Biomedical subjects

D M Robins

Publications and source records attributed to D M Robins.

At least 37 records · Page 2Linked to original sources

PubMed1995

In vivo footprinting of an androgen-dependent enhancer reveals an accessory element integral to hormonal response.

A hormonally responsive enhancer that is specifically activated by androgens resides 2 kilobases upstream of the transcription start site of the mouse sex-limited protein (Slp) gene. We have previously shown that strong androgen induction in transfection requires a consensus hormone response element as well as several nonreceptor factor binding sites within this complex enhancer. To determine which accessory elements are required for androgen-dependent transcription, we have examined binding of nuclear proteins to the enhancer both in vitro and in vivo. In vitro footprinting assays demonstrated that multiple factors present in mouse liver and kidney nuclear extracts bound the enhancer, with tissue-specific but not sex-dependent differences in pattern. In contrast, examination of DNA sites occupied in liver chromatin identified a footprint (FPIV) that is well protected in males but sensitive to DNase I in females. FPIV was occupied in males in other sites of Slp expression, such as kidney, but not in tissues lacking expression, such as lung. FPIV protection was induced in females treated with androgen, abrogated in castrated males, and absent in immature mice, implying hormonal and developmental regulation of FPIV binding. Protection of the hormone response element, in contrast to FPIV, was not obvious but was discerned by analysis of densitometry data. Together with results from in vivo protein-DNA interactions determined for other steroid-dependent enhancers, this suggests that in some cases receptor may permit transcriptional activation by altering chromatin structure to allow access to other factors, which may not necessitate tight binding of receptor itself. This further emphasizes the crucial role of the nonreceptor factors in hormone response. The ubiquitous transcription factor Oct-1 forms complexes with an octamer motif present within FPIV by gel shift analysis with liver and kidney extracts, making Oct-1 an intriguing candidate for partnership in androgen regulation.

Androgens↗

PubMed1994

Specific steroid response from a nonspecific DNA element.

A fundamental dilemma of steroid hormone regulation is how specific transcription is attained in vivo when several receptors recognize the same DNA sequence in vitro. We have identified an enhancer of the mouse sex-limited protein (Slp) gene that is activated by androgens but not by glucocorticoids in transfection. Induction requires a consensus hormone response element (HRE) and multiple auxiliary elements within 120 base pairs. Androgen specificity relies on a dual function to augment androgen but prevent glucocorticoid action from a site that both receptors can bind. The nonreceptor factors are the dominant force in transcriptional specificity, although HRE sequence variations can affect the stringency and magnitude of hormonal response. The effect of HRE variations suggests that receptor position is altered relative to the other factors. Thus protein interactions that elicit specific gene regulation are established by the array of DNA elements in a complex enhancer and can be modulated by subtle sequence differences that may influence precise protein contacts.

Androgens↗

PubMed1993

The stringency and magnitude of androgen-specific gene activation are combinatorial functions of receptor and nonreceptor binding site sequences.

The mechanism by which specific hormonal regulation of gene expression is attained in vivo is a paradox in that several of the steroid receptors recognize the same DNA element in vitro. We have characterized a complex enhancer of the mouse sex-limited protein (Slp) gene that is activated exclusively by androgens but not by glucocorticoids in transfection. Potent androgen induction requires both the consensus hormone response element (HRE) and auxiliary elements residing within the 120-bp DNA fragment C' delta 9. Multiple nonreceptor factors are involved in androgen specificity, with respect to both the elevation of androgen receptor activity and the inactivity of glucocorticoid receptor (GR), since clustered base changes at any of several sites reduce or abolish androgen induction and do not increase glucocorticoid response. However, moving the HRE as little as 10 bases away from the rest of the enhancer allows GR to function, suggesting that GR is repressed by juxtaposition to particular factors within the androgen-specific complex. Surprisingly, some sequence variations of the HRE itself, within the context of C' delta 9, alter the stringency of specificity, as well as the magnitude, of hormonal response. These HRE sequence effects on expression correspond in a qualitative manner with receptor binding, i.e., GR shows a threefold difference in affinities for HREs amongst which androgen receptor does not discriminate. Altering the HRE orientation within the enhancer also affects hormonal stringency, increasing glucocorticoid but not androgen response. The effect of these subtle variations suggests that they alter receptor position with respect to other factors. Thus, protein-protein interactions that elicit specific gene regulation are established by the array of DNA elements in a complex enhancer and can be modulated by sequence variations within these elements that may influence selection of precise protein contacts.

Animals↗

PubMed1992

Androgen-specific gene activation via a consensus glucocorticoid response element is determined by interaction with nonreceptor factors.

A fundamental issue in steroid hormone regulation is the question of how specific transcription is attained in vivo when several receptors can bind the same DNA sequence in vitro. We report an enhancer of the mouse sex-limited protein (Slp) gene that, unlike previously characterized enhancers, is activated by androgens but not by glucocorticoids or progestins. Potent androgen induction requires both a consensus glucocorticoid (hormone) response element and auxiliary elements also present within a 120-base-pair DNA fragment. Cotransfection assays with wild-type and mutant receptors reveal that glucocorticoid receptor can bind, but not transactivate from, the hormone response element within the enhancer. The positive effect of androgen and the null effect of glucocorticoid appear to require the amino-terminal domains of the respective receptors. Thus, exclusive transcriptional response to androgens, and lack of response to glucocorticoids, derives from factor interactions that are determined by the context of the receptor binding site rather than by its distinct sequence.

Androgens↗

PubMed1992

Retrotransposons and the evolution of mammalian gene expression.

Transposable elements, and retroviral-like elements in particular, are a rich potential source of genetic variation within a host's genome. Many mutations of endogenous genes in phylogenetically diverse organisms are due to insertion of elements that affect gene expression by altering the normal pattern of regulation. While few such associations are known to have been maintained over time, two recently elucidated examples suggest transposable elements may have a significant impact in evolution of gene expression. The first example, concerning the mouse sex-limited protein (Slp), clearly establishes that ancient retroviral enhancer sequences now confer hormonal dependence on the adjacent gene. The second example shows that within the human amylase gene family, salivary specific expression has arisen due to inserted sequences, deriving perhaps from a conjunction of two retrotransposable elements.

Amylases↗

PubMed1991

Multiple components of a complex androgen-dependent enhancer.

Sex-limited protein (Slp) is expressed in adult male mice. A 160-basepair fragment 2 kilobases upstream of the gene serves as an androgen-dependent enhancer of chloramphenicol acetyltransferase expression in transient transfection assays in cells with endogenous or cotransfected androgen receptor. One element that is necessary, but not sufficient, for induction is a consensus glucocorticoid (or hormone) response element (HRE). This element binds to the mouse androgen receptor in vitro, but with apparent weak affinity. Induction by the HRE is greatly augmented by an accessory sequence within the 160 basepairs, suggesting that cooperative interactions confer strong response to androgen. Additional elements within the enhancer modulate induction, positively or negatively, and exhibit cell-specific behavior. Of particular interest are two degenerate HREs that are adjacent to the consensus sequence; they show no independent activity, but are functionally significant in conjunction with other elements. The complexity of this enhancer may reflect biological mechanisms that ensure specificity of hormonal response and allow gene expression to respond to changes in hormone concentration.

Animals↗

PubMed1989

Trans-regulatory genes affect Slpa and Slpo expression and act in a tissue-specific manner.

The plasma protein C4 and its androgen-dependent homologue Slp are encoded by genes located in the mouse major histocompatibility complex, H-2. The C4 and Slp protein levels and liver mRNA levels are influenced by non-H-2-linked regulatory genes. The allele-specific regulation of C4 expression and the androgen regulation of Slp expression are manifest only in some of the tissues where these genes are expressed. Therefore, we studied tissues in which the effects of the non-H-2 regulatory genes are apparent. We show that these genes only affect the Slp expression in tissues where it is androgen-dependent. This indicates that the non-H-2 regulatory genes most likely act through interaction with the androgen regulation of Slp expression. We also show that liver cells of mice with the Slpo allele, which do not produce Slp protein, do express Slp mRNA; this expression is also androgen-induced and regulated by non-H-2 genes. Thus, both the Slpa and Slpo alleles appear to be regulated in the same way.

Androgens↗

PubMed1989

Allele-specific occurrence of multiple C4 and Slp mRNAs.

The H-2 S region genes C4 (fourth complement component) and Slp (sex-limited protein) are highly homologous. In males with C4high, Slpa alleles, both proteins are expressed in plasma and the two genes are each transcribed in a single species of mRNA which is indistinguishable in length. The Sk region of the H-2k haplotype carries C4low, Slpo alleles, characterized by very low C4 protein levels in plasma and absence of Slp protein in plasma. We show that mice carrying the Sk region express multiple C4 and Slp mRNAs (RNA doublets) in contrast to mice with any other S region. This represents a genetic polymorphism at the level of RNA multiplicity. The RNA doublets do not result from the use of an alternative upstream initiation site of transcription. We hypothesize that their existence is caused by alternative splicing; however, the use of alternative polyadenylation signals cannot be ruled out. The occurrence of multiple RNA species in strains which carry the C4low, Slpo alleles is possibly related to the very low level of C4 protein expression characteristic for these alleles.

Alleles↗

PubMed1988

An ancient provirus has imposed androgen regulation on the adjacent mouse sex-limited protein gene.

The mouse sex-limited protein (Slp) gene is dependent on androgen for expression, unlike its homologous neighbor, which encodes the fourth component of complement (C4). We have found that the extensive identity of Slp and C4 is disrupted by an endogenous provirus inserted 2 kb upstream of Slp. The 5' LTR of this element corresponds to the previously characterized hormone-responsive enhancer associated with Slp regulation, leading to the conclusion that the provirus has conferred androgen response on the adjacent Slp gene. The provirus is extremely old, based on LTR sequence divergence, the accumulation of mutations in former retroviral-like coding regions, and its stability within the mouse genome. The association of this transposable element with Slp regulation thus provides a long-sought example of an insertional mutation that has been maintained in evolution.

Androgens↗

PubMed1988

Tissue-specific variation in C4 and Slp gene regulation.

C4 and Slp are highly homologous mouse genes that differ in function and regulation. Allelic variants exist in quantitative regulation of C4 and in hormonal regulation of Slp. We have examined expression in several tissues, including liver and peritoneal macrophages which are the major sites of synthesis, using a probe that allows direct comparison of C4 and Slp mRNAs. Correctly-sized and initiated RNA, within an order of magnitude of liver levels, is found in mammary gland, lung, spleen, and kidney; lower levels are detectable in testis, brain, heart and submaxillary gland. By comparing expression in congenic mouse strains differing in C4 and Slp loci, regulation of these genes is seen to vary in different tissues. This provides a well-defined genetic system in which to examine cis-acting sequences and trans-acting factors that result in tissue-specific patterns of gene regulation.

Alleles↗

PubMed1988

A complex androgen-responsive enhancer resides 2 kilobases upstream of the mouse Slp gene.

Neighboring genes encoding the mouse sex-limited protein (Slp) and fourth component of complement (C4) show extensive homology. In contrast to C4, however, Slp is regulated by androgen. One region of the Slp gene capable of hormonal response following transfection was located about 2 kilobases upstream of the transcription start site, where the C4 and Slp sequences diverge. This region, delimited here to a 0.75-kilobase fragment, showed cryptic promoter activity as well as androgen responsiveness in either orientation in front of the bacterial chloramphenicol acetyltransferase coding region. When this fragment was placed upstream of a viral long terminal repeat, increased chloramphenicol acetyltransferase expression derived from the viral promoter. Proteins from nuclear extracts specifically bound to four sequences within the region, near sites that are DNase I hypersensitive in vivo and reflect the hormonal and developmental regulation of Slp. Like several other cellular enhancers, this androgen-responsive element seems to be modular in nature and complex in its function.

Acetyltransferases↗

PubMed1987

Constitutive expression of Slp genes in mouse strain B10.WR directed by C4 regulatory sequences.

The murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins that exhibit very disparate patterns of gene expression: all mice constitutively express C4, whereas only adult male mice from a limited number of standard inbred strains express Slp. Several exceptional strains exhibit constitutive (C4-like) Slp expression, a phenotype that correlates with multiple copies of the Slp gene. To determine the molecular basis for constitutive Slp expression we have isolated genomic clones and compared the sequences of 1.5 kb of 5' flanking DNA from 1 C4 gene and three different Slp genes from the Slp-constitutive strain B10.WR. These sequence comparisons demonstrate C4-like regulatory sequences adjacent to two of the Slp genes. By analysis of cDNA clones isolated from a B10.WR liver library we demonstrate that the constitutive Slp phenotype is due primarily to expression of one of these C4/Slp hybrid genes. It appears likely that Slp gene duplication in strain B10.WR came about via homologous unequal crossover events between C4 and Slp genes; this would accommodate both the gene sequence data and the pattern of C4-like Slp expression in mouse strain B10.WR.

Animals↗

PubMed1987

DNase I-hypersensitive sites associated with expression and hormonal regulation of mouse C4 and Slp genes.

There are four major regions of DNase I hypersensitivity in the 5' regions of the genes for the murine fourth component of complement (C4) and its homologous neighbor, Slp (sex-limited protein). Hypersensitivity around the start site of transcription and approximately equal to 0.5 kilobases upstream correlates qualitatively with expression of these genes. Two hypersensitive sites, at -2.3 and -2.0 kilobases, map specifically to the Slp gene and correlate with its hormonal regulation. That is, these sites are more prominent in male liver chromatin and become more apparent in chromatin from females treated with testosterone. Further, these sites are established in males to a greater extent than in females prior to expression of Slp and may reflect gene-commitment events. Comparison of chromatin from mouse strains differing in C4 and Slp alleles indicates that the four regions of hypersensitivity may be necessary but are not sufficient for high levels of expression.

Animals↗

PubMed1987

Molecular genetics of androgen-dependent and -independent expression of mouse sex-limited protein.

Genes of the mouse S locus encoding C4 (the fourth component complement) and Slp (sex-limited protein) show extensive homology but are distinct in their function and regulation. In some mouse strains, such as B10.D2, Slp is androgen regulated, whereas in others, such as B10.W7R, expression of Slp is constitutive. We have previously shown that the B10.W7R strain has multiple Slp genes. In this report, we present the structure of the single C4 and four Slp genes of the B10.W7R S locus and compare the upstream flanking regions by partial sequence analysis and function in transfection assays. Of the four Slp genes, three (Slpw7.A, Slpw7.B, and Slpw7.C) have upstream and promoter regions very similar to those of C4. The fourth Slp gene (Slpw7.D) is instead virtually identical to the androgen-regulated allele (Slpd from the B10.D2 mouse) in upstream regions. In particular, far-upstream sequences from both Slpd and Slpw7.D render the bacterial chloramphenicol acetyltransferase gene hormonally responsive upon transfection into mammary carcinoma cell lines. The upstream sequences between 2 to 3 kilobases of the Slp promoter initiate transcription from multiple sites when fused proximal to the chloramphenicol acetyltransferase gene, and these transcripts are threefold more abundant in the presence of androgen. This behavior is similar for Slpd and Slpw7.D, which suggests that Slpw7.D may be androgen regulated but that this is masked in vivo by constitutive expression of the other Slp genes. Nonhomologous recombination is implicated not only in expanding the copy number of C4 and Slp genes in the B10.W7R mouse but also in creating hybrid genes with regulatory features of C4 and structural features of Slp.

Androgens↗

PubMed1986

Multiple C4/Slp genes distinguished by expression after transfection.

The S region of the murine major histocompatibility complex contains two closely related genes: C4, encoding the fourth component of complement, and Slp, encoding sex-limited protein. We cloned these genes from a cosmid library of the B10.W7R strain that does not show androgen regulation of the Slp protein. Restriction site polymorphisms revealed at least four C4-like genes within the Sw7 locus, indicating evolutionary amplification of this region. Transfection of these genes into L cells resulted in expression, processing, and secretion of immunologically correct C4 and Slp proteins. At least two different Slp genes and one C4 gene were capable, after transfection, of expressing C4 and Slp indistinguishable from macrophage-derived protein. A third Slp gene exists within this locus whose recombinant cognate did not express in L cells. Thus, the B10.W7R S region includes one C4 gene and at least three Slp-like genes.

Animals↗

PubMed1982

Regulated expression of human growth hormone genes in mouse cells.

We have asked whether there are sequences around the human growth hormone gene that render this gene responsive to induction by glucocorticoid hormones. Recombinant clones encoding human growth hormone were introduced into the chromosome of murine fibroblasts by cotransformation. Exposure of cotransformants to glucocorticoids results in a three to five fold induction of human growth hormone mRNA and a similar induction in secreted human growth hormone protein. The DNA sequences required for induction reside within 500 nucleotides of 5'-flanking DNA. Fusion of this segment of 5'-flanking DNA to the structural gene sequences of a hormone-insensitive gene, such as thymidine kinase, now renders this gene responsive to glucocorticoid induction.

Animals↗

PubMed1981

Transforming DNA integrates into the host chromosome.

A series of rat liver cotransformed cell lines have been constructed containing from 5 to 100 copies of a variant human growth hormone gene. We have used hybridization in situ to demonstrate that most, if not all, cotransformed sequences reside in a chromosome of the host cell. In each of four cell lines examined, hybridization was restricted to a single chromosomal site with no extrachromosomal sites apparent. The site was invariant within each line; however, each line revealed a different site of integration for transforming sequences. In two of the four lines, transforming DNA resided at or near the site of gross chromosomal rearrangements, in one line near an rDNA site, and in one line in the middle of an apparently normal chromosome. Thus, insertion is not restricted to a unique chromosome or chromosomal region.

Animals↗

PubMed1981

Chromosome structure and DNA sequence alterations associated with mutation of transformed genes.

We have constructed a series of tk+ cell lines by DNA-mediated gene transfer to correlate chromosomal behavior and DNA sequence alterations associated with reversion to the tk- phenotype. Tk- revertants were selected from each of four well-characterized transformed cell lines containing the viral tk gene and multiple human growth hormone genes (HGH). Tk- colonies were analyzed for the presence of tk and HGH sequences by blot hybridization to restriction endonuclease cleaved DNA. Revertants were further characterized by detailed karyotype analysis and hybridization in situ. Blot hybridization of forty tk- revertants indicates that over half of the revertants delete all of the transforming DNA from the recipient chromosome. In fifteen additional revertants, significant deletion has occurred, although transforming DNA is retained. The analysis of chromosomes by Giemsa banding together with hybridization in situ reveals that the deletion of transforming DNA is never associated with loss of an entire chromosome. Reversion to the tk- phenotype, therefore, seems to involve discrete deletions of transforming DNA without apparent chromosome loss. In this restricted set of mutants, it thus seems crucial to maintain the diploid chromosomal complement.

Animals↗