Reproductive morphology of three species of neotropical harvestmen (Opiliones, Laniatores, Gonyleptoidea).

Illustrations of penis morphology are essential components of species descriptions for harvestmen belonging to the suborder Laniatores. Male genitalia are important sources of taxonomic characters and are generally assumed to exhibit relatively little intraspecific variation. In contrast, descriptions of female reproductive morphology are rarely included in taxonomic descriptions of laniatorean harvestmen. As a result, relatively little is known about variation in the external features of the ovipositor. In this study, we used scanning electron microscopy to examine variation in male and female reproductive morphology among three species of harvestmen that are members of the superfamily Gonyleptoidea. Specifically, we examined the microanatomy of penises and ovipositors of Cynortula granulata (Cosmetidae), Phareicranaus calcariferus (Cranaidae), and Rhopalocranaus albilineatus (Manaosbiidae). Our results support the general observation that male reproductive morphology is conservative and displays little intraspecific variation. We observed considerable intraspecific variation in the number and shape of marginal setae on the ventral plate of the penis, but little or no variation in the morphology of the distal border of the ventral plate or the shape of the glans penis or stylus. With respect to female genitalia, we observed considerable intraspecific variation in the number of peripheral setae on the distal tip of the ovipositor. We also observed interspecific variation in the morphology of the peripheral setae (surface of the shaft and at shape of the distal tip), the distribution and morphology of smaller setae on the external surface of the ovipositor, and the surface texture of the external lobes. Our results indicate that there are several features associated with ovipositor morphology among laniatorean harvestmen that may represent potentially informative taxonomic characters.

Selective binding of sterol regulatory element-binding protein isoforms and co-regulatory proteins to promoters for lipid metabolic genes in liver.

Mice were subjected to different dietary manipulations to selectively alter expression of hepatic sterol regulatory element-binding protein 1 (SREBP-1) or SREBP-2. mRNA levels for key target genes were measured and compared with the direct binding of SREBP-1 and -2 to the associated promoters using isoform specific antibodies in chromatin immunoprecipitation studies. A diet supplemented with Zetia (ezetimibe) and lovastatin increased and decreased nuclear SREBP-2 and SREBP-1, respectively, whereas a fasting/refeeding protocol dramatically altered SREBP-1 but had modest effects on SREBP-2 levels. Binding of both SREBP-1 and -2 increased on promoters for 3-hydroxy-3-methylglutaryl-CoA reductase, fatty-acid synthase, and squalene synthase in livers of Zetia/lovastatin-treated mice despite the decline in total SREBP-1 protein. In contrast, only SREBP-2 binding was increased for the low density lipoprotein receptor promoter. Decreased SREBP-1 binding during fasting and a dramatic increase upon refeeding indicates that the lipogenic "overshoot" for fatty-acid synthase gene expression known to occur during high carbohydrate refeeding can be attributed to a similar overshoot in SREBP-1 binding. SREBP co-regulatory protein recruitment was also increased/decreased in parallel with associated changes in SREBP binding, and there were clear distinctions for different promoters in response to the dietary manipulations. Taken together, these studies reveal that there are alternative molecular mechanisms for activating SREBP target genes in response to the different dietary challenges of Zetia/lovastatin versus fasting/refeeding. This underscores the mechanistic flexibility that has evolved at the individual gene/promoter level to maintain metabolic homeostasis in response to shifting nutritional states and environmental fluctuations.

A key role for orphan nuclear receptor liver receptor homologue-1 in activation of fatty acid synthase promoter by liver X receptor.

Liver X receptor (LXR) activates fatty acid synthase (FAS) gene expression through binding to a DR-4 element in the promoter. We show that a distinct nuclear receptor half-site 21 bases downstream of the DR-4 element is also critical for the response of FAS to LXR but is not involved in LXR binding to DNA. This half-site specifically binds liver receptor homologue-1 (LRH-1) in vitro and in vivo, and we show LRH-1 is required for maximal LXR responsiveness of the endogenous FAS gene as well as from promoter reporter constructs. We also demonstrate that LRH-1 stimulation of the FAS LXR response is blocked by the addition of small heterodimer partner (SHP) and that FAS mRNA is overexpressed in SHP knock-out animals, providing evidence that FAS is an in vivo target of SHP repression. Taken together, these findings identify the first direct lipogenic gene target of LRH-1/SHP repression and provide a mechanistic explanation for bile acid repression of FAS and lipogenesis recently reported by others.

Coordinated control of bile acids and lipogenesis through FXR-dependent regulation of fatty acid synthase.

We discovered a nuclear receptor element in the FAS promoter consisting of an inverted repeat spaced by one nucleotide (IR-1) and located 21 bases downstream of a direct repeat sequenced by 4 nucleotides (DR-4) oxysterol liver X receptor response element. An IR-1 is present in promoters of several genes of bile acid and lipid homeostasis and binds farnesoid X receptor/retinoid X receptor (FXR/RXR) heterodimers to mediate bile acid-dependent transcription. We show that FXR/RXRalpha specifically binds to the FAS IR-1 and that the FAS promoter is activated approximately 10-fold by the addition of a synthetic FXR agonist in transient transfection assays. We also demonstrate that endogenous FXR binds directly to the murine FAS promoter in the hepatic genome using a tissue-based chromatin immunoprecipitation procedure. Furthermore, we show that feeding wild-type mice a chow diet supplemented with the natural FXR agonist chenodeoxycholic acid results in a significant induction of FAS mRNA expression. Thus, we have identified a novel IR-1 in the FAS promoter and demonstrate that it mediates FXR/bile acid regulation of the FAS gene. These findings provide the first evidence for direct regulation of lipogenesis by bile acids and also provide a mechanistic rationale for previously unexplained observations regarding bile acid control of FAS expression.

Selective association of sterol regulatory element-binding protein isoforms with target promoters in vivo.

The mRNAs for all three members of the sterol regulatory element-binding protein (SREBP) family are widely expressed, and the proteins are highly similar. They have potential to both hetero- and homodimerize through their bHLHLZ domains, so it has been difficult to definitively study the role of each one apart from the other two. In the current study, we have utilized cell lines that express only one functional SREBP and the chromatin immunoprecipitation technique to analyze individual SREBP binding to three specific target genes: hydroxymethylglutaryl-CoA reductase (Red), fatty acid synthase (FAS), and squalene synthase (SQS). Our studies show that SREBP-2 binds to promoters for all three genes, and in agreement with the original report using these cells, all three mRNAs are also induced. In the line expressing only SREBP-1a, mRNAs for Red and FAS are induced, but SQS is not. Chromatin immunoprecipitation also shows that SREBP-1a is recruited efficiently to Red and FAS promoters but not to SQS. This observation indicates SREBP-2 selectively binds the SQS promoter and is sufficient to explain the lack of SQS mRNA induction in the SREBP-1a-expressing cells. SREBP-1c protein was not stably recruited to any SREBP target promoter despite being fully active in DNA binding when purified from extracts of the corresponding cells. This is also sufficient to explain the lack of SREBP target gene induction by the singular expression of SREBP-1c. We also show that whereas SREBP-1a and -2 proteins interact efficiently with transcriptional co-activators that modify cellular chromatin, SREBP-1c does not. Taken together, our data support a model suggesting that chromatin modification is required during the initial stage of specific site recognition by SREBPs in native chromatin in vivo.

A role for cyclic AMP response element-binding protein (CREB) but not the highly similar ATF-2 protein in sterol regulation of the promoter for 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Sterol regulatory element-binding proteins (SREBPs) activate promoters for key genes of metabolism to keep pace with the cellular demand for lipids. In each SREBP-regulated promoter, at least one ubiquitous co-regulatory factor that binds to a neighboring recognition site is also required for efficient gene induction. Some of these putative co-regulatory proteins are members of transcription factor families that all bind to the same DNA sequence elements in vitro and are often expressed in the same cells. These two observations have made it difficult to assign specific and redundant functions to the unique members of a specific gene family. We have used the chromatin immunoprecipitation (ChIP) technique coupled with a transient complementation assay in Drosophila SL2 cells to directly compare the ability of two members of the CREB/ATF family to function as co-regulatory proteins for SREBP-dependent activation of the HMG-CoA reductase promoter. Results from both of these experimental systems demonstrate that CREB is an efficient SREBP co-regulator but ATF-2 is not.