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Biomedical subjects

R S Buckle

Publications and source records attributed to R S Buckle.

8 recordsLinked to original sources
PubMed1998

Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1.

The estrogen receptor (ER) is an important regulator of growth and differentiation of breast epithelium. Transactivation by ER depends on a leucine-rich motif, which constitutes a ligand-regulated binding site for steroid receptor coactivators (SRCs). Cyclin D1 is frequently amplified in breast cancer and can activate ER through direct binding. We show here that cyclin D1 also interacts in a ligand-independent fashion with coactivators of the SRC-1 family through a motif that resembles the leucine-rich coactivator binding motif of nuclear receptors. By acting as a bridging factor between ER and SRCs, cyclin D1 can recruit SRC-family coactivators to ER in the absence of ligand. A cyclin D1 mutant that binds to ER but fails to recruit coactivators preferentially interferes with ER activation in breast cancer cells that have high levels of cyclin D1. These data support that cyclin D1 contributes significantly to ER activation in breast cancers in which the protein is overexpressed. Our present results reveal a novel route of coactivator recruitment to ER and establish a direct role for cyclin D1 in regulation of transcription.

Animals↗
PubMed1995

Programming of a repressed but committed chromatin structure during early development.

The determination of chromatin for transcription during early development as well as the requirement for trans-acting factors during this period has been analysed in Xenopus. Basal transcription is repressed both during oogenesis and after the mid-blastula transition (MBT), and transactivators are required to relieve this repression. In contrast, transactivators cannot overcome the generalized transcriptional repression which occurs in embryos before MBT. However, they do bind to promoters leading to a repressed but preset chromatin structure. Experiments involving the pre-binding of TATA binding protein (TBP) or of the strong transactivator GAL4-VP16 further show that there is no limiting factor before the MBT, and that it is the recruitment and stabilization of the basal transcription machinery and not of transactivators which is repressed during early development. This multi-step process in gene activation, with activation of promoters temporally uncoupled from their commitment, may be of importance in the regulation of early embryonic events by providing molecular signposts for future determinations.

Animals↗
PubMed1995

Analysis of c-Myc and Max binding to the c-myc promoter: evidence that autosuppression occurs via an indirect mechanism.

c-myc negatively autoregulates its expression at the level of transcriptional initiation, although the precise mechanism remains debated. While conclusive evidence for c-Myc binding in its own promoter has not been found, it has been proposed that c-Myc binds to a site upstream of the human c-myc gene which may also be a component of a replication origin (Ariga et al., 1989). In an attempt to clarify this issue, sequences flanking the c-myc gene were screened for c-Myc or Max binding sites using a novel procedure to facilitate the detection of DNA binding dependent upon long distance interactions or DNA secondary structure. Since the sequence specificity of DNA binding proteins may also be mediated by interaction with other proteins or by protein modification, this affinity capture assay was used in conjunction with nuclear extracts, potentially allowing the selection of novel in vivo DNA binding specificities. Using conditions that gave strong binding to an internal control sequence, c-Myc or Max binding elements were not detected in genomic sequences extending 5.4 kb upstream of the Xenopus c-myc gene. Identical results were obtained using both purified proteins and a variety of nuclear extracts, suggesting c-myc autosuppression most likely involves an indirect pathway.

Animals↗
PubMed1995

Neither ERK nor JNK/SAPK MAP kinase subtypes are essential for histone H3/HMG-14 phosphorylation or c-fos and c-jun induction.

The effects of EGF, TPA, UV radiation, okadaic acid and anisomycin on ERK and JNK/SAPK MAP kinase cascades have been compared with their ability to elicit histone H3/HMG-14 phosphorylation and induce c-fos and c-jun in C3H 10T1/2 cells. EGF and UV radiation activate both ERKs and JNK/SAPKs but to markedly different extents; EGF activates ERKs more strongly than JNK/SAPKs, whereas UV radiation activates JNK/SAPKs much more strongly than ERKs. Anisomycin and okadaic acid activate JNK/SAPKs but not ERKs, and conversely, TPA activates ERKs but not JNK/SAPKs. Nevertheless, all these agents elicit phosphorylation of ribosomal and pre-ribosomal S6, histone H3 and HMG-14, and the induction of c-fos and c-jun, showing that neither cascade is absolutely essential for these responses. We then analysed the relationship between ERKs, JNK/SAPKs and the transcription factors Elk-1 and c-Jun, implicated in controlling c-fos and c-jun, respectively. JNK/SAPKs bind to GST-cJun1-79, and ERKs, particularly ERK-2, to GST-Elk1(307-428); there is no cross-specificity of binding. Further, GST-Elk1(307-428) binds preferentially to active rather than inactive ERK-2. In vitro, JNK/SAPKs phosphorylate both GST-cJun1-79 and GST-Elk1(307-428), whereas ERKs phosphorylate GST-Elk1(307-428) but not GST-cJun1-79. Thus, neither ERKs nor JNK/SAPKs are absolutely essential for nuclear signalling and c-fos and c-jun induction. The data suggest either that activation of a single MAP kinase subtype is sufficient to elicit a complete nuclear response, or that other uncharacterised routes exist.

3T3 Cells↗
PubMed1992

Site-directed mutagenesis studies on the binding of the globular domain of linker histone H5 to the nucleosome.

The globular domain of the linker histone H5 has been expressed in Escherichia coli. The purified peptide is functional as it permits chromatosome protection during micrococcal nuclease digestion of chromatin reconstituted with the peptide, indicating that it binds correctly at the dyad axis of the nucleosomal core particle. The globular domain residue lysine 64 is highly conserved within the linker histone family, and site-directed mutagenesis has been used to assess the importance of this residue in the binding of the globular domain of linker histone H5 to the nucleosome. Recombinant peptides mutated at lysine 64 are unable to elicit chromatosome protection to the same degree as the wild-type peptide, and since they appear to be fully folded, these observations confirm a major role for this residue in determining the effective interaction between the globular domain of histone H5 and the nucleosome.

Amino Acid Sequence↗
PubMed1991

A chicken red cell inhibitor of transcription associated with the terminally differentiated state.

When a red cell nuclear extract (RCE) from adult chickens was injected into Xenopus oocytes along with the chicken beta globin gene, transcript levels were dramatically reduced compared to injection of DNA alone. The inhibitory action of the RCE was not specific to the beta globin gene since the Herpes thymidine kinase and Xenopus 5S RNA gene transcript levels were similarly reduced. Transcriptional repression was observed even after passage of the RCE through oocyte cytoplasm to the nucleus. The inhibitory activity binds to DNA cellulose, which suggests that the inhibitor either binds to DNA or associates with DNA-binding proteins. Nuclease digestion of the chromatin assembled on injected beta globin DNA revealed that inhibition was not associated with local changes in chromatin structure. Extracts from 9-d chicken embryonic erythroid cells, in which the endogenous beta globin gene is actively expressed, did not inhibit transcription. The inhibitory activity is, therefore, restricted to transcriptionally quiescent, adult erythrocytes. Since the inhibitory effects were seen with both polymerase II and III directed genes, we speculate that the activity may be part of the extreme transcriptional repression which occurs in the terminally differentiated erythrocyte.

Animals↗