineage development and differentiation. In Aim 1 the structure, function and expression of Bright will be further characterized. In Aim 2 a similar analysis will be performed on the Cux and 40kd proteins. In Aim 3 the mechanism of the genetic switch involving these factors, and the role of MARs in this process will be investigated. In Aim 4 the consequences of the genetic switch on Emu cell type restriction and B lineage development will be investigated.
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2001 — 2005 |
Tucker, Philip W. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Bcl11 Genes in Normal and Malignant B Cell Development @ University of Texas Austin
DESCRIPTION (provided by applicant): Molecular cloning of chromosomal translocations targeted to the immunoglobulin (IG) loci allows the identification of genes of importance in the genesis of normal and malignant B-cells. We have cloned a highly conserved zinc finger gene locus BCL11A, from a chromosomal translocation, t(2;14)(p13;q32.3), that occurs as the sole cytogenetic abnormality in rare and clinically aggressive subset of CLL. All breakpoints involved IG gamma switch regions and clustered 5' of a CpG island associated with BCL11A. BCL11A maps closely telomeric to REL and also appears to be a target gene for amplifications and gains of 2p13 observed frequently in Hodgkin's disease and in extranodal B-NHL. Together the data implicate deregulated expression of BCL1 1A in the pathogenesis of divergent subtypes of aggressive human cancers. There are three common BCL11A isoforms; each is a transcriptional repressor and varies in the number of zinc fingers. BCL11A interacts physically with and shares several similarities with BCL6, a gene frequently translocated to both IG and non-IG associated sites. BCL1 1A shares high identity with a human family member, BCL11B, on chromosome 14q32.1 and with homologues across metazoan evolution. We propose to study the clinical significance of deregulation BCL11 expression in malignancies with abnormalities of chromosome 2p13 and to determine the function of BCL11 in normal and malignant B-cell development Specific approaches include screening for additional (BCL1 1A) and initial (BCL11B) cases containing breaks/amplifications a these loci; functional analysis of transcriptional mechanisms an downstream targets; assessing transforming activities using in vitro and transgenic models; and inactivating the gene by targeted disruption.
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2002 — 2011 |
Tucker, Philip W. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mar Mediated Control of the Igh Enhancer @ University of Texas Austin
The analysis of matrix attachment regions (MARs) associated with the lymphocyte receptors has provided a link between nuclear matrix attachment and a number of activities that regulate gene expression. We identified a nuclear matrix- associated, B cell-restricted regulator of IgH transcription, Bright. Bright binds as a tetramer to MARs necessary for matrix attachment of the IgH intronic enhancer (Emu) and the VHS107 promoter. These interactions lead to DNA bending, enhanced chromatin accessibility, and transcriptional activation. As with the nuclear matrix, transcription take place in defined subnuclear topological domains. We have identified and characterized several heteromeric interactions of Bright that provide a link between one such domain, the PML nuclear body (NB), matrix attachment and transcription of the IgH locus. We propose to extend our work by (1) further characterizing the structure, function, and expression of Bright; (2) determining the functional consequences for interactions of Bright with PML NB proteins that lead to alteration of its activity and localization; and (3) investigating the mechanism by which Bright contributes to chromatin remodeling.
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0.904 |
2004 — 2005 |
Tucker, Philip W. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Interaction of Cis-Acting Elements in Cd8 Regulation @ University of Texas Austin
DESCRIPTION (Applicant's Abstract): The regulation of CD8 expression during the differentiation of CD4-CD8+ (CD8SP) and intestinal intraepithelial lymphocytes (IELs) is still poorly understood. Results from laboratories using transgenic approaches have described an enhancer (called E81) approximately 16 kb upstream of the CD8a gene that drives reporter gene expression in IELs and mature CD8SP cells but not in thymocytes at earlier stages of differentiation. In vitro transfection studies in our laboratory have defined an approximately 200 nt cis-acting element (called L2a) located 4-5 kb upstream of the CD8a gene that appears to be the target of negative regulation in hybridomas of CD8-positive T-cells fused with the thymic lymphoma cell line, BW5 147. The L2a element is a nuclear matrix associated region (or MAR). Interestingly, inclusion of both the E81 enhancer and a 4.3 kb Hindlil/Hindlil fragment containing the L2a element in the transgenic construct results in E8I enhancer function in CD4+CD8+ double-positive (DP) thymocytes as well as in CD8SP T-cells and IELs. We hypothesize that it is the L2a element that imparts to the E81 enhancer the ability to function in DP thymocytes. To test this hypothesis, transgenic studies using a human CD2 reporter gene and 4.3 kb HindIII/HmdIII fragments with various deletions will be used to localize the region that imparts DP thymocyte function upon the E81 enhancer. The effect of these cis-acting sequences on the activity of other CD8 enhancers will be evaluated using the same approach knock-out and knock-in studies using the Cre/loxP system will be performed to test the effect of deletion or modification of L2a element (or other cis-acting sequences) in situ upon CD8 expression in thymic subpopulations and peripheral T-cells. If the L2a element proves to harbor the cis-acting sequences as hypothesized, we will specifically modify sites within L2a shown to bind the MAR-binding proteins, SATBI and CDP/Cux, suggested by us to play positive and negative roles, respectively, in CD8a gene regulation. Complementary studies in which expression of SATB1 or CDP/Cux is compromised will be attempted to determine whether the effect observed is similar to that seen when each protein's binding site in L2a is abolished. Finally, chromatin cross-linking and immunoprecipitation studies will be performed to test whether SATB 1 and CDP/Cux bind to the L2a element with cell specificity consistent with the roles we have hypothesized for them in CD8 gene regulation. These studies will contribute to our knowledge of CD8 gene regulation during T-cell differentiation, and to the roles played by MARs and their cognate binding proteins in modulating the activities of cis-acting sequences in vivo.
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0.904 |
2004 — 2006 |
Tucker, Philip W. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Bop in Cardiac Development and Function @ University of Texas Austin
DESCRIPTION (provided by applicant): Congenital heart disease and acquired heart disease are the leading non-infectious causes of death in children and adults, respectively. The long term objectives of this project are to understand the role of m-Bop proteins in cardiac myocyte differentiation and cardiac development. Bop encodes m-Bop proteins specifically expressed in the heart field and myotome of the mouse and chick early in development as well as in fetal and adult mouse myocardium and skeletal muscle. m-Bop proteins contain both a MYND domain, shown in other proteins to recruit histone deacetylases (HDACs), and a S-ET domain, shown elsewhere to affect chromatin structure, sometimes through intrinsic histone methyltransferase (HMT) activity. Both activities can repress gene expression through epigenetic effects involving chromatin modifications. Targeted inactivation of Bop in mice leads to death at embryonic day 10 (El0 0), and hearts of Bop-null fetuses lack a right ventricle and have abnormal cardiomyocyte differentiation. Absence of the transcription factor, Hand2, from the heart primordia of E7 75 Bop-null embryos suggests a role for m-Bop in an early gene expression cascade that leads to right ventricular development. m-Bop has repressive activity in vitro due to recruitment of HDACs, and it physically interacts directly or indirectly with HDACs. m-Bop interacts with skNAC, a heart- and skeletal muscle-specific transcription factor, and co-localizes with skNAC during myogenesis in vitro and during cardiogenesis in vivo. m-Bop also interacts with MITR, a co-repressor of myogenesis, and HRT2, a heart ventricle-specific transcription factor. We plan to test the hypotheses that 1) m-Bop is a cardiac-specific regulator of chromatin modifications early in cardiomyocyte development and functions through direct and indirect interactions with DNA-binding proteins, and 2) that m-Bop and skNAC interact in a physiologically meaningful manner during myogenesis in vitro and cardiac development in vivo. Specific Aims are 1) To define the mechanisms by which the MYND, SET and other domains of m-Bop promote alterations in chromatin structure and regulate transcription, and 2) To determine the biological significance of m-Bop/skNAC interaction and the role of skNAC during cardiogenesis. These studies are relevant to mechanisms that may underlie ventricular hypoplasia in congenital heart disease. That m-Bop appears to specifically affect heart development by promoting histone modifications and hence chromatin reorganization in a lineage-specific fashion places it among a very few proteins known to operate in this way. This gives a broader relevance to the mechanisms to be investigated in the proposed studies.
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0.904 |
2004 — 2008 |
Tucker, Philip W. [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Earids in Lymphocyte Function and Development @ University of Texas Austin
ARID (AT- Rich Interaction Domain) transcription factors have been implicated in chromatin remodeling and growth deregulation. Of the 13 members of the ARID family, two are termed eARIDs because they show extended (e) identity beyond the ARID DNA binding domain: Bright (for B cell specific regulator of IgH transcription) and Bdp (for Bright-Dri-like protein). Bright functions as a positive transcriptional activator of specific motifs (P sites) within nuclear matrix associated regions (MARs) flanking the IgH intronic enhancer (Em) and 5' to the V1 member of the VH $107 family. In human (h) and mouse (m) B cell differentiation, Bright is restricted to early preB and germinal center B cells. Little is known about Bdp in either species. Aim 1 proposes a full characterization of Bdp expression, function, and localization relative to Bright. In Aim 2 we propose to identify and validate genes in additional to IgH that are activated by the eARIDs. Our initial focus will include 5 targets that define a potential role for Bright in cell survival. There have been no ARID knockouts reported. Conventional targeted disruption of the Bright gene in mice leads to embryonic lethality (Progress Report). In Aim 3, we propose a conditional knockout approach for eliminating Bright only in B cells. We will evaluate Bright null mice in the context of normal and malignant B cell biology and with regard to the developmental and repertoire dysfunction proposed for Bright in X-linked immunodeficiency disease. We propose to create Bdp null mice using one of the above strategies.
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0.904 |
2010 |
Tucker, Philip W. [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
A Transcription Factor Within Lipid Rafts Modulates B Cell Signaling Via the Bcr @ University of Texas, Austin
DESCRIPTION (provided by applicant): How variant signaling strength of the B cell antigen receptor (BCR) complex is transformed into biochemical signals is not well understood. A growing body of evidence indicates that lipid rafts function as platforms for signalling through the BCR. Bright is a B cell-restricted transcription factor that transactivates the immunoglobulin heavy chain (IgH) locus by binding to nuclear matrix attachment regions flanking the IgH intronic enhancer. Bright's DNA binding and transcriptional activities are stimulated by its direct association with Bruton's tyrosine kinase (Btk), the implicated molecule in XLA/xid. Bright undergoes a cell cycle- dependent shuttle between the nucleus and the cytoplasm and is implicated in G1/S cell cycle progression. We discovered that a small (1-2%) pool of Bright resides constitutively within lipid rafts. There it associates with Btk and the BCR signaling complex. Following BCR stimulation by surrogate antigen, Bright is discharged from rafts at a rate proportional to signal strength. An inducible association of Bright with sumoylation E2 and E3 components leads to Sumo-I-modification of Bright and its subsequent discharge from rafts into plasma membranes. BCR stimulation induces a transient, rafts-specific association and subsequent co-discharge of Bright and the F-actin linker protein Ezrin, suggesting a potential role for Bright in microfilament depolymerization - a key event in BCR signaling. This is the first functional demonstration of a transcription factor in lipid rafts. We hypothesize that Bright functions to attenuate BCR signaling;i.e., Bright-depleted signalosomes are more active than Bright-containing signalosomes. In this R21 application, we propose (1) to define the requirements for specification of Bright to lipid rafts;(2) to construct and initiate analysis of mice specifically altered for lipid-rafts-localized Bright;and (3) to identify pathways engaged by lipid rafts-localized Bright to regulate BCR signaling strength. These studies suggest another avenue for the transport of cargo between the membrane and the nucleus. They provide long-term significance for immunological tolerance, autoimmunity and immunodeficiency. Transcription factors are proteins that bind to DNA within the nucleus to initiate gene expression. We discovered that a transcription factor (Bright), which is known to possess this property, also has the unexpected property of localizing within specific structures of the cell membrane (lipid rafts). There Bright functions in an unknown manner to regulate the ability of the B lymphocyte to respond to antigen. Understanding how lipid rafts-localized Bright modulates immune responses will impact on our understanding of immunological tolerance, the immune mechanism that prevents the immune system from reacting against self;i.e., autoimmunity.
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