Biomedical subjects

A K Ryan

Publications and source records attributed to A K Ryan.

23 records · Page 2Linked to original sources

PubMed1995

Development and survival of the endocrine hypothalamus and posterior pituitary gland requires the neuronal POU domain factor Brn-2.

Neurons comprising the endocrine hypothalamus are disposed in several nuclei that develop in tandem with their ultimate target the pituitary gland, and arise from a primordium in which three related class III POU domain factors, Brn-2, Brn-4, and Brn-1, are initially coexpressed. Subsequently, these factors exhibit stratified patterns of ontogenic expression, correlating with the appearance of distinct neuropeptides that define three major endocrine hypothalamic cell types. Strikingly, deletion of the Brn-2 genomic locus results in loss of endocrine hypothalamic nuclei and the posterior pituitary gland. Lack of Brn-2 does not affect initial hypothalamic developmental events, but instead results in a failure of differentiation to mature neurosecretory neurons of the paraventricular and supraoptic nuclei, characterized by an inability to activate genes encoding regulatory neuropeptides or to make correct axonal projections, with subsequent loss of these neurons. Thus, both neuronal and endocrine components of the hypothalamic-pituitary axis are critically dependent on the action of specific POU domain factors at a penultimate step in the sequential events that underlie the appearance of mature cellular phenotypes.

Animals↗

PubMed1995

Isolation and characterization of the chicken homeodomain protein AKR.

Expression of the avian apoVLDLII gene is liver specific and completely dependent on estrogen. Previous analyses of protein binding sites in the apoVLDLII promoter revealed interactions between liver-enriched and ubiquitous factors at a location, site F', between nucleotides -229 and -260 relative to the major transcriptional start site. Site-directed mutagenesis of G residues contacted by these factors decreased expression from the promoter approximately 5-fold in the avian hepatoma cell line LMH2A. We have used this site to screen a cDNA expression library constructed from day 9 embryonic liver RNA. One of the two DNA binding factors isolated is a novel homeodomain protein. With the exception of the homeodomain itself, which is atypically located close to the protein N-terminus, the factor displays little similarity to any known DNA binding protein. Its homeodomain is most similar to that of the maize protein Knotted-1, while the most closely related vertebrate domain is that of the human proto-oncoprotein Pbx1. We demonstrate that the DNA binding specificity of the factor is consistent with its involvement in the ubiquitous complex formed with site F' and that it is capable of suppressing expression from the apoVLDLII promoter in short-term transfection experiments. These studies, combined with its DNA binding specificity, the tissue distribution of its mRNA and its developmental regulation, suggest a role as a negative regulator of gene expression in non-hepatic tissues and in the liver early during embryogenesis.

Amino Acid Sequence↗

PubMed1994

Characterization of protein interactions with positive and negative elements regulating the apoVLDLII gene.

Synthesis of avian apo very-low-density lipoprotein (apoVLDL)II is estrogen dependent and liver specific. Competence to express the apoVLDLII gene is not acquired until days 7-9 of embryogenesis and thus lags 5-6 days behind appearance of the liver primordial bud. It is not known whether the delayed ability to activate the gene is attributable to hepatic estrogen receptor profiles, or a requirement for other transcription factors not expressed at earlier stages of embryogenesis. The latter possibility is supported by developmental alterations in nuclease hypersensitivity flanking the gene that occur independently of estrogen administration. We have examined the influence of these hypersensitive regions on expression from the apoVLDLII promoter and have characterized novel protein-DNA interactions at two of them. One is located in a copy of the CR1 family of middle repetitive elements approximately 3.0 kb upstream from the start of the gene. We demonstrate by DNase I footprinting that the site contains an element which matches a predicted consensus silencer sequence. The other site contains no previously identified binding motifs. It is located between nucleotides -228 and -245 and is adjacent to an imperfect estrogen response element (ERE) that we demonstrate acts additively with a canonical ERE 30 nucleotides downstream. We have identified ubiquitous and liver-specific factors that display overlapping DNA contacts with the site. Mutation of G residues contacted by these proteins decreases hormone-inducible expression from the promoter 5- to 8-fold. Hepatic levels of the liver-enriched factor interacting with this site increase abruptly between days 7 and 9 of embryogenesis, suggesting that it may be an important determinant of the ability to express the apoVLDLII and possibly other liver-specific genes.

Animals↗

PubMed1992

Characterization of liver-enriched proteins binding to a developmentally demethylated site flanking the avian apoVLDLII gene.

Although the avian apoVLDLII gene is normally expressed exclusively in the liver of the laying hen, the gene can be activated by estrogen in birds of either sex beginning between days 7-9 of embryogenesis. Developmentally programmed demethylation of sites in the 5'- and 3'-flanking regions of the gene have been shown to occur during this period of embryogenesis, suggesting that they may reflect changes in protein-DNA interactions that are involved in the acquisition of competence to activate the apoVLDLII gene. We have detected specific protein interactions at one location approximately 2.6 kb upstream from the apoVLDLII gene, that includes an Msp I site whose methylation status changes between days 7 and 9 of embryogenesis. The sequence of this region bears significant similarity to binding sites of members of the bZIP family of liver-enriched or -specific factors such as C/EBP, DBP, and LAP, that are characteristically produced relatively late during liver development. In the studies described here, we demonstrate that proteins binding to the upstream apoVLDLII site do not correspond to previously identified liver-enriched or -specific factors. They also display a pattern of activity during development and in human and avian hepatoma cell lines indicating that their expression is increased in proliferating cells. Southwestern blotting and UV cross-linking studies indicate that two proteins of approximately 60 kD are capable of binding to the site and we describe the purification of these factors from crude nuclear protein extracts obtained from rooster liver.

Animals↗

PubMed1990

Developmental regulation of specific protein interactions with an enhancerlike binding site far upstream from the avian very-low-density apolipoprotein II gene.

Expression of the avian very-low-density apolipoprotein II (apoVLDLII) gene is completely dependent on estrogen and restricted to the liver. We have identified binding sites for nonhistone nuclear proteins located between -1.96 and -2.61 kilobases. One of these sites, located at -2.6 kilobases (designated site 1), was found to span an MspI site that becomes demethylated between days 7 and 9 of embryogenesis, the stage of development at which competence to express the apoVLDLII gene begins to be acquired. Levels of the factor(s) involved were high at day 7 of embryogenesis, decreased two- to threefold by days 9 to 11, and continued to decline more slowly until hatching. Furthermore, the mobility of the complex formed underwent a well-defined shift between days 11 to 13 embryogenesis. Methylation interference studies showed that modification of the outer guanosines of the MspI site resulted in marked inhibition of the formation of the protein-DNA complex. Competition studies, fractionation of nuclear extracts, and tissue distribution indicated that the factor was not the avian homolog of hepatocyte nuclear factor 1, nuclear factor 1, or CCAAT/enhancer-binding protein (C/EBP). However, site 1 could complete for binding to an oligonucleotide, previously shown to be recognized by C/EBP, in a nonreciprocal fashion. These studies demonstrate that the sequence recognized by the protein includes a C/EBP consensus sequence but that elements in addition to the core enhancer motif are essential for binding.

Age Factors↗