through a cAMP-dependent mechanism (Hui et al., John R. Yates III, 2 Hiroshi Takemori, 3, * 2003). The ability of GLP-1 to modulate islet cell gene
The cAMP responsive factor CREB stimulates gene expression, following its phosphorylation at Ser133, via recruitment of the coactivator CBP. In certain cell types, CREB also functions as a constitutive activator, although the underlying mechanisms are not understood. Here, we characterize a conserved family of coactivators, designated TORCs, for Transducers of Regulated CREB activity, that enhances CRE-dependent transcription via a phosphorylation-independent interaction with the bZIP DNA binding/dimerization domain of CREB. TORC recruitment does not appear to modulate CREB DNA binding activity, but rather enhances the interaction of CREB with the TAF(II)130 component of TFIID following its recruitment to the promoter. Remarkably, in certain mucoepidermoid carcinomas, a chromosomal translocation fuses the CREB binding domain of TORC1 to the Notch coactivator Mastermind (MAML2). As expression of the TORC1-MAML2 chimera strongly induced target gene expression via CREB, our results reveal a mechanism by which CREB stimulates transcription in normal and transformed cells.
Recent studies have demonstrated roles for Spt4, Spt5, and Spt6 in the regulation of transcriptional elongation in both yeast and humans. Here, we show that Drosophila Spt5 and Spt6 colocalize at a large number of transcriptionally active chromosomal sites on polytene chromosomes and are rapidly recruited to endogenous and transgenic heat shock loci upon heat shock. Costaining with antibodies to Spt6 and to either the largest subunit of RNA polymerase II or cyclin T, a subunit of the elongation factor P-TEFb, reveals that all three factors have a similar distribution at sites of active transcription. Crosslinking and immunoprecipitation experiments show that Spt5 is present at uninduced heat shock gene promoters, and that upon heat shock, Spt5 and Spt6 associate with the 5 and 3 ends of heat shock genes. Spt6 is recruited within 2 minutes of a heat shock, similar to heat shock factor (HSF); moreover, this recruitment is dependent on HSF. These findings provide support for the roles of Spt5 in promoter-associated pausing and of Spt5 and Spt6 in transcriptional elongation in vivo.
Human DNA profiling using PCR at polymorphic short tandem repeat (STR) loci followed by capillary electrophoresis (CE) size separation and length-based allele typing has been the standard in the forensic community for over 20 years. Over the last decade, Next-Generation Sequencing (NGS) matured rapidly, bringing modern advantages to forensic DNA analysis. The MiSeq FGx™ Forensic Genomics System, comprised of the ForenSeq™ DNA Signature Prep Kit, MiSeq FGx™ Reagent Kit, MiSeq FGx™ instrument and ForenSeq™ Universal Analysis Software, uses PCR to simultaneously amplify up to 231 forensic loci in a single multiplex reaction. Targeted loci include Amelogenin, 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs and three classes of single nucleotide polymorphisms (SNPs). The ForenSeq™ kit includes two primer sets: Amelogenin, 58 STRs and 94 identity informative SNPs (iiSNPs) are amplified using DNA Primer Set A (DPMA; 153 loci); if a laboratory chooses to generate investigative leads using DNA Primer Set B, amplification is targeted to the 153 loci in DPMA plus 22 phenotypic informative (piSNPs) and 56 biogeographical ancestry SNPs (aiSNPs). High-resolution genotypes, including detection of intra-STR sequence variants, are semi-automatically generated with the ForenSeq™ software. This system was subjected to developmental validation studies according to the 2012 Revised SWGDAM Validation Guidelines. A two-step PCR first amplifies the target forensic STR and SNP loci (PCR1); unique, sample-specific indexed adapters or "barcodes" are attached in PCR2. Approximately 1736 ForenSeq™ reactions were analyzed. Studies include DNA substrate testing (cotton swabs, FTA cards, filter paper), species studies from a range of nonhuman organisms, DNA input sensitivity studies from 1ng down to 7.8pg, two-person human DNA mixture testing with three genotype combinations, stability analysis of partially degraded DNA, and effects of five commonly encountered PCR inhibitors. Calculations from ForenSeq™ STR and SNP repeatability and reproducibility studies (1ng template) indicate 100.0% accuracy of the MiSeq FGx™ System in allele calling relative to CE for STRs (1260 samples), and >99.1% accuracy relative to bead array typing for SNPs (1260 samples for iiSNPs, 310 samples for aiSNPs and piSNPs), with >99.0% and >97.8% precision, respectively. Call rates of >99.0% were observed for all STRs and SNPs amplified with both ForenSeq™ primer mixes. Limitations of the MiSeq FGx™ System are discussed. Results described here demonstrate that the MiSeq FGx™ System meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities.
The second messenger cAMP stimulates transcription with burst-attenuation kinetics that mirror the PKA-dependent phosphorylation and subsequent protein phosphatase 1 (PP1)-mediated dephosphorylation of the cAMP responsive element binding protein (CREB) at Ser133. Phosphorylation of Ser133 promotes recruitment of the co-activator histone acetylase (HAT) paralogs CBP and P300, which in turn stimulate acetylation of promoter-bound histones during the burst phase. Remarkably, histone deacetylase (HDAC) inhibitors seem to potentiate CREB activity by prolonging Ser133 phosphorylation in response to cAMP stimulus, suggesting a potential role for HDAC complexes in silencing CREB activity. Here we show that HDAC1 associates with and blocks Ser133 phosphorylation of CREB during pre-stimulus and attenuation phases of the cAMP response. HDAC1 promotes Ser133 dephosphorylation via a stable interaction with PP1, which we detected in co-immunoprecipitation and co-purification studies. These results illustrate a novel mechanism by which signaling and chromatin-modifying activities act coordinately to repress the activity of a phosphorylation-dependent activator.
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