Daniel Reines

Acetylated histones are associated with FMR1 in normal but not fragile X-syndrome cells.

Mutation of FMR1 results in fragile X mental retardation. The most common FMR1 mutation is expansion of a CGG repeat tract at the 5´ end of FMR1 (refs 2, 3, 4), which leads to cytosine methylation and transcriptional silencing. Both DNA methylation and histone deacetylation have been associated with transcriptional inactivity. The finding that the methyl cytosine-binding protein MeCP2 binds to histone deacetylases and represses transcription in vivo supports a model in which MeCP2 recruits histone deacetylases to methylated DNA, resulting in histone deacetylation, chromatin condensation and transcriptional silencing. Here we demonstrate that the 5´ end of FMR1 is associated with acetylated histones H3 and H4 in cells from normal individuals, but acetylation is reduced in cells from fragile X patients. Treatment of fragile X cells with 5-aza-2´-deoxycytidine (5-aza-dC) resulted in reassociation of acetylated histones H3 and H4 with FMR1 and transcriptional reactivation, whereas treatment with trichostatin A (TSA) led to almost complete acetylated histone H4 and little acetylated histone H3 reassociation with FMR1, as well as no detectable transcription. Our results represent the first description of loss of histone acetylation at a specific locus in human disease, and advance understanding of the mechanism of FMR1 transcriptional silencing.

Transcription elongation factor SII.

RNA chain elongation by RNA polymerase II (pol II) is a complex and regulated process which is coordinated with capping, splicing, and polyadenylation of the primary transcript. Numerous elongation factors that enable pol II to transcribe faster and/or more efficiently have been purified. SII is one such factor. It helps pol II bypass specific blocks to elongation that are encountered during transcript elongation. SII was first identified biochemically on the basis of its ability to enable pol II to synthesize long transcripts.(1) Both the high resolution structure of SII and the details of its novel mechanism of action have been refined through mutagenesis and sophisticated in vitro assays. SII engages transcribing pol II and assists it in bypassing blocks to elongation by stimulating a cryptic, nascent RNA cleavage activity intrinsic to RNA polymerase. The nuclease activity can also result in removal of misincorporated bases from RNA. Molecular genetic experiments in yeast suggest that SII is generally involved in mRNA synthesis in vivo and that it is one type of a growing collection of elongation factors that regulate pol II. In vertebrates, a family of related SII genes has been identified; some of its members are expressed in a tissue‐specific manner. The principal challenge now is to understand the isoform‐specific functional differences and the biology of regulation exerted by the SII family of proteins on target genes, particularly in multicellular organisms. BioEssays 22:327–336, 2000.

Structural Characterization of RNA Polymerase II Complexes Arrested by a Cyclobutane Pyrimidine Dimer in the Transcribed Strand of Template DNA

We have characterized the properties of immunopurified transcription complexes arrested at a specifically located cyclobutane pyrimidine dimer (CPD) using enzymatic probes and an in vitro transcription system with purified RNA polymerase II (RNAP II) and initiation factors. To help understand how RNAP II distinguishes between a natural impediment and a lesion in the DNA to initiate a repair event, we have compared the conformation of RNAP II complexes arrested at a CPD with complexes arrested at a naturally occurring elongation impediment. The footprint of RNAP II arrested at a CPD, using exonuclease III and T4 DNA polymerase’s 3′→5′ exonuclease, covers ∼35 base pairs and is asymmetrically located around the dimer. A similar footprint is observed when RNAP II is arrested at the human histone H3.3 arrest site. Addition of elongation factor SII to RNAP II arrested at a CPD produced shortened transcripts of discrete lengths up to 25 nucleotides shorter than those seen without SII. After addition of photolyase and exposure to visible light, some of the transcripts could be reelongated beyond the dimer, suggesting that SII-mediated transcript cleavage accompanied significant RNAP II backup, thereby providing access of the repair enzyme to the arresting CPD.

Saccharomyces cerevisiae transcription elongation mutants are defective in PUR5 induction in response to nucleotide depletion.

ABSTRACT IMP dehydrogenase (IMPDH) is the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. It is a target of therapeutically useful drugs and is implicated in the regulation of cell growth rate. In the yeast Saccharomyces cerevisiae, mutations in components of the RNA polymerase II (Pol II) transcription elongation machinery confer increased sensitivity to a drug that inhibits IMPDH, 6-azauracil (6AU), by a mechanism that is poorly understood. This phenotype is thought to reflect the need for an optimally functioning transcription machinery under conditions of lowered intracellular GTP levels. Here we show that in response to the application of IMPDH inhibitors such as 6AU, wild-type yeast strains induce transcription of PUR5, one of four genes encoding IMPDH-related enzymes. Yeast elongation mutants sensitive to 6AU, such as those with a disrupted gene encoding elongation factor SII or those containing amino acid substitutions in Pol II subunits, are defective inPUR5 induction. The inability to fully inducePUR5 correlates with mutations that effect transcription elongation since 6AU-sensitive strains deleted for genes not related to transcription elongation are competent to induce PUR5. DNA encompassing the PUR5 promoter and 5′ untranslated region supports 6AU induction of a luciferase reporter gene in wild-type cells. Thus, yeast sense and respond to nucleotide depletion via a mechanism of transcriptional induction that restores nucleotides to levels required for normal growth. An optimally functioning elongation machinery is critical for this response.

The RNA polymerase II general elongation factors

Synthesis of eukaryotic messenger RNA by RNA polymerase II is governed by the concerted action of a set of general transcription factors that control the activity of polymerase during both the initiation and elongation stages of transcription. To date, five general elongation factors [P-TEFb, SII, TFIIF, Elongin (SIII) and ELL] have been defined biochemically. Here, we discuss these transcription factors and their roles in controlling the activity of the RNA polymerase II elongation complex.

Mechanism and regulation of transcriptional elongation by RNA polymerase II

Over the past few years, biochemical and genetic studies have shed considerable light on the structure and function of the RNA polymerase II (pol II) elongation complex and the transcription factors that control it. Novel elongation factors have been identified and their mechanisms of action characterized in increasing detail; new insights into the biological roles of elongation factors have been gained from genetic studies of the regulation of mRNA synthesis in yeast; and intriguing links between the pol II elongation machinery and the pathways of DNA repair and recombination have emerged.

Properties of an Intergenic Terminator and Start Site Switch That Regulate IMD2 Transcription in Yeast

ABSTRACT The IMD2 gene in Saccharomyces cerevisiae is regulated by intracellular guanine nucleotides. Regulation is exerted through the choice of alternative transcription start sites that results in synthesis of either an unstable short transcript terminating upstream of the start codon or a full-length productive IMD2 mRNA. Start site selection is dictated by the intracellular guanine nucleotide levels. Here we have mapped the polyadenylation sites of the upstream, unstable short transcripts that form a heterogeneous family of RNAs of ≈200 nucleotides. The switch from the upstream to downstream start sites required the Rpb9 subunit of RNA polymerase II. The enzymes ability to locate the downstream initiation site decreased exponentially as the start was moved downstream from the TATA box. This suggests that RNA polymerase IIs pincer grip is important as it slides on DNA in search of a start site. Exosome degradation of the upstream transcripts was highly dependent upon the distance between the terminator and promoter. Similarly, termination was dependent upon the Sen1 helicase when close to the promoter. These findings extend the emerging concept that distinct modes of termination by RNA polymerase II exist and that the distance of the terminator from the promoter, as well as its sequence, is important for the pathway chosen.

Large-scale screening of yeast mutants for sensitivity to the IMP dehydrogenase inhibitor 6-azauracil

Mutations in several genes encoding components of the RNA polymerase II elongation machinery render S. cerevisiae cells sensitive to the drug 6‐azauracil (6AU), an inhibitor of IMP dehydrogenase and orotidylate decarboxylase. It is thought that a reduction in nucleotide levels following drug treatment causes transcriptional elongation to be more dependent on a fully functional RNA polymerase. To gain insight into the basis of the 6AU‐sensitive phenotype and discern its specificity, we screened almost 3000 deletion mutants for growth in the presence of drug; 42 (1.5%) were reproducibly sensitive to the drug. The sensitive mutants included several missing known transcription elongation factors, but the majority were in genes involved in other cellular processes. Not all of the 6AU‐sensitive strains displayed cross‐sensitivity to mycophenolic acid (MPA), another drug that inhibits IMP dehydrogenase and has been employed as a screening agent for elongation mutants, showing that these two drugs are mechanistically distinct. Several of the mutants were tested for the ability to induce transcription of IMP dehydrogenase‐encoding genes, in response to 6‐AU and MPA treatment. As expected, mutants defective in transcriptional elongation factors were unable to fully induce IMPDH expression. However, most of the 6AU‐sensitive strains had normal levels of IMPDH expression. Thus, although 6AU‐sensitivity often results from defects in the elongation machinery, mutations that compromise processes other than transcription and induction of IMPDH also lead to sensitivity to this drug. Copyright

T7 RNA polymerase bypass of large gaps on the template strand reveals a critical role of the nontemplate strand in elongation

We show that T7 RNA polymerase can efficiently transcribe DNA containing gaps from one to five bases in the template strand. Surprisingly, broken template strands missing up to 24 bases can still be transcribed, although at reduced efficiency. The resulting transcripts contain the full template sequence with the RNA deleted for the gapped region missing on the template strand. These findings indicate that the end of a downstream template strand can be brought into the polymerase and transcribed as if it were a part of an intact polynucleotide chain by utilizing the unpaired nontemplate strand. This, as well as transcription of an intact template strand, relies heavily upon the non-template strand, suggesting that a duplex DNA-binding site on the leading edge of RNA polymerase is required for RNA chain elongation on DNA templates. This work contributes substantially to the emerging picture that the nontemplate strand is an important element of the transcription elongation complex.

Mutations in RNA Polymerase II and Elongation Factor SII Severely Reduce mRNA Levels in Saccharomyces cerevisiae

ABSTRACT Elongation factor SII interacts with RNA polymerase II and enables it to transcribe through arrest sites in vitro. The set of genes dependent upon SII function in vivo and the effects on RNA levels of mutations in different components of the elongation machinery are poorly understood. Using yeast lacking SII and bearing a conditional allele of RPB2, the gene encoding the second largest subunit of RNA polymerase II, we describe a genetic interaction between SII and RPB2. An SII gene disruption or therpb2-10 mutation, which yields an arrest-prone enzyme in vitro, confers sensitivity to 6-azauracil (6AU), a drug that depresses cellular nucleoside triphosphates. Cells with both mutations had reduced levels of total poly(A)+ RNA and specific mRNAs and displayed a synergistic level of drug hypersensitivity. In cells in which the SII gene was inactivated, rpb2-10 became dominant, as if template-associated mutant RNA polymerase II hindered the ability of wild-type polymerase to transcribe. Interestingly, while 6AU depressed RNA levels in both wild-type and mutant cells, wild-type cells reestablished normal RNA levels, whereas double-mutant cells could not. This work shows the importance of an optimally functioning elongation machinery for in vivo RNA synthesis and identifies an initial set of candidate genes with which SII-dependent transcription can be studied.