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D M Robins

Publications and source records attributed to D M Robins.

At least 19 recordsLinked to original sources
PubMed1999

AML3/CBFalpha1 is required for androgen-specific activation of the enhancer of the mouse sex-limited protein (Slp) gene.

A complex 120-base pair enhancer, derived from the mouse sex-limited protein (Slp) gene, is activated solely by the androgen receptor (AR) in specific tissues, although it contains a hormone response element recognized by several steroid receptors. The generation of this transcriptional specificity has been ascribed to the interactions of the receptor with tissue-specific nonreceptor factors bound to accessory sites within the enhancer. Protein-DNA interaction assays revealed two factors binding the 5' part of the enhancer that differ widely in abundance between cells showing AR-specific activation of the Slp element compared with those that also permit activation by glucocorticoid receptor (GR). The factor designated B formed a complex centered on the sequence TGTGGT, a core motif recognized by members of the AML/CBFalpha transcription factor family. This complex was competed by a high affinity binding site specific for AML/CBFalpha and was specifically supershifted by an antibody to AML3/CBFalpha1, placing factor B within the AML3/CBFalpha1 subclass. Interestingly, this factor was shown to bind to a second site in the 3' part of the enhancer, positioned between the two critical AR binding sites. Transfection studies revealed that AML1-ETO, a dominant-negative AML/CBFalpha construct, abrogated AR induction of the enhancer, but not of simple hormone response elements. Furthermore, overexpression of AML3/CBFalpha1 could rescue the AML1-ETO repression. Finally, glutathione S-transferase-AML/CBFalpha fusion proteins demonstrated direct interaction between AML/CBFalpha and steroid receptors. Although this interaction was equivalent between AML1/CBFalpha2 and AR or GR, AML3/CBFalpha1 showed stronger interaction with AR than with GR. These data demonstrate that AML3/CBFalpha1 is functionally required for hormonal induction of the Slp enhancer and that direct, preferential protein-protein interactions may contribute to AR-specific activation. These results demonstrate an intriguing role of AML3/CBFalpha1 in steroid- as well as tissue-specific activation of target genes.

Animals↗
PubMed1999

Sex-limited protein: in vitro and in vivo functions.

Mouse complement component C4 exists in two isoforms, C4 and a protein with expression restricted to male animals called sex-limited protein (Slp). Although Slp is about 95% homologous to C4, it is generally believed to be non-functional, at least in conventional haemolytic complement assays. In a previous study, however, we showed that Slp is haemolytically active in a C1-inhibitor (C1INH)-regulated, EDTA-resistant mouse complement activation pathway. To study other possible implications of this finding, we generated constitutively expressing Slp-transgenic mice. The transgene was crossed into otherwise Slp-deficient C57Bl/6J and NZB mice. Members of the third backcross generation of C57Bl/6J mice were tested for functional Slp and classical and alternative complement pathway activities (CH50 and AP50 levels, respectively). Slp-transgenic C57Bl/6J mice showed enhanced CH50, but normal AP50 levels when compared with non-transgenic littermates. To discover a possible protective role for Slp in spontaneous systemic lupus erythematosus (SLE) in NZBxNZW (NZBxW) mice, the third backcross generation of Slp-transgenic NZB mice was mated with NZW mice and the development of SLE in the female offspring was followed. In these introductory experiments, Slp-transgenic NZBxW animals presented with a significantly extended life span. Our results imply that Slp is a mouse complement component with functions which partially resemble some of those of human C4A.

Animals↗
PubMed1998

Multiple receptor domains interact to permit, or restrict, androgen-specific gene activation.

A critical problem within transcription factor families is how diverse regulatory programs are directed by highly related members. Androgen and glucocorticoid receptors (AR, GR) recognize a consensus DNA hormone response element (HRE), but they activate target genes with precise specificity, largely dependent on the promoter and cell context. We have assessed the role of different receptor domains in hormone-specific response by testing chimeras of AR and GR for their ability to activate the androgen-specific enhancer of the mouse sex-limited protein (Slp) gene. Although all of the mutant receptors activated simple HREs, only a few activated the androgen-specific element. One component shared by receptors functional on the AR-specific target was the AR DNA binding domain. Activation was not due to differential DNA affinity but rather to the AR DNA binding domain escaping suppression directed at the GR DNA binding domain in this enhancer context. A further mechanism increasing specific activation was cooperation of receptors at multiple and weak HREs, which was accentuated in the presence of both the AR N terminus and ligand binding domain. These domains together increased recognition of weak HREs, as demonstrated by in vitro DNase I footprinting and transactivation of mutant enhancers. Further, AR N-terminal subdomains reported to interact directly with the ligand binding domain relieved an inhibitory effect imposed by that domain. Therefore, functions intrinsic to AR augment steroid-specific gene activation, by evading negative regulation operating on the domains of other receptors and by enhancing cooperativity through intra- and inter-receptor domain interactions. These subtle distinctions in AR and GR behavior enforce transcriptional specificity established by the context of nonreceptor factors.

Amino Acid Substitution↗
PubMed1997

Regulatory capacity of an androgen-specific enhancer of the mouse Slp gene in transgenic mice.

Different steroid hormone receptors can activate transcription from the same hormone response element (HRE) in vitro, but in vivo the effects of each hormone on gene activity are distinct. To determine sequences mediating androgen-specific response in a physiological setting, we placed the androgen-responsive mouse sex-limited protein gene (Slp) enhancer before a tkCAT reporter in transgenic mice. The enhancer contains a consensus HRE plus accessory factor binding sites that act in concert to direct transcription in response to androgen. A 160 bp fragment, C'delta2, is responsive to several steroids in transfection; in transgenic mice, this enhancer was active in several tissues of male and female mice, in four of six transgenic lines. In striking contrast, C'delta9, a 120 bp sub-fragment of C'delta2 that responds only to androgen in transfection, showed activity in testes, prostate and kidney, where it was strongly androgen-inducible in females. However, expression was obtained in only one transgenic line. Multimerization of the C'delta9 enhancer conferred expression in prostate, but again in only one line. The greater penetrance of C'delta2 expression was not driven by glucocorticoids, as adrenalectomy had little effect, but may be dependent on the NF-kappaB-like element absent from the C'delta9 fragment. That two transgenic lines showed expression in androgen target sites driven by enhancers that are androgen-specific in vitro suggested that activation of this enhancer, when it could occur, was in response to androgen. The dramatically different behavior of the two related enhancer sequences underscores the importance of chromosomal context to the activity and specificity of regulatory elements.

Adrenal Glands↗
PubMed1997

Steroid hormone responsiveness of a family of closely related mouse proviral elements.

Regulation of the mouse sex-limited protein (Slp) gene in unusual in that hormone response is conferred by the 5' LTR of an upstream inserted provirus, dubbed the imposon (imp1). In a search for additional genes whose regulation has been affected by retrotransposition events, we isolated two partial proviral elements by stringent screening of a mouse genomic library. One clone (imp2) contained a portion of the envelope gene and a 3' LTR that was nearly identical to the 3' LTR of imp1; this similarity extended to insertion into a B1 repetitive element. The second proviral clone (imp3) contained a 5' LTR and associated coding sequences, but lacked its 3' LTR; the LTR of imp3 differed by 12% from the imp1 sequence. To assess potential hormone response, proviral enhancer regions cloned into reporter vectors were tested in transfection. The imp2 enhancer was similar in behavior to imp1, conferring both androgen and glucocorticoid induction in one fragment context and an androgen-specific response in another. In contrast, the imp3 enhancer allowed high expression in the absence of hormone and was less responsive to steroids in general and androgen in particular. These three proviral elements define a small family of steroid responsive proviruses in the mouse genome, and at least one member has had a lasting impact on an endogenous gene's regulation.

Animals↗
PubMed1997

Involvement of an octamer-like sequence within a crucial region of the androgen-dependent Slp enhancer.

Androgen dependence of the mouse sex-limited protein (Slp) gene is conferred by an enhancer encompassing a consensus hormone response element (HRE) and sites for several nonreceptor factors. The footprint IV (FPIV) region of the enhancer plays a key role in hormone- and tissue-specific response, both in vitro and in vivo. We characterized FPIV-binding factors by methylation interference analysis and UV cross-linking of several complexes evident in gel mobility-shift assays. The footprinting analysis revealed that distinct base contacts within the multiple nuclear protein-DNA complexes occurred primarily within a sequence similar to an octamer transcription factor (Oct-1) binding site. With additional data on approximate molecular weights from UV cross-linking, several plausible candidates were tested for their DNA binding and functional activity at FPIV. Oct-like protein binding in gel-shift assays with several cell and tissue extracts was evident using specific competitors and antibodies, but was lower in affinity for FPIV than for an Oct-1 consensus site. Site-directed mutation of the FPIV sequence to a consensus Oct-1 element within the Slp enhancer context increased Oct-1 binding in vitro, but greatly reduced hormonal induction in vivo. This suggested that Oct-1 is not directly involved in response, or alternatively, that Oct-1 bound to the lower-affinity site interacts with neighboring factors significantly differently than Oct-1 bound to a consensus sequence. A sequence overlapping the Oct-like element that was similar to a hepatic nuclear factor-4 (HNF-4) site showed no ability to bind HNF-4 in vitro, nor the related orphan receptor, chicken ovalbumin upstream promoter factor (COUP-TF). Intriguingly, however, expression of COUP-TF in transfection had a dramatic inhibitory effect on response of the androgen-specific enhancer (C' delta9), but did not affect other enhancer configurations that can also be induced by glucocorticoid (C 'delta2). This underscores that, despite extensive sequence identity of C' delta9 and C' delta2, components of the androgen-specific transcription complex differ significantly from that of one that is more generally steroid responsive.

Androgens↗
PubMed1997

Repression of cAMP-induced expression of the mouse P450 17 alpha-hydroxylase/C17-20 lyase gene (Cyp17) by androgens.

In primary cultures of mouse Leydig cells, testosterone represses the cAMP-induced de novo synthesis of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) protein and the accumulation of P450c17 mRNA, via an androgen receptor (AR)-mediated mechanism. To examine the mechanism by which androgens repress the cAMP-induced expression of the mouse Cyp17 gene, constructs containing 5'-flanking sequences of the mouse Cyp17 linked to the chloramphenicol acetyltransferase (CAT) reporter gene were cotransfected into MA-10 tumor Leydig cells with a mouse AR expression plasmid. In the presence of dihydrotestosterone, the cAMP-induced expression of a reporter construct containing -1021 bp of Cyp17 promoter sequences was repressed. In contrast, no repression by dihydrotestosterone was observed when the -1021 bp Cyp17-CAT construct was cotransfected with a human AR expression plasmid missing the second zinc finger of the DNA-binding domain, indicating that DNA binding is involved in AR-mediated repression of Cyp17 expression. Analysis of deletions -346 bp of 5'-flanking region of the mouse Cyp17 promoter are sufficient to confer androgen repression of the cAMP-induced expression of Cyp17. Deoxyribonuclease I footprinting analysis indicated that the AR interacts with sequences between -330. and -278 bp of the Cyp17 promoter. This region overlaps with the previously identified cAMP-responsive region located between -346 and -245 bp of the Cyp17 promoter. These results suggest that AR-mediated repression involves binding of the AR to sequences in the cAMP-responsive region of the Cyp17 promoter, possibly interfering with the binding of the protein(s) that mediate cAMP induction of Cyp17.

Animals↗
PubMed1997

Two distinct mechanisms elicit androgen-dependent expression of the mouse sex-limited protein gene.

The mouse sex-limited protein (Slp) gene is expressed in liver and kidney of adult males and is testosterone-inducible in females, indicative of androgen dependence. Analysis of mRNA levels and chromatin configuration reveals that this androgen regulation is achieved by distinct means in the two tissues. In the liver, Slp expression requires pituitary function, and specifically, as shown by others, a pulsatile pattern of GH secretion that is itself determined by androgen. After hypophysectomy, Slp synthesis cannot be reestablished in liver by testosterone, although mRNA decline can be slowed. In contrast, in the kidney Slp mRNA is directly induced by androgen in hypophysectomized mice. In vivo footprinting was used to examine the role of the Slp enhancer, which directs androgen-specific transcription in transfection and contains a factor-binding site, FPIV, whose protection in vivo has been correlated with Slp expression. In kidney, FPIV was protected in intact males and hypophysectomized mice supplemented with testosterone, but not in females or untreated hypophysectomized mice, corroborating FPIV's association with androgen-driven transcription. Surprisingly, protection of FPIV also occurred in liver of hypophysectomized males treated with testosterone, despite the lack of Slp expression. Thus androgen directly affects the Slp enhancer in kidney, where steroid is sufficient for gene activation, as well as in liver, where chromatin remodeling occurs in response to androgen, although GH is clearly required for expression. This may indicate that both GH and androgen signal transduction pathways target the Slp enhancer to elicit precise gene regulation.

Animals↗
PubMed1996

Hsp90 regulates androgen receptor hormone binding affinity in vivo.

The regulation of human androgen receptor (AR) by the molecular chaperone Hsp90 was investigated using the yeast Saccharomyces cerevisiae as a model system. These studies were performed in strains expressing a conditional temperature-sensitive mutant allele of the hsp82 gene, which encodes Hsp90 protein. At the restrictive temperature in the mutant, there is a decrease in hormone-dependent transactivation by the AR, although steady state levels of AR protein are unchanged. Quantitative hormone binding studies at the permissive temperature revealed the presence of both high affinity and low affinity hormone binding states. At the restrictive temperature in the hsp82 mutant, the high affinity state was abolished, and only the low affinity state was observed. The change in hormone binding affinity was further investigated by a competition assay with the anti-androgen hydroxyflutamide. Under permissive conditions, hydroxyflutamide competes poorly for the synthetic androgen R1881, but under restrictive conditions in the hsp82 mutant strain, hydroxyflutamide was shown to be a potent competitive inhibitor. Our findings indicate that Hsp90 participates in the activation process by maintaining apoAR in a high affinity ligand binding conformation which is important for efficient response to hormone.

Androgen Antagonists↗
PubMed1996

Contextual dependence of steroid receptor function on an androgen-responsive enhancer.

The enhancer of the mouse sex-limited protein (Slp) gene includes a consensus hormone response element (HRE) that interacts with several auxiliary elements for steroid induction. The 160-bp fragment. C' delta 2, confers response to androgen or glucocorticoid in transfection, while a 120-bp subfragment, C' delta 9, is activated only by androgen in some cells. Site-directed mutants were tested to identify elements affecting differential response of androgen or glucocorticoid receptors (AR, GR). While most mutations of C' delta 2 affected induction by either steroid similarly, disruptions of the consensus HRE or an octamer-like sequence were more severe for GR than AR activity. An HRE half-site was critical to androgen-specific induction of C' delta 9 but had little impact in the nonspecific C' delta 2 context. In DNase I footprinting, full-length AR and GR bound similarly to the consensus HRE but dissimilarly to nonconsensus sites. Intriguingly, NF-kappa B bound the region of C' delta 2 absent from C' delta 9. Expression of I kappa B decreased response of C' delta 2, but not C' delta 9, confirming a permissive role of NF-kappa B in steroid activation. In this case, different factors may associate with receptors in the presence of NF-kappa B than those that confer androgen specificity in NF-kappa B's absence, suggesting that exclusion of some factors from a specific transcription complex is as crucial as inclusion of others. This dissection of C' delta 2 and C' delta 9 in vitro reveals subtle distinctions in AR and GR interactions that may underlie specific hormonal response in vivo.

Androgens↗
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↗