Nuclear factor 90 (NF90) was originally isolated in a complex that binds to the antigen recognition response element (ARRE-2) present in the interleukin-2 promoter. To characterize the transcriptional properties of NF90 in mammalian cells, we examined its ability to modulate promoter function in cellular transfection assays. NF90-Gal4 fusion proteins inhibited transcription from the adenovirus major late promoter in a fashion that was dependent on Gal4 targeting. Conversely, NF90 activated the cytomegalovirus immediateearly promoter, to which it was not targeted. These effects required distinct but overlapping domains in the C terminus of NF90, which contains a functional nuclear localization signal and two double-stranded-RNA binding motifs. NF90 is present in cellular complexes together with the NF45 protein. Transfection assays showed that NF45 binds NF90 strongly and stimulates its ability to activate but not to inhibit gene expression. This report characterizes NF90 as both a positive and negative regulator of gene expression, depending on the promoter context, and suggests a role for NF45 as a regulator of NF90.

3-Untranslated regions (UTRs) of genes often contain key regulatory elements involved in gene expression control. A high degree of evolutionary conservation in regions of the 3-UTR suggests important, conserved elements. In particular, we are interested in those elements involved in regulation of 3 end formation. In addition to canonical sequence elements, auxiliary sequences likely play an important role in determining the polyadenylation efficiency of mammalian pre-mRNAs. We identified highly conserved sequence elements upstream of the AAUAAA in three human collagen genes, COL1A1, COL1A2, and COL2A1, and demonstrate that these upstream sequence elements (USEs) influence polyadenylation efficiency. Mutation of the USEs decreases polyadenylation efficiency both in vitro and in vivo, and inclusion of competitor oligoribonucleotides representing the USEs specifically inhibit polyadenylation. We have also shown that insertion of a USE into a weak polyadenylation signal can enhance 3 end formation. Close inspection of the COL1A2 3-UTR reveals an unusual feature of two closely spaced, competing polyadenylation signals. Taken together, these data demonstrate that USEs are important auxiliary polyadenylation elements in mammalian genes.Poly(A) tails are found on the 3Ј end of nearly every fully processed eukaryotic mRNA. The poly(A) tail has been suggested to influence mRNA stability, translation, and transport (for review, see Refs. 1-4). Polyadenylation is a two-step process that first involves specific endonucleolytic cleavage at a site determined by binding of polyadenylation factors (for review, see Refs. 5-10). The second step involves polymerization of an adenosine tail to an average length of ϳ200 residues. These steps are tightly coupled processes since reaction intermediates are not detectable under normal conditions.The vast majority of eukaryotic polyadenylation signals contain the consensus sequence AAUAAA between 10 and 35 nucleotides upstream of the actual cleavage and polyadenylation site. In addition, sequences 10 -30 nucleotides downstream of the cleavage site are known to be involved in directing polyadenylation (Refs. 11-13 and references therein). These downstream elements (DSEs) 1 can be characterized as a block containing 4 of 5 uracil (U) residues. These two sequence elements recruit cleavage and polyadenylation specificity factor (CPSF) and cleavage stimulatory factor (CstF), respectively, to define the cleavage site; therefore, mutations within these sequences abolish polyadenylation.The intricate nature of this process implies that polyadenylation might be a useful mechanism to regulate gene expression. The efficiency of 3Ј end processing is a level at which regulation can occur. Because most pre-mRNAs in the cell are not efficiently processed, even small changes in the overall processing efficiency of a particular pre-mRNA may have a substantial effect on gene expression. Experimental evidence has demonstrated that poly(A) signal strength directly influences the amount of mature, exported mR...

Cyclin D2 (Ccnd2) is an essential gene for folliculogenesis, as null mutation in mice impairs granulosa cell proliferation in response to FSH. Ccnd2 mRNA is induced during the estrus cycle by FSH and is rapidly inhibited by LH. Yet, the responsive elements and transcription factors accounting for the gene expression of cyclin D2 in the ovary have not been fully characterized. Using primary cultures of rat granulosa cells and immortalized mouse granulosa cells, we demonstrate a mechanism for the regulation of cyclin D2 at the level of transcription via a PKA-dependent signaling mechanism. The promoter activity of cyclin D2 was shown to be induced by FSH and the catalytic alpha subunit of PKA (PRKACA), and this activity was repressible by inducible cAMP early repressor (ICER), a cAMP response element (CRE) modulator isoform. In silico analysis of the mouse, rat, and human cyclin D2 promoters identified two CRE-binding protein sites, a conserved proximal element and a less conserved distal element relative to the translation start site. The mutation on the proximal element drastically decreases the effects of PRKACA and ICER on the promoter activity, whereas the mutation on the distal element did not contribute to the decrease in the promoter activity. Electrophoretic mobility shift assays and deoxyribonuclease footprint analysis confirmed ICER binding to the proximal element, and chromatin immunoprecipitation analysis demonstrated the occurrence of this binding in vivo. These results showed a CRE within the upstream region of Ccnd2 that is (at least partly) implicated in the stimulation and repression of cyclin D2 transcription. Finally, our data suggest that ICER involvement in the regulation of granulosa cell proliferation as overexpression of ICER results in the inhibition of PRKACA-induced DNA synthesis.

The events culminating in ovulation are controlled by the cyclical actions of hormones such as Follical Stimulating Hormone (FSH) and Luteinizing Hormone (LH). The secondary messenger, cyclic AMP (cAMP) conveys the intracellular activity of these hormones. It is well established that a family of transcription factors facilitate cAMP mediated gene expression, yet it remains unknown how these factors directly affect ovulation. One of these factors, Inducible cAMP Early Repressor (ICER) has been implicated in the transcriptional regulation of cAMP inducible genes during folliculogenesis and ovulation. In order to better determine the role of ICER in ovarian function we have identified novel targets using a genome-wide approach. Using a modification of the chromatin immunoprecipitation (ChIP) assay we directly cloned and sequenced the immunoprecipitated ICER-associated DNAs from an immortalized mouse granulose cell line (GRMO2). The analysis of the immunoprecipitated DNA fragments has revealed that ICER’s binding to DNA has the following distribution; 16% within the promoter region, 31% within an intron, 14% were not within a gene, 6% were within 20kb of a promoter and 3% were within the 3’ end of genes.