Biomedical subjects

Terace M Fletcher

Publications and source records attributed to Terace M Fletcher.

10 recordsLinked to original sources

PubMed2005

DNA structure-dependent recruitment of telomeric proteins to single-stranded/double-stranded DNA junctions.

Telomeres protect chromosome ends by assembling unique protein-DNA complexes. TRF2 is a telomere binding protein that is involved in protecting the G-strand overhang, a 3', guanine-rich, overhang at the telomere terminus. TRF2 may protect the G-strand overhang by recognizing some organizational aspect of the telomeric single-stranded/double-stranded (ss/ds) DNA junction. This work demonstrates that TRF2, purified or in crude extracts, recognizes telomeric ss/ds DNA junctions containing wild type telomeric sequence in the ds region and a G-strand overhang with at least one telomeric repeat. Telomeric complexes containing TRF2 and pot1 assemble less efficiently when the G-strand overhang is in the form of an intramolecular G-quadruplex. However, recruitment of the DNA repair proteins, WRN, Mre11, and Ku86, is not inhibited by a G-quadruplex. This suggests that an intramolecular G-quadruplex has the potential to disrupt certain telomeric assemblies, but efficient recruitment of appropriate DNA repair proteins provides the means to overcome this obstacle.

Binding Sites↗

PubMed2005

Ligand-specific dynamics of the progesterone receptor in living cells and during chromatin remodeling in vitro.

Progesterone receptor (PR), a member of the nuclear receptor superfamily, is a key regulator of several processes in reproductive function. We have studied the dynamics of the interaction of PR with a natural target promoter in living cells through the use of fluorescence recovery after photobleaching (FRAP) analysis and also have characterized the dynamics of the interaction of PR with the mouse mammary tumor virus (MMTV) promoter reconstituted into chromatin in vitro. In photobleaching experiments, PR in the presence of the agonist R5020 exhibits rapid exchange with the MMTV promoter in living cells. Two PR antagonists, RU486 and ZK98299, have opposite effects on receptor dynamics in vivo. In the presence of RU486, PR binds to the promoter and is exchanged more slowly than the agonist-activated receptor. In contrast, PR bound to ZK98299 is not localized to the promoter and exhibits higher mobility in the nucleoplasm than the agonist-bound receptor. Significantly, PR bound to R5020 or RU486 can recruit the SWI/SNF chromatin remodeling complex to the promoter, but PR activated with ZK98299 cannot. Furthermore, we found ligand-specific active displacement of PR from the MMTV promoter during chromatin remodeling in vitro and conclude that the interaction of PR with chromatin is highly dynamic both in vivo and in vitro. We propose that factor displacement during chromatin remodeling is an important component of receptor mobility and that ligand-specific interactions with remodeling complexes can strongly influence receptor nuclear dynamics and rates of exchange with chromatin in living cells.

Cell Nucleus↗

PubMed2005

Telomerase: a potential therapeutic target for cancer.

Telomeres are complex structures which serve to protect chromosome ends. Telomere shortening occurs in normal somatic cells reaching a point in which cells senesce. Senescence can be counteracted by activating telomerase. Telomerase activity is present in a majority of cancer cells and requires the upregulation of the reverse transcriptase component called hTERT. Because telomerase activity is essential for proliferation of most cancer cells, therapeutic strategies have been developed to inhibit its activity. These strategies centre on targeting the active site, hTERT and hTERC expression, core enzyme stability and telomeric DNA. Successful approaches involve a combination of traditional drugs with telomerase inhibitors. Disrupting the functional expression of hTERT is particularly effective in agreement with evidence that hTERT is an antiapoptotic factor in some cancer cells. In addition, approaches that stabilise DNA secondary structures may disrupt telomere maintenance through a variety of routes making them, potentially, very potent in attack-ing cancer cells.

Antineoplastic Agents↗

PubMed2004

Subnuclear trafficking and gene targeting by steroid receptors.

Through the use of novel imaging techniques, we have observed direct steroid receptor binding to a tandem array of a hormone-responsive promoter in living cells. We found that the glucocorticoid receptor (GR) exchanges rapidly with regulatory elements in the continued presence of ligand. We have also reconstituted a GR-dependent nucleoprotein transition with chromatin assembled on promoter DNA, and we discovered that GR is actively displaced from the chromatin template during the chromatin remodeling process. Using high-intensity UV laser crosslinking, we have observed highly periodic interactions of GR with promoter chromatin. These periodic binding events are dependent on GR-directed hSWI/SNF remodeling of the template and require the presence of ATP. Both the in vitro and in vivo results are consistent with a dynamic model ("hit-and-run") in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is simultaneously lost from the template. We also find that receptor mobility in the nucleoplasm is strongly enhanced by molecular chaperones. These observations indicate that multiple mechanisms are involved in transient receptor interactions with nucleoplasmic targets.

Animals↗

PubMed2003

Formation of higher-order secondary and tertiary chromatin structures by genomic mouse mammary tumor virus promoters.

Agarose multigel electrophoresis has been used to characterize the structural features of isolated genomic mouse mammary tumor virus (MMTV) promoters. The mouse 3134 cells used for these studies contain approximately 200 stably integrated tandem repeats of a 2.4-kb MMTV promoter fragment. Inactive, basally active, and hormonally activated genomic promoters were liberated by restriction digestion of isolated nuclei, recovered in low-salt nuclear extracts, and electrophoresed in multigels consisting of nine individual agarose running gels. Specific bands were detected and characterized by Southern and Western blotting. We find that transcriptionally inactive promoters contain TBP and high levels of histone H1, and are present to varying extents in both untreated and dexamethasone (DEX)-treated 3134 cells. In contrast, the basally active promoter, present in untreated cells, is bound to RNA Pol II, TBP, and Oct1, contains acetylated H3 tail domains, and is depleted of histone H1. The DEX-activated promoter possessed similar composition as the basal promoter, but also contains stably bound Brg1. Strikingly, all forms of the MMTV promoter condense into higher-order secondary and/or tertiary chromatin structures in vitro in the presence of Mg2+. Thus, genomic MMTV promoter chromatin retains the ability to form classical higher-order structures under physiological salt conditions, even after dissociation of H1 and binding of several transcription factors and multiprotein complexes. These results suggest that transcriptionally active eukaryotic promoters may function in a locally folded chromatin environment in vivo.

Acetylation↗

PubMed2003

Telomere higher-order structure and genomic instability.

Telomeres, nucleoprotein complexes at the end of eukaryotic chromosomes, have vital roles in chromosome integrity. Telomere chromatin structure is both intricate and dynamic allowing for a variety of responses to several stimuli. A critical determinant in telomere structure is the G-strand overhang. Facilitated by telomeric proteins, the G-strand overhang stabilizes telomere higher-order assemblies most likely by adopting unusual DNA structures. These structures influence activities that occur at the chromosome end. Dysfunctional telomeres induce signals resulting in cell growth arrest or death. To overcome telomere dysfunction, cancer cells activate the DNA polymerase, telomerase. The presence of telomerase at the telomere may establish a particular telomeric state. If the chromosome ends of cancer and normal cells exist in different states, cancer-specific telomere structures would offer a unique chemotherapeutic target.

Animals↗

PubMed2002

Glucocorticoid receptor domain requirements for chromatin remodeling and transcriptional activation of the mouse mammary tumor virus promoter in different nucleoprotein contexts.

The glucocorticoid receptor (GR) contains several activation domains, tau1 (AF-1), tau2, and AF-2, which were initially defined using transiently transfected reporter constructs. Using domain mutations in the context of full-length GR, this study defines those domains required for activation of the mouse mammary tumor virus (MMTV) promoter in two distinct nucleoprotein configurations. A transiently transfected MMTV template with a disorganized, accessible chromatin structure was largely dependent on the AF-2 domain for activation. In contrast, activation of an MMTV template in organized, replicated chromatin requires both domains but has a relatively larger dependence on the tau1 domain. Domain requirements for GR-induced chromatin remodeling of the latter template were also investigated. Mutation of the AF-2 helix 12 domain partially inhibits the induction of nuclease hypersensitivity, but the inhibition was relieved in the absence of tau1, suggesting the occurrence of an important interaction between the two domains. Further mutational analysis indicates that GR-induced chromatin remodeling requires the ligand-binding domain in the region of helix 3. Our study shows that the GR activation surfaces required for transcriptional modulation of a target promoter were determined in part by its chromatin structure. Within a particular cellular environment the GR appears to possess a significant degree of versatility in the mechanism by which it activates a target promoter.

Alanine↗

PubMed2002

ATP-dependent mobilization of the glucocorticoid receptor during chromatin remodeling.

Chromatin remodeling by the glucocorticoid receptor (GR) is associated with activation of transcription at the mouse mammary tumor virus (MMTV) promoter. We reconstituted this nucleoprotein transition with chromatin assembled on MMTV DNA. The remodeling event was ATP dependent and required either a nuclear extract from HeLa cells or purified human Swi/Snf. Through the use of a direct interaction assay (magnetic bead pull-down), we demonstrated recruitment of human Swi/Snf to MMTV chromatin by GR. Unexpectedly, we found that GR is actively displaced from the chromatin template during the remodeling process. ATP-dependent GR displacement was reversed by the addition of apyrase and was specific to chromatin templates. The disengagement reaction could also be induced with purified human Swi/Snf. Although GR apparently dissociated during chromatin remodeling by Swi/Snf, it participated in binding of the secondary transcription factor, nuclear factor 1. These results are paralleled by a recent discovery that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment in living cells. Both the in vitro and in vivo results are consistent with a dynamic model (hit and run) in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, facilitates transcription factor binding, and is simultaneously lost from the template.

Adenosine Triphosphate↗

PubMed2002

Acetylation of nucleosomal histones by p300 facilitates transcription from tax-responsive human T-cell leukemia virus type 1 chromatin template.

Expression of human T-cell leukemia virus type 1 (HTLV-1) is regulated by the viral transcriptional activator Tax. Tax activates viral transcription through interaction with the cellular transcription factor CREB and the coactivators CBP/p300. One key property of the coactivators is the presence of histone acetyltransferase (HAT) activity, which enables p300/CBP to modify nucleosome structure. The data presented in this manuscript demonstrate that full-length p300 and CBP facilitate transcription of a reconstituted chromatin template in the presence of Tax and CREB. The ability of p300 and CBP to activate transcription from the chromatin template is dependent upon the HAT activity. Moreover, the coactivator HAT activity must be tethered to the template by Tax and CREB, since a p300 mutant that fails to interact with Tax did not facilitate transcription or acetylate histones. p300 acetylates histones H3 and H4 within nucleosomes located in the promoter and 5' proximal regions of the template. Nucleosome acetylation is accompanied by an increase in the level of binding of RNA polymerase II transcription factor TFIID and RNA polymerase II to the promoter. Interestingly, we found distinct transcriptional activities between CBP and p300. CBP, but not p300, possesses an N-terminal activation domain which directly activates Tax-mediated HTLV-1 transcription from a naked DNA template. Finally, using the chromatin immunoprecipitation assay, we provide the first direct experimental evidence that p300 and CBP are associated with the HTLV-1 long terminal repeat in vivo.

Acetylation↗

PubMed2001

Telomerase - strategies to exploit an important chemotherapeutic target.

Telomeres, unique protein-DNA complexes located at the chromosome ends, have important functions involving both DNA protection and cellular signalling. Telomere structure is very dynamic yet tightly controlled. One important factor is the presence of telomerase, a telomere-specific DNA polymerase activated in a majority of cancer cells. Cancer and normal cell telomeres may have dissimilar structures due to variances in telomere length, telomerase activity and levels of telomere binding proteins. In designing compounds to strictly target cancer cells, these distinctions should be investigated. Much of the recent focus has been on the development of highly effective telomerase inhibitors. Another novel group of small molecules target telomere DNA, thereby disrupting both telomerase activity and telomere structure. This class of compounds should have an immediate impact on cell growth and viability. Since many molecular characteristics of telomeres are unknown, small molecules should also be useful in probing differences in telomere dynamics unique to cancer cells.

Journal Article↗