PubMed Health⌕ Search

Biomedical subjects

M D Jonsen

Publications and source records attributed to M D Jonsen.

5 recordsLinked to original sources
PubMed2005

Pit-1beta reduces transcription and CREB-binding protein recruitment in a DNA context-dependent manner.

Many transcription factors are expressed as multiple isoforms with distinct effects on the regulation of gene expression, and the functional consequences of structural differences between transcription factor isoforms may allow for precise control of gene expression. The pituitary transcription factor isoforms Pit-1 and Pit-1beta differentially regulate anterior pituitary hormone gene expression. Pit-1 is required for the development of and appropriate hormone expression by anterior pituitary somatotrophs and lactotrophs. Pit-1beta differs structurally from Pit-1 by the splice-insertion of the 26-residue beta-domain in the trans-activation domain, and it differs functionally from Pit-1 in that it represses expression of the prolactin promoter in a cell-type specific manner. In order to identify signal and promoter context requirements for repression by Pit-1beta, we examined its function in the presence of physiological regulatory signals as well as wild-type and mutant Pit-1-dependent target promoters. Here, we demonstrate that Pit-1beta impairs recruitment of cAMP response element-binding protein (CREB)-binding protein to the promoters that it represses. In addition, we show that repression of target promoter activity, reduction in promoter histone acetylation, and decrease of CREB-binding protein recruitment all depend on promoter context. These findings provide a mechanism for promoter-specific repression by Pit-1beta.

Acetylation↗
PubMed1998

Equilibrium analysis of high affinity interactions using BIACORE.

BIACORE biosensors are useful for measuring reaction kinetics and calculating affinity constants for macromolecular interactions. However, one drawback with the flow system used in these instruments is that the standard injection procedures limit the amount of time available to collect association-phase data. This is especially problematic during equilibrium analysis of high affinity interactions. Using protein-DNA interactions as a model system, we demonstrate a simple method for overcoming this limitation. By placing the analyte directly into the running buffer we were able to deliver a continuous supply of protein to the sensor surfaces for greater than 12 h at a time. Complete equilibrium binding profiles were generated by changing the concentration of analyte and allowing the surface reactions to reequilibrate. Analyte concentrations were also decreased to demonstrate that the binding reactions were fully reversible. This method of analysis is a simple and convenient way of directly measuring equilibrium dissociation constants for very high affinity interactions.

Base Sequence↗
PubMed1996

Characterization of the cooperative function of inhibitory sequences in Ets-1.

DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated by an intramolecular mechanism. Quantitative binding assays compared the DNA-binding activities of native Ets-1, three deletion mutants, and three tryptic fragments. Ets-1 and activated Ets-1 polypeptides differed in DNA-binding affinity as much as 23-fold. Inhibition was mediated by two regions flanking the minimal DNA-binding domain. Both regions regulated affinity by enhancing dissociation of the protein-DNA complex. Three lines of evidence indicated that inhibition requires cooperative interaction between the two regions: first, the two inhibitory regions acted through a common mechanism; second, neither region functioned independently of the other; finally, mutation of the C-terminal inhibitory region altered the conformation of the N-terminal inhibitory region. In addition, partial proteolysis detected an identical altered conformation in the N-terminal inhibitory region of Ets-1 bound to DNA. This finding suggested that repression is transiently disrupted during DNA binding. These results provide evidence that the two inhibitory regions of Ets-1 are structurally, as well as functionally, coupled. In addition, conformational change is shown to be a critical component of the inhibition mechanism. A cooperative, allosteric model of autoinhibition is described. Autoinhibition of Ets-1 could be relieved by either protein partner(s) or posttranslational modifications.

Allosteric Regulation↗
PubMed1992

Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif.

The proto-oncogene ets-1 is the founding member of a new family of eukaryotic transcriptional regulators. Using deletion mutants of murine ets-1 cDNA expressed in Escherichia coli, we show that the DNA-binding domain corresponds closely to the ETS domain, an 85-amino-acid region that is conserved among ets family members. To investigate the specificity of DNA binding of the ETS domain, we mapped the DNA contacts of a monomeric Ets-1 fragment by chemical protection and interference assays. DNA backbone interactions span a 20-nucleotide region and are localized on one face of the helix. Close phosphate and base contacts are restricted to 10 central nucleotides. Contacts map to the major groove in the center of the site. Flanking minor groove interactions also are predicted. To determine the sequence preference in the close contact zone, we selected a pool of high-affinity binding sites using a purified Ets-1 carboxy-terminal fragment. Our Ets-1-selected consensus, 5'-A/GCCGGAA/TGT/C-3', differs from the binding consensus for the Drosophila ETS domain protein E74A, suggesting that specificity of action of ets family members is mediated by the ETS domain. Compared to other well-characterized classes of DNA-binding proteins, Ets-1 produces a unique pattern of DNA contacts. These studies demonstrate that the ETS domain proteins bind DNA in a novel manner.

Animals↗