Impact of meltwater flow intensity on the spatiotemporal heterogeneity of microbial mats in the McMurdo Dry Valleys, Antarctica.

The meltwater streams of the McMurdo Dry Valleys are hot spots of biological diversity in the climate-sensitive polar desert landscape. Microbial mats, largely comprised of cyanobacteria, dominate the streams which flow for a brief window of time (~10 weeks) over the austral summer. These communities, critical to nutrient and carbon cycling, display previously uncharacterized patterns of rapid destabilization and recovery upon exposure to variable and physiologically detrimental conditions. Here, we characterize changes in biodiversity, transcriptional responses and activity of microbial mats in response to hydrological disturbance over spatiotemporal gradients. While diverse metabolic strategies persist between marginal mats and main channel mats, data collected from 4 time points during the austral summer revealed a homogenization of the mat communities during the mid-season peak meltwater flow, directly influencing the biogeochemical roles of this stream ecosystem. Gene expression pattern analyses identified strong functional sensitivities of nitrogen-fixing marginal mats to changes in hydrological activities. Stress response markers detailed the environmental challenges of each microhabitat and the molecular mechanisms underpinning survival in a polar desert ecosystem at the forefront of climate change. At mid and end points in the flow cycle, mobile genetic elements were upregulated across all mat types indicating high degrees of genome evolvability and transcriptional synchronies. Additionally, we identified novel antifreeze activity in the stream microbial mats indicating the presence of ice-binding proteins (IBPs). Cumulatively, these data provide a new view of active intra-stream diversity, biotic interactions and alterations in ecosystem function over a high-flow hydrological regime.

Molecular dissection of DNA sequences and factors involved in slow muscle-specific transcription.

Transcription is a major regulatory mechanism for the generation of slow- and fast-twitch myofibers. We previously identified an upstream region of the slow TnI gene (slow upstream regulatory element [SURE]) and an intronic region of the fast TnI gene (fast intronic regulatory element [FIRE]) that are sufficient to direct fiber type-specific transcription in transgenic mice. Here we demonstrate that the downstream half of TnI SURE, containing E box, NFAT, MEF-2, and CACC motifs, is sufficient to confer pan-skeletal muscle-specific expression in transgenic mice. However, upstream regions of SURE and FIRE are required for slow and fast fiber type specificity, respectively. By adding back upstream SURE sequences to the pan-muscle-specific enhancer, we delineated a 15-bp region necessary for slow muscle specificity. Using this sequence in a yeast one-hybrid screen, we isolated cDNAs for general transcription factor 3 (GTF3)/muscle TFII-I repeat domain-containing protein 1 (MusTRD1). GTF3 is a multidomain nuclear protein related to initiator element-binding transcription factor TF II-I; the genes for both proteins are deleted in persons with Williams-Beuren syndrome, who often manifest muscle weakness. Gel retardation assays revealed that full-length GTF3, as well as its carboxy-terminal half, specifically bind the bicoid-like motif of SURE (GTTAATCCG). GTF3 expression is neither muscle nor fiber type specific. Its levels are highest during a period of fetal development that coincides with the emergence of specific fiber types and transiently increases in regenerating muscles damaged by bupivacaine. We further show that transcription from TnI SURE is repressed by GTF3 when overexpressed in electroporated adult soleus muscles. These results suggest a role for GTF3 as a regulator of slow TnI expression during early stages of muscle development and suggest how it could contribute to Williams-Beuren syndrome.

Fiber-type-specific transcription of the troponin I slow gene is regulated by multiple elements.

The regulatory elements that restrict transcription of genes encoding contractile proteins specifically to either slow- or fast-twitch skeletal muscles are unknown. As an initial step towards understanding the mechanisms that generate muscle diversity during development, we have identified a 128-bp troponin I slow upstream element (SURE) and a 144-bp troponin I fast intronic element (FIRE) that confer fiber type specificity in transgenic mice (M. Nakayama et al., Mol. Cell. Biol. 16:2408-2417, 1996). SURE and FIRE have maintained the spatial organization of four conserved motifs (3' to 5'): an E box, an AT-rich site (A/T2) that binds MEF-2, a CACC site, and a novel CAGG motif. Troponin I slow (TnIs) constructs harboring mutations in these motifs were analyzed in transiently and stably transfected Sol8 myocytes and in transgenic mice to assess their function. Mutations of the E-box, A/T2, and CAGG motifs completely abolish transcription from the TnI SURE. In contrast, mutation of the CACC motif had no significant effect in transfected myocytes or on the slow-specific transcription of the TnI SURE in transgenic mice. To assess the role of E boxes in fiber type specificity, a chimeric enhancer was constructed in which the E box of SURE was replaced with the E box from FIRE. This TnI E box chimera, which lacks the SURE NFAT site, confers essentially the same levels of transcription in transgenic mice as those conferred by wild-type SURE and is specifically expressed in slow-twitch muscles, indicating that the E box on its own cannot determine the fiber-type-specific expression of the TnI promoter. The importance of the 5' half of SURE, which bears little homology to the TnI FIRE, in muscle-specific expression was analyzed by deletion and linker scanning analyses. Removal of the 5' half of SURE (-846 to -811) results in the loss of expression in stably transfected but not in transiently expressing myocytes. Linker scanning mutations identified sequences in this region that are necessary for the function of SURE when integrated into chromatin. One of these sites (GTTAATCCG), which is highly homologous to a bicoid consensus site, binds to nuclear proteins from several mesodermal cells. These results show that multiple elements are involved in the muscle-specific activity of the TnIs promoter and that interactions between upstream and downstream regions of SURE are important for transcription in the context of native chromatin.

Conformationally defined retinoic acid analogues. 4. Potential new agents for acute promyelocytic and juvenile myelomonocytic leukemias.

We recently synthesized several conformationally constrained retinoic acid (RA) analogues [8-(2'-cyclohexen-1'-ylidene)-3, 7-dimethyl-2,4,6-octatrienoic acids with different alkyl substituents at 2' (R1) and 3' (R2) positions on the cyclohexene ring] (Muccio et al. J. Med. Chem. 1996, 39, 3625) as cancer chemopreventive agents. UAB8 (R1 = Et; R2 = iPr), which contains sufficient steric bulk at the terminal end of the polyene chain to mimic the trimethylcyclohexenyl ring of RA, displayed biological properties similar to those of RA. To explore the efficacy of this retinoid in acute promyelocytic leukemia (APL) and juvenile myelomonocytic leukemia (JMML), we evaluated UAB8 isomers in in vitro assays which measure the capacity of retinoids to inhibit aberrant myeloid colony growth from blood or bone marrow cells obtained from human JMML patients and in assays measuring the potential of retinoids to differentiate NB4 cells (an APL cell line). Both (all-E)- and (13Z)-UAB8 were 2-fold more active than RA in the NB4 cell differentiation assay; however, only (all-E)-UAB8 had comparable activity to the natural retinoids in the JMML cell assays. These results were compared to the biological effectiveness of a new retinoid, UAB30 [8-(3', 4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4, 6-octatrienoic acid], which had different nuclear receptor binding and transactivational properties than UAB8. Relative to (all-E)-RA and (all-E)-UAB8, (all-E)-UAB30 bound well to RARalpha but did not activate transcription-mediated RARalpha homodimers, even though it was effective in RARbeta- and RARgamma-mediated transactivational assays. In APL assays, this retinoid had much reduced activity and was only moderately effective in JMML assays and in cancer chemoprevention assays.

Analysis of homo- and heterodimerization of retinoid receptors in solution.

To characterize the dimerization of retinoid receptors in solution, RAR alpha homodimers and RAR alpha-RXR alpha heterodimers, formed in the absence or the presence of a naturally occurring RA response element (betaRARE) under different ionic conditions, were analyzed by size-exclusion fast protein liquid chromatography and sucrose density gradient sedimentation. In the presence of [3H]RA both RAR alpha and RXR alpha existed primarily as monomers of 50 kDa in solutions containing 80 mM KCl. However, when betaRARE was included in these incubations, a 40-fold increase in the occurrence of both the RAR alpha homodimers and the RAR alpha-RXR alpha heterodimers (125 kDa) was observed. The presence of RAR alpha and RXR alpha in the betaRARE-associated homo- and heterodimers was confirmed by the positive interaction of the receptors with the specific antibodies. Both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers bound betaRARE even in the absence of the ligand RA with the heterodimer showing a 2- to 4-fold greater affinity than the homodimer for the DNA binding element. When the receptors were incubated in solutions of increasing ionic concentration (50-300 mM KCl), a decrease in the amount of both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers was accompanied by a corresponding increase in the monomeric fraction even in the presence of betaRARE, suggesting that the high salt concentrations inhibit the surface to surface interactions between the monomers. These observations suggest that in vivo, as in solution, the formation of a stable retinoid receptor dimer complex is dependent upon both receptor-receptor and receptor-RARE interactions.

Didehydroretinoic acid: retinoid receptor-mediated transcriptional activation and binding properties.

All-trans-3,4-Didehydroretinoic acid (vitamin A2 acid; DDRA) is one of the retinoids present in human skin, the most responsive tissue to retinoid treatment. To understand the mechanism of action of DDRA in the control of differentiation and tumorigenesis, we studied its interaction with cellular retinoic acid-binding proteins (CRABPs) and nuclear all-trans-retinoic acid (RA) receptors (RARs), and 9-cis-retinoic acid receptors (RXRs). The IC50 plots of DDRA for inhibition of [3H]RA binding to CRABP I and II and to RAR alpha, beta and gamma illustrate that this retinoid binds with the same affinity as RA to these proteins. DDRA, however, showed higher affinity than RA for RXR alpha. Evaluation of the transcriptional activation potential of DDRA in CV-1 cells showed that this retinoid induced RAR alpha-mediated transcription to the same magnitude as RA in the 10(-9) to 10(-6) M concentration range. However, in comparison to RA, DDRA produced a 2- to 3-fold higher activation of the transcription mediated by RXR alpha homodimers, as well as RAR beta-RXR alpha heterodimers. These results suggest that the biological activity of retinoids in the skin may be attained through the joint potential of both RA and DDRA.

Characterization of cellular retinoic acid-binding protein I from chick embryo and its ligand binding properties.

In order to characterize cellular retinoic acid-binding protein (CRABP) from chick embryos, we resolved the [3H]RA-binding activity of embryo extracts by Mono Q anion-exchange chromatography. Fractions containing [3H]RA-binding proteins eluted in two peaks at 7 and 10 min with the latter showing 10-fold higher activity than the former. When the [3H]RA-binding activity in the major peak was resolved on a Superose-12 size-exclusion column, a protein of about 15,000 Da, similar in size to CRABP I or II, was eluted. The identity of this RA-binding component as CRABP I was confirmed by its immunopositive reaction with a CRABP I-specific monoclonal antibody. The chick embryo CRABP I, upon electrophoresis on native gel, however, showed slower migration than the mouse CRABP I, although both exhibited similar isoelectric pH (pI) of about 4.5. Equilibrium binding studies performed under saturating levels of RA indicated that the retinoid bound to the chick CRABP I with a Kd of 27 nM, a value similar to that reported for the native form of this protein from other species. Moreover, as indicated by their IC50 values, the relative binding affinities of various RA analogs for chick CRABP I are consistent with those obtained with human and mouse CRABP I. These results demonstrate that the major RA-binding protein expressed in chick embryo, while having a different charge as judged by electrophoretic mobility, is similar to mouse CRABP I in its size, pI, antigenic specificity, and ligand binding properties.

Conformationally defined 6-s-trans-retinoic acid analogs. 3. Structure-activity relationships for nuclear receptor binding, transcriptional activity, and cancer chemopreventive activity.

We recently demonstrated that conformationally defined 6-s-trans-retinoic acid (RA) analogs were effective in the prevention of skin papillomas (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) and selective agonists for nuclear receptor binding and activation (Alam et al. J. Med. Chem. 1995, 38, 2302-2310). In order to probe important structure-activity relationships, we evaluated a homologous series of four 6-s-trans-retinoids that are 8-(2'-cyclohexen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acids with different substituents at 2' (R2) and 3' (R1) positions on the cyclohexene ring. UAB1 (R1 = R2 = H), UAB4 (R1 = R2 = Me), UAB7 (R1 = Me, R2 = iPr), and UAB8 (R1 = Et, R2 = iPr) contain alkyl R groups that mimic, to different extents, portions of the trimethylcyclohexenyl ring of RA. Both 9Z- and all-E-isomers of these retinoids were evaluated in binding assays for cellular retinoic acid-binding proteins (CRABP-I and CRABP-II), a nuclear retinoic acid receptor (RAR alpha), and a nuclear retinoid X receptor (RXR alpha). The all-E-isomers of UAB retinoids bound tightly to CRABPs and RAR alpha, the binding affinity of the all-E-isomer increased systematically from UAB1 to UAB8, and binding for the latter was comparable to that of all-E-RA. In contrast to RA, the (9Z)-UAB retinoids were at least 200-fold less active than the all-E-isomers in binding to RAR alpha. The (9Z)-UAB isomers exhibited increasingly stronger binding to RXR alpha, and (9Z)-UAB8 was nearly as effective as (9Z)-RA in binding affinity. The retinoids were also evaluated in gene expression assays mediated by RAR alpha and RXR alpha homodimers or RAR alpha/RXR alpha heterodimers. Consistent with the binding affinities, the (all-E)-UAB retinoids activated gene transciption mediated by RAR alpha homodimers or RAR alpha/RXR alpha heterodimers, while the (9Z)-UAB isomers activated only the RXR alpha homodimer-mediated transcription. The all-E- and 9Z-isomers of the UAB retinoids were further evaluated for their capacity to prevent the induction of mouse skin papillomas. When compared to RA, only the (all-E)-UAB retinoids containing bulky R1 and R2 groups were effective in this chemoprevention assay. (9Z)-RA displayed equal capacity as RA to prevent papillomas, while the 9Z-isomers of the UAB retinoids were much less effective. Taken together, these studies demonstrate that the cyclohexenyl ring substituents of 6-s-trans-UAB retinoids are important for their biological activities and that the chemopreventive effect of the all-E-isomers of these retinoids correlates well with their capacity to bind to RARs and activate RAR/RXR-mediated transcription.

Analysis of the effects of CRABP I expression on the RA-induced transcription mediated by retinoid receptors.

The involvement of cellular retinoic acid-binding protein I (CRABP I) in the RA signaling was investigated by examining its effects on the interaction of retinoid receptors (RARs and RXRs) with RA-response elements (RAREs) as well as on the RA-induced transcription mediated by retinoid receptors. Analysis of the expression of mouse CRABP I from a cDNA expression plasmid in COS-1 cells revealed that this protein was about 5-fold more abundant in cytosol than in nuclei. The identity and the localization of CRABP I in the cytoplasm as well as the nuclei were also confirmed by the immunoperoxidase staining of the transfected COS-1 cells with CRABP I-specific antibody. When the nuclear extract containing a 10-fold molar excess of CRABP I was incubated with RAR alpha extract in the presence of [3H]RA and resolved on an FPLC size-exclusion column, a 20% decrease in the bound radioactivity in the RAR alpha fraction was accompanied by a proportional increase in the CRABP I fraction. In contrast, the addition of CRABP I did not significantly affect the interaction of RAR alpha or RAR alpha-RXR alpha heterodimers with RAREs. Moreover, the coexpression of CRABP I in CV-1 cells did not markedly inhibit or enhance the transcription activated by RARs and RAR alpha-RXR alpha heterodimers under RA concentrations ranging from 10(-10) to 10(-6) M. These results demonstrate that CRABP I, while it might be important for RA homeostasis, is not directly involved in the retinoid receptor-mediated RA-signaling pathway.

Biochemical characteristics and differentiating activity of 4-oxo analogs of retinoic acid.

3-Methyl-4-oxoretinoic acid and 3-cinnamyl-4-oxoretinoic acid bind to a cellular retinoic acid-binding protein (CRABP-II) and to a retinoic acid-receptor protein (RARa). These analogs of 4-oxoretinoic acid, as well as the parent compound, have less binding affinity than retinoic acid. Cotransfection assays in CV-1 cells with plasmids containing cDNAs for RAR alpha, RAR beta and RAR gamma (homodimers) and RAR alpha-RXR alpha and RAR beta-RXR alpha (heterodimers), indicate that 3-cinnamyl-4-oxoretinoic acid induces relatively less transcriptional activity than 4-oxoretinoic acid and its 3-methyl analog, both of which are less effective than retinoic acid. In differentiating mouse F9 embryocarcarcinoma cells, the order of effectiveness is retinoic acid > 4-oxoretinoic acid = 3-methyl-4-oxoretinoic acid > 3-cinnamyl-4-oxoretinoic acid. This order of potency is similar to that for inhibition of induction of ornithine decarboxylase (ODC) activity and for prevention of papillomas on the skin of mice. Binding to CRABP-II and activation of RARs appear to be important factors for expression of differentiating activity, inhibition of induction of ODC activity and prevention of papillomas on the skin of mice.

Two Sp1-binding sites mediate cAMP-induced transcription of the bovine CYP11A gene through the protein kinase A signaling pathway.

Two sequence elements located at -111 to -100 base pairs and -70 to -50 base pairs in the 5'-flanking region of the bovine CYP11A gene and in closely related positions in CYP11A of other species contain G-rich regions that are similar to the consensus Sp1-binding site. These sequences bind the purified transcription factor Sp1 as well as nuclear proteins from mouse Y1 adrenal cells that interact with an antibody specific for Sp1. Both of these CYP11A sequences support basal and cAMP-dependent transcription of reporter gene plasmids transfected into Y1 cells, and mutations within the G-rich -111/-100-base pair sequence that reduce or eliminate the binding of Sp1-related Y1 nuclear proteins also markedly reduce cAMP-induced transcription. cAMP-dependent transcription supported by both CYP11A sequence elements is mediated by protein kinase A at levels comparable to that promoted by different cAMP-response sequences and transcription factors in other genes involved in steroidogenesis. These results indicate that ACTH-dependent regulation of cholesterol side chain cleavage cytochrome P450 levels in the adrenal cortex which is mediated through cAMP involves the ubiquitous transcription factor Sp1.

Conformationally defined 6-s-trans-retinoic acid analogs. 2. Selective agonists for nuclear receptor binding and transcriptional activity.

We recently demonstrated in animal models that a new conformationally defined RA isomer (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) was as effective as RA in the prevention of skin papillomas but was less toxic. In order to provide more details concerning this improved action, we report here the preparation of a homologous conformationally defined 6-s-trans-retinoid (1) and investigate its ability to interact with proteins and to activate gene expression. Four configurational isomers of 1 were evaluated in binding assays for cellular retinoic acid binding protein, CRABP (isolated from chick skin); CRABP-I and CRABP-II (cloned from mouse); nuclear retinoic acid receptors (RARs); and nuclear retinoid X receptors (RXRs). In each assay the all-E-isomer of this retinoid had an activity that was comparable to that of (all-E)-RA. However, the 9Z-isomer was at least 200-fold less active than (all-E)-RA in binding to different RARs, while it was only 6-20 times less active than (9Z)-RA in binding to different RXRs. In an in vivo transient transfection assay, the all-E-isomer activated a reporter gene containing a retinoic acid response element (RARE) with efficiency similar to (all-E)-RA when expression vectors for either RAR alpha, RAR beta, RAR gamma alone or RAR alpha together with RXR alpha were cotransfected. In contrast, the 9Z-isomer was much less active than (9Z)-RA in the same assay systems. However, (9Z)-1 efficiently enhanced the DNA binding and transactivational activity of RXR alpha homodimers. Taken together, these studies demonstrate that the all-E- and 9Z-isomers of this retinoid are selective and potent agonists of RAR and RXR binding and activation.

Transcriptional regulatory elements for basal expression of cytochrome P450IIC genes.

To analyze the transcriptional regulatory elements in rabbit cytochrome P450IIC genes, varying lengths of the 5'-flanking regions of CYP2C1 and CYP2C2 were fused to a luciferase reporter gene. Promoter activity was assayed by transfection into HepG2 cells, a hepatic cell line, and monkey kidney COS-1 cells, a nonhepatic cell line. Activity of the CYP2C1 promoter in HepG2 cells increased slightly with progressive 5' deletions of the 5'-flanking region from nucleotide -3600 to -1500 relative to the transcription start site. Additional deletions to -900, -358, and -116 each reduced activity by about 50%, and deletion of the sequence from -116 to -67 reduced activity by a factor of 12. Activity of the CYP2C2 promoter increased about 3-fold with progressive 5' deletions of sequence from nucleotide -3500 to -410. In contrast, deletions of sequences from -251 to -193 and from -133 to -64 reduced promoter activity by factors of 2 and 8, respectively. In COS-1 cells, the maximum activities of the CYP2C1 and CYP2C2 promoters normalized to a Rous sarcoma viral promoter were about 10-20% of that in the HepG2 cells. The changes in activity between different constructions in COS-1 cells largely paralleled those in the HepG2 cells except for deletions of the sequences -133 to -64 and -116 to -67 for CYP2C1 and CYP2C2, respectively, which produced the largest reduction of promoter activity in HepG2 cells but had little effect in COS-1 cells. These results show that HepG2-specific regulatory elements are present in the regions between -120 and -65 in both genes. Nuclear proteins from HepG2 cells, but not from COS-1 cells, bound to sequences within these regions, and the binding was inhibited by an oligonucleotide containing a sequence conserved in rabbit P450IIC genes which has been designated the HepG2-specific P450 2C factor-1 (HPF1) motif. Mutation of this sequence eliminated the binding of nuclear proteins and reduced transcriptional activity 25-fold. The HPF1 binding sequence is conserved in CYP2A, CYP2C, and CYP2D genes and resembles the binding motif for hepatic nuclear factor-4. These results demonstrate that CYP2C1 and CYP2C2 contain several potential regulatory elements for basal expression, including one HepG2-specific sequence that may be important for liver expression.