Search PubMed⌕ Search

Biomedical subjects

R Renkawitz

Publications and source records attributed to R Renkawitz.

At least 55 records · Page 3Linked to original sources
PubMed1993

NeP1. A ubiquitous transcription factor synergizes with v-ERBA in transcriptional silencing.

One of the chicken lysozyme gene silencers binds two transcription factors, v-ERBA or the thyroid hormone receptor and NeP1 (negative protein 1), a new silencer binding protein. NeP1 is neutral on a monomeric binding site, but mediates weak repression on a multimerized site and strong synergistic repression in conjunction with v-ERBA on the wild-type silencer. Depending on the presence or absence of ligand, synergistic induction or repression is seen with the thyroid hormone receptor. This synergism is not based on cooperative DNA-binding as measured in vitro. The NeP1 DNA-binding activity is dependent on zinc ions, the binding site is characterized by a footprint of approximately 50 bp. NeP1 has a molecular weight of 140 to 160 kDa and has been enriched by affinity columns.

Animals↗
PubMed1993

Repression mechanisms of v-ERBA and other members of the steroid receptor superfamily.

Members of the steroid receptor superfamily, like other transcription factors, can function as transcriptional inducers as well as repressors of transcription. The mechanisms by which repression is achieved seem to be specific for the factors and [table: see text] regulatory sequences involved. Many and perhaps all of the steps required for transcriptional activation can be interfered with by nuclear receptors. Binding of a receptor dimer immediately adjacent to a transcription factor leads to synergistic transactivation (Fig. 6A). Binding of the GR to a nGRE displaces a positive transcription factor but has no or weak transactivation potential because no synergizing factor is nearby (Fig. 6B). The DNA-AP1 complex may bind GR, TR, or RAR so that the transactivating functions of both partners are inhibited (Fig. 6C). These negative effects (Fig. 6B and C) inhibit transactivating factor mediated gene activation, whereas the following examples show a reduction below the activity of a minimal promoter, thus acting very likely on general factors in the transcription initiation complex. v-ERBA competes with TR or RAR for DNA binding and in this respect resembles the mechanism described in Figure 6B. Silencing activity is then conferred by the bound v-ERBA, which is able to repress the activity of a complete or of a minimal promoter (Fig. 6D). Removal of the ligands T3 or RA converts the activating T3R or RAR into a silencing conformation (Fig. 6E). Ligand-free T3R, RAR, or v-ERBA bind to a silencer sequence and synergize with other silencer modules in repression (Fig. 6F).

Animals↗
PubMed1992

The mouse M-lysozyme gene domain: identification of myeloid and differentiation specific DNasel hypersensitive sites and of a 3'-cis acting regulatory element.

The mouse M-lysozyme gene is exclusively expressed in myeloid cells of the blood system being progressively turned on upon cell differentiation. In this study the mechanism controlling this tissue- and differentiation stage-specific gene expression was analyzed at the level of chromatin structure. A complex pattern consisting of constitutive and differentiation dependent DNasel hypersensitive sites (HSs) was found in a set of various myeloid cell lines, representing different stages of maturity. The chromatin of a lymphoid cell line, which does not express the lysozyme gene, is completely insensitive to DNasel digestion. Chromatin analysis of two multipotent hematopoietic stem cell lines which can be differentiated in vitro to mature myeloid cells confirmed that these identified DNasel HSs are specific for distinct differentiation stages, rather than being a characteristic feature of the cell lines. Additionally, the stem cell studies revealed that the hypersensitivity of the chromatin domain is already established at the multipotent stage. DNA fragments spanning a cell type- and differentiation stage-specific cluster of HSs in the 3' region of the gene showed enhancer activity in all cell types tested. In the light of this lack of specificity, we suggest that cell type-specific modification of the chromatin structure in this region may play a role in determining the binding of a widespread transcription factor, and hence contribute to the time specificity of lysozyme M gene expression.

Animals↗
PubMed1992

The involvement of demethylation in the myeloid-specific function of the mouse M lysozyme gene downstream enhancer.

Lysozyme gene expression is a specific marker for the macrophage/granulocyte lineage of hematopoietic differentiation in mammals, its expression being gradually increased during maturation. Analysis of the mechanisms regulating mouse M lysozyme gene expression during myeloid differentiation revealed a complicated pattern of DNase I hypersensitive sites (HS sites) within the flanking regions of the gene. The HS-3 site, located in the 3'-flanking region of the gene, overlapped with an enhancer element, which is the only strong enhancer identified in the vicinity of the gene. We demonstrate a positive correlation between undermethylation of the entire 3'-flanking region, the appearance of the HS-3 site, and M lysozyme gene expression during in vitro differentiation of hematopoietic stem cells. We furthermore show that methylation of a single CpG site within the enhancer core element, only observed in immature macrophage cells in vivo, is sufficient to inhibit nuclear factor binding to this element in vitro and to inhibit its transactivation potential in DNA transfection experiments.

Animals↗
PubMed1992

A transferable silencing domain is present in the thyroid hormone receptor, in the v-erbA oncogene product and in the retinoic acid receptor.

Inhibition of gene transcription is brought about by several mechanisms. The least understood mechanism is probably silencing, the analogue to transcriptional enhancing. We provide evidence that the silencing function of the oncogene product v-ERBA or the cellular counterpart, the thyroid hormone receptor (TR, c-erbA) is located in the C-terminal part and is transferable to a heterologous DNA binding domain. Deletion analyses suggest an important role for a basic and hydrophilic amino acid stretch on both ends of the domain. In addition we show that the related retinoic acid receptor (RAR) also contains a functional silencing domain similar in size and amino acid sequence. However, the activity of this domain can be neutralized by an additional domain in the C-terminus which functions cell specifically.

Alpharetrovirus↗
PubMed1991

Co-operative binding of the glucocorticoid receptor DNA binding domain is one of at least two mechanisms for synergism.

Steroid induction of responsive genes functions through the synergistic activity of steroid receptor binding sequences with adjacent binding sites either for other transcription factors or for further steroid receptors. Analysis of the human glucocorticoid receptor revealed that the DNA-binding domain of the receptor is sufficient to mediate co-operative binding to adjacent receptor binding sites. This is a novel feature of the domain in addition to its DNA-binding, trans-activating and trans-repressing properties. Chimaeric proteins containing the N- or C-terminal receptor halves fused to the GAL4 DNA-binding domain do not co-operate in DNA-binding, however they do functionally synergize. Thus, at least two mechanisms contribute to the synergism of the human glucocorticoid receptor bound to two adjacent receptor binding sites.

Base Sequence↗
PubMed1991

Multiple domains of the glucocorticoid receptor involved in synergism with the CACCC box factor(s).

Steroid induction of responsive genes functions through the synergistic activity of steroid receptor-binding sequences with adjacent transcription factor-binding sites. To analyze the mechanism of synergy we tested different human glucocorticoid receptor mutants for synergistic function with another transcription factor in comparison with intrinsic trans-activation obtained with a single receptor binding site (glucocorticoid response element). Multiple domains were found to be involved in synergistic activity of the glucocorticoid receptor with the CACCC box factor. Deletions within the N-terminal receptor half affected simultaneously intrinsic trans-activation and synergism. However, deletion of the hormone-binding domain mainly impaired synergism rather than intrinsic trans-activation, clearly showing that this domain synergizes by a mechanism independent of intrinsic activation. A chimeric protein where the DNA-binding domain of the glucocorticoid receptor was replaced by that of the yeast GAL4 protein also showed functional synergism. These data suggest that some of the receptor domains outside the DNA-binding domain synergize by their intrinsic trans-activating property, but the hormone-binding domain contributes to synergism by a different mechanism.

Animals↗
PubMed1990

Modular structure of a chicken lysozyme silencer: involvement of an unusual thyroid hormone receptor binding site.

Silencer elements, by analogy to enhancer elements, function independently of their position and orientation. We show that the chicken lysozyme silencer S-2.4 kb has many other characteristics in common with enhancer elements. The silencer is comprised of modules that independently repress gene activity--repression being increased synergistically when different or identical modules are combined. Repression is effective both on a complete and on a minimal promoter consisting of a TATA box only. One silencer module is bound in vitro by a 75-93 kd protein, termed NeP1; the other can be bound either by the product of the oncogene v-erbA or by the thyroid hormone receptor. This erbA binding site is unusual in that the palindromic sequence is inverted.

Animals↗
PubMed1990

Repetitive sequence involvement in the duplication and divergence of mouse lysozyme genes.

Mouse M and P lysozymes are the products of separate genes, are specifically expressed in separate tissues, and are adapted to different functions. The lysozyme genes have assumed these markedly different characteristics following their generation by gene duplication 30-50 million years ago. The discovery of the lysozyme P gene only 5 kb upstream from the M gene in tandem repeat has enabled an investigation of the molecular basis of their duplication and subsequent divergence. The duplication is shown to have involved recombination between two B2 repeat sequences flanking the original gene. The resulting downstream copy has retained the myeloid specificity of expression along with just 1.7 kb of upstream sequences, while the upstream copy is inactive in macrophages and has become expressed instead in the small intestine. Although multiple gene conversion events have served to maintain a generally high homology between the genes, certain regions have been found to be specific for either one of the gene pair: two repetitive sequences peculiar to the P region may serve to protect the coding regions from gene conversion, while sequences unique to the M gene may be more directly involved in differential regulation.

Amino Acid Sequence↗
PubMed1990

Transcriptional repression in eukaryotes.

Gene transcription can be controlled by positive or negative regulatory mechanisms; a combination of both is frequently responsible for the observed expression patterns. Analysis of a number of eukaryotic systems suggests that repressors can interfere with many and perhaps all the steps required for transcriptional activation. Transcriptional repression mechanisms can usefully be divided into three major classes: inhibition of DNA binding, blocking of activation and silencing.

Animals↗
PubMed1989

Cooperative interaction of chicken lysozyme enhancer sub-domains partially overlapping with a steroid receptor binding site.

Expression of the lysozyme gene is a marker for the differentiation of macrophages, lysozyme transcription being gradually increased during maturation. We have analyzed the fine structure and function of two macrophage-specific enhancer elements of the chicken lysozyme gene (E-2.7 kb and E-0.2 kb). Both increase their activities upon LPS induction, both contain multiple binding sites for similar or identical nuclear factors and both can be divided into two functional modules. For the E-0.2 kb enhancer we found a synergistic activity of the modules to be dependent on their distance. Binding sites for nuclear proteins within enhancer E-0.2 kb overlap substantially with the previously identified progesterone/glucocorticoid receptor binding site, which is required for steroid induction of lysozyme transcription in the oviduct.

Animals↗
PubMed1988

Many transcription factors interact synergistically with steroid receptors.

Progesterone (PRE) or glucocorticoid receptor (GRE) DNA binding sites are often found clustered with binding sites for other transcription factors. Individual protein binding sites were tested without the influence of adjacent factors by analyzing isolated combinations of several transcription factor binding sites with PREs or GREs. All show strong synergistic effects on steroid induction. The degree of synergism is inversely related to the strength of the GRE. Thus, a steroid responsive unit can be composed of several modules that, if positioned correctly, act synergistically.

Base Sequence↗
PubMed1988

Cooperativity of the glucocorticoid receptor and the CACCC-box binding factor.

Glucocorticoid receptor binding sites (GRE) are often tightly clustered with other transcription factor binding sequences. Examples of this occur upstream of the genes for chicken lysozyme and human metallothionein IIA (ref. 3), in several retroviral LTRs and upstream of the rat tryptophan oxygenase (TO) gene. In the TO gene, sequences immediately upstream of a glucocorticoid receptor binding site are required for steroid induction and contain a CACCC-box identical to that found in the beta globin gene. Here we demonstrate specific binding to this TO-CACCC element and show that it will also act cooperatively with a MMTV glucocorticoid receptor binding site. The response to dexamethasone is independent of the order and relative orientation of these elements but does depend on their precise spacing. Optimal induction occurs at a periodicity of approximately 10 base pairs (bp) indicating a requirement for stereospecific alignment. Binding to the CACCC box, however, is not affected by its distance from the glucocorticoid receptor site. We conclude that the observed cooperativity is mediated by protein:protein interactions and does not depend on cooperative DNA binding.

Acetyltransferases↗
PubMed1988

Mouse lysozyme M gene: isolation, characterization, and expression studies.

We have isolated and characterized both cDNA and genomic DNA of the mouse lysozyme M gene. Derivation of the amino acid sequence from the nucleotide sequences revealed six positions in the carboxyl terminus that differ from partial sequences previously published. The differential detection of specific mRNAs from the closely related lysozyme M and P genes has revealed different but overlapping tissue specificities of expression. The M gene is expressed weakly in myeloblasts, moderately in immature macrophages, and strongly in both mature macrophages and macrophage-rich tissues, while high levels of P transcripts are present only in small intestine. Sites of protein accumulation, rather than gene expression, have been identified by comparative quantitation of mRNA and enzyme levels.

Amino Acid Sequence↗
PubMed1987

Lysozyme gene activity in chicken macrophages is controlled by positive and negative regulatory elements.

The chicken lysozyme gene is constitutively active in macrophages and under the control of steroid hormones in the oviduct. To investigate which DNA elements are involved in the control of its expression in macrophages we performed transient DNA transfer experiments with two different types of plasmids: 5'-deletion mutants of the upstream region of the chicken lysozyme gene and different fragments from this area in front of the thymidine kinase promoter (herpes simplex virus), each placed in front of the CAT (chloramphenicol acetyl transferase) coding sequence. Two enhancers (E-2.7 kb and E-0.2 kb) were characterized. They are active in macrophages, but not in chicken fibroblasts. Furthermore a negative element (N-2.4 kb) was identified, which is active in fibroblasts and promyelocytes, but not in mature macrophages. The combined action of all three elements contributes to the observed lysozyme gene activities: no activity in fibroblasts, moderate activity in promyelocytes and high activity in mature macrophages.

Animals↗
PubMed1987

A new method for constructing linker scanning mutants.

A new procedure for the construction of linker scanning mutants is described. A plasmid containing the target DNA is randomly linearized and slightly shortened by a novel combination of established methods. After partial apurination with formic acid a specific nick or small gap is introduced at the apurinic site by exonuclease III, followed by nuclease S1 cleavage of the strand opposite the nick/gap. Synthetic linkers are ligated to the ends and plasmids having the linker inserted in the target DNA are enriched. Putative linker scanning mutants are identified by their topoisomer patterns after relaxation with topoisomerase I. This technique allows the distinction of plasmids differing in length by a single basepair. We have used this rapid and efficient strategy to generate a set of 32 linker scanning mutants covering the chicken lysozyme promoter from -208 to +15.

Animals↗
PubMed1987

Activity of two different silencer elements of the chicken lysozyme gene can be compensated by enhancer elements.

The chicken lysozyme gene is constitutively expressed in macrophages. Transfection of recombinant genes containing different portions of the lysozyme 5' upstream region revealed the existence of two negative transcriptional elements within 1 kb upstream of the start sites. Both elements placed upstream or downstream of a heterologous promoter-gene unit repress transcription independent of their orientation and are therefore called silencer elements, although their repressing activities 3' of the gene are reduced. One silencer (N-1.0 kb) at position -1 kb consists of the central region of the chicken middle repetitive sequence element CR1 and can be divided into two functional domains. N-1.0 kb is active in all cell types tested. The other silencer (N-0.25 kb) at position -0.25 kb shows reduced activity in primary macrophages. Despite their different specificities, the activity of both silencer elements can be influenced similarly. An inverse linear relationship between the transcriptional activity of the tested constructs and the potential inhibition by the silencer elements was found: weak transcription units can be strongly repressed, whereas strong transcription units can be only weakly repressed. Such a mechanism may help to turn off completely a particular gene in situations or tissues where strong positive regulators are inactive.

Animals↗