Intergenic transcriptional interference is blocked by RNA polymerase III transcription factor TFIIIB in Saccharomyces cerevisiae

The major function of eukaryotic RNA polymerase III is to transcribe transfer RNA, 5S ribosomal RNA, and other small non-protein-coding RNA molecules. Assembly of the RNA polymerase III complex on chromosomal DNA requires the sequential binding of transcription factor complexes TFIIIC and TFIIIB. Recent evidence has suggested that in addition to producing RNA transcripts, chromatin-assembled RNA polymerase III complexes may mediate additional nuclear functions that include chromatin boundary, nucleosome phasing, and general genome organization activities. This study provides evidence of another such extratranscriptional activity of assembled RNA polymerase III complexes, which is the ability to block progression of intergenic RNA polymerase II transcription. We demonstrate that the RNA polymerase III complex bound to the tRNA gene upstream of the Saccharomyces cerevisiae ATG31 gene protects the ATG31 promoter against readthrough transcriptional interference from the upstream noncoding intergenic SUT467 transcription unit. This protection is predominately mediated by binding of the TFIIIB complex. When TFIIIB binding to this tRNA gene is weakened, an extended SUT467-ATG31 readthrough transcript is produced, resulting in compromised ATG31 translation. Since the ATG31 gene product is required for autophagy, strains expressing the readthrough transcript exhibit defective autophagy induction and reduced fitness under autophagy-inducing nitrogen starvation conditions. Given the recent discovery of widespread pervasive transcription in all forms of life, protection of neighboring genes from intergenic transcriptional interference may be a key extratranscriptional function of assembled RNA polymerase III complexes and possibly other DNA binding proteins. © 2014 by the Genetics Society of America

Novel Transactivation Domain in Erythroid Kruppel-like Factor (EKLF)

Erythroid Kruppel-like Factor (EKLF) is an erythroid-specific transcription factor that plays a critical role in γ- to β-globin gene switching during development. To identify essential domains required for EKLF transactivation function, we cotransfected a human erythroleukemia cell line (K562) with a locus control region γ/Luc-β/Cat reporter and an EKLF expression vector. In this assay EKLF mediates a 500-fold induction of β/CAT expression compared with controls. To map essential transactivation domains, progressive NH2-terminal and internal deletion mutants of EKLF were constructed. All EKLF mutants were expressed at wild-type levels, localized to the nucleus, and bound DNA. When mutant EKLF proteins were tested for β/CAT activation, a novel transactivation domain was identified. This novel domain, encompassing amino acids (aa) 140-358, is sufficient for maximal β/CAT activation. An 85-amino acid subdomain within this region (aa 140-225) is essential for its activity. Interestingly, this central transactivation subdomain is functionally redundant with the amino-terminal domain (aa 1-139). Thus, EKLF possesses at least two potent transactivation domains that appear to function in a redundant manner

Activation of δ-globin gene expression by erythroid Krupple-like factor: A potential approach for gene therapy of sickle cell disease

Hemoglobin A2 (HbA2; α2δ2) is a powerful inhibitor of HbS (α2β2/(S) polymerization. However, HbA2 levels are normally low in sickle cell patients. We show that a major reason for low δ-globin gene expression is the defective CACCC box at -90 in the δ-globin promoter. When the CACCC box defect in δ is corrected, expression of an HS2 δ/Luciferase reporter is equivalent to HS2 β/Luciferase. Erythroid Krupple-like factor (EKLF), which binds to the CACCC box of the β-globin gene and activates high-level expression, does not bind to the normal δ-globin promoter. Our goal is to design a modified EKLF that binds to the defective δ-globin promoter and enhances δ-globin gene expression. To test the feasibility of this strategy, we inserted the β-globin CACCC box at -90 of the δ-globin gene promoter to produce an HS2 δ(CAC)-β construct and quantitated human δ- and β-globin mRNA in stably transformed murine erythroleukemia (MEL) cells. δ-Globin mRNA in these cells was 22.0% ± 9.0% of total human globin mRNA (δ/δ + β) as compared with 3.0% ± 1.3% in the HS2 δ-β control. In a second set of experiments a GAL4 DNA-binding site was inserted at -90 of the δ-globin gene to produce an HS2 δ(GAL4)-β construct. This construct and a GAL4((1-147))/EKLF expression vector were stably transfected into MEL cells. δ-Globin mRNA in these cells was 27.8% ± 7.1% of total human globin mRNA as compared with 9.9% ± 2.5% in the HS2 δ(GAL4)-β plus GAL4((1-147)) control. These results show that δ-globin gene expression can be significantly increased by a modified EKLF. Based on these results, we suggest that modified EKLFs, which contain zinc fingers designed to bind specifically to the defective δ-globin CACCC box, may be useful in gene therapy approaches to increase HbA2 levels and inhibit HbS polymerization

Role of erythroid Kruppel-like factor in human γ- to β-globin gene switching

Erythroid Kruppel-like factor (EKLF) is an erythroid-specific transcription factor that contains zinc finger domains similar to the Kruppel protein of Drosophila melanogaster. Previous studies demonstrated that EKLF binds to the CACCC box in the human β-globin gene promoter and activates transcription. CACCC box mutations that cause severe β-thalassemias in humans inhibit EKLF binding. Results described in this paper suggest that EKLF functions predominately in adult erythroid tissue. The EKLF gene is expressed at a 3-fold higher level in adult erythroid tissue than in fetal erythroid tissue, and the EKLF protein binds to the human β-globin promoter 8-fold more efficiently than to the human γ-globin promoter. Co-transfection experiments in the human fetal-like erythroleukemia cell line K562 demonstrate that over-expression of EKLF activates a β-globin reporter construct 1000-fold; a linked γ-globin reporter is activated only 3-fold. Mutation of the β-globin CACCC box severely inhibits activation. These results demonstrate that EKLF is a developmental stage-enriched protein that preferentially activates human β-globin gene expression. The data strongly suggest that EKLF is an important factor involved in human γ- to β-globin gene switching

Epigenetic Chromatin Silencing: Bistability and Front Propagation

The role of post-translational modification of histones in eukaryotic gene regulation is well recognized. Epigenetic silencing of genes via heritable chromatin modifications plays a major role in cell fate specification in higher organisms. We formulate a coarse-grained model of chromatin silencing in yeast and study the conditions under which the system becomes bistable, allowing for different epigenetic states. We also study the dynamics of the boundary between the two locally stable states of chromatin: silenced and unsilenced. The model could be of use in guiding the discussion on chromatin silencing in general. In the context of silencing in budding yeast, it helps us understand the phenotype of various mutants, some of which may be non-trivial to see without the help of a mathematical model. One such example is a mutation that reduces the rate of background acetylation of particular histone side-chains that competes with the deacetylation by Sir2p. The resulting negative feedback due to a Sir protein depletion effect gives rise to interesting counter-intuitive consequences. Our mathematical analysis brings forth the different dynamical behaviors possible within the same molecular model and guides the formulation of more refined hypotheses that could be addressed experimentally.Comment: 19 pages, 5 figure

Co-infection with opportunistic pathogens promotes human immunodeficiency virus type 1 infection in macrophages

Human immunodeficiency virus type 1 (HIV-1) infection is dependent on susceptible host cells that express both CD4 and chemokine co-receptors. The co-receptor CCR5 is associated with primary infection by macrophage-tropic virus isolates, whereas CXCR4 is commonly associated with T cell- and dual- tropic viruses. Once infected, lymphocytes and macrophages may replicate HIV- 1 or harbor latent virus, depending on environmental factors and cellular activation. Immune activation is often associated with viremia, which is consistent with enhanced infection and viral replication in activated cells harboring virus. In this regard, opportunistic infections activate the immune system with the detrimental sequelae of enhanced viral replication and viremia. Under these conditions, viral expansion extends beyond T cells to tissue macrophages, many of which are co-infected with opportunistic pathogens. The opportunistic infections promote macrophage susceptibility to HIV-1 through cytokine modulation and altered chemokine co-receptors, potential targets for intervention

Compromised RNA polymerase III complex assembly leads to local alterations of intergenic RNA polymerase II transcription in Saccharomyces cerevisiae

© 2014 Wang et al. Background: Assembled RNA polymerase III (Pol III) complexes exert local effects on chromatin processes, including influencing transcription of neighboring RNA polymerase II (Pol II) transcribed genes. These properties have been designated as \u27extra-transcriptional\u27 effects of the Pol III complex. Previous coding sequence microarray studies using Pol III factor mutants to determine global effects of Pol III complex assembly on Pol II promoter activity revealed only modest effects that did not correlate with the proximity of Pol III complex binding sites. Results: Given our recent results demonstrating that tDNAs block progression of intergenic Pol II transcription, we hypothesized that extra-transcriptional effects within intergenic regions were not identified in the microarray study. To reconsider global impacts of Pol III complex binding, we used RNA sequencing to compare transcriptomes of wild type versus Pol III transcription factor TFIIIC depleted mutants. The results reveal altered intergenic Pol II transcription near TFIIIC binding sites in the mutant strains, where we observe readthrough of upstream transcripts that normally terminate near these sites, 5\u27- and 3\u27-extended transcripts, and de-repression of adjacent genes and intergenic regions. Conclusions: The results suggest that effects of assembled Pol III complexes on transcription of neighboring Pol II promoters are of greater magnitude than previously appreciated, that such effects influence expression of adjacent genes at transcriptional start site and translational levels, and may explain a function of the conserved ETC sites in yeast. The results may also be relevant to synthetic biology efforts to design a minimal yeast genome

Multiple elements in human β-globin locus control region 5′ HS 2 are involved in enhancer activity and position independent, transgene expression

The human β-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human ε-, γ- and β-giobin genes and, secondly, these sequences function as a powerful enhancer of ε-, γ- and β-globin gene expression. Erythrold-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5′ HS 2 site confers position-independent expression in transgenic mice and enhances human β-giobin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease β-globin gene expression. In contrast, no single site is required for position- independent transgene expression; all mice with site- specific mutations in 5′ HS 2 express human β-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5′ HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression. © 1994 Oxford University Press

Functional Characterization of the Chlamydomonas reinhardtii ERG3 Ortholog, a Gene Involved in the Biosynthesis of Ergosterol

The predominant sterol in the membranes of the alga Chlamydomonas reinhardtii is ergosterol, which is commonly found in the membranes of fungi, but is rarely found in higher plants. Higher plants and fungi synthesize sterols by different pathways, with plants producing cycloartenol as a precursor to end-product sterols, while non-photosynthesizing organisms like yeast and humans produce lanosterol as a precursor. Analysis of the C. reinhardtii genome sequence reveals that this algae is also likely to synthesize sterols using a pathway resembling the higher plant pathway, indicating that its sterols are synthesized somewhat differently than in fungi. The work presented here seeks to establish experimental evidence to support the annotated molecular function of one of the sterol biosynthetic genes in the Chlamydomonas genome.A gene with homology to the yeast sterol C-5 desaturase, ERG3, is present in the Chlamydomonas genome. To test whether the ERG3 ortholog of C. reinhardtii encodes a sterol C-5 desaturase, Saccharomyces cerevisiae ERG3 knockout strains were created and complemented with a plasmid expressing the Chlamydomonas ERG3. Expression of C. reinhardtii ERG3 cDNA in erg3 null yeast was able to restore ergosterol biosynthesis and reverse phenotypes associated with lack of ERG3 function.Complementation of the yeast erg3 null phenotypes strongly suggests that the gene annotated as ERG3 in C. reinhardtii functions as a sterol C-5 desaturase