Publications by authors named "Rosa Luna"

32 Publications

Remediation by means of EDTA of an agricultural calcareous soil polluted with Pb.

Environ Geochem Health 2021 Jun 22;43(6):2231-2242. Epub 2020 Oct 22.

Departamento de Energía, Unidad Azcapotzalco, Universidad Autónoma Metropolitana, Av. San Pablo 180, 02200, Ciudad de México, México.

The dispersion of mine tailings affects ecosystems due to their high content of potentially toxic elements. Environmental risk increases when the soil impacted by tailings is used for agriculture; this use may result in health impacts. This study analyzes the feasibility of remediating a calcareous soil (used for maize cultivation) polluted with lead in the semiarid zone of Zimapán, México, by using EDTA as an extractant. Total geoavailable and bioaccessible concentrations in the gastric and intestinal phases were determined to evaluate lead availability and health risk. The soil was then washed with EDTA, and the geochemical fractionation (interchangeable, carbonates, Fe/Mn oxy-hydroxides, organic matter-sulfides, and residual) and impact on the mesophile bacteria and fungi/yeast populations were analyzed. The results showed total Pb concentrations up to 647 ± 3.50 mg/kg, a 46% bioaccessible fraction (297 ± 9.90 mg/kg) in the gastric phase and a 12.2% (80 ± 5 mg/kg) bioaccessible fraction in the intestinal phase, indicating a health and environmental risk. Meanwhile, the geochemical fractionation before washing showed a Pb fraction mainly consisting of Fe/Mn oxy-hydroxides (69.6%); this reducible fraction may progressively increase its bioaccessibility. Geochemical fractionation performed in the washed soil showed differences from that determined before the treatment; however, the iron and manganese fraction, at 42.4%, accounted for most of the Pb. The soil microbiology was also modified by EDTA, with an increase in aerobic bacteria and a decrease in fungi/yeast populations. Although 44% total lead removal was achieved, corresponding to a final concentration of 363.50 ± 43.50 mg/kg (below national and USEPA standards), washing with EDTA increased the soluble and interchangeable lead concentrations. Statistical analysis indicated a significant effect (p < 0.05) of EDTA on the soil's geochemical fractionation of lead.
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http://dx.doi.org/10.1007/s10653-020-00754-5DOI Listing
June 2021

The THO Complex as a Paradigm for the Prevention of Cotranscriptional R-Loops.

Cold Spring Harb Symp Quant Biol 2019 3;84:105-114. Epub 2020 Jun 3.

Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, 41092 Seville, Spain.

Different proteins associate with the nascent RNA and the RNA polymerase (RNAP) to catalyze the transcription cycle and RNA export. If these processes are not properly controlled, the nascent RNA can thread back and hybridize to the DNA template forming R-loops capable of stalling replication, leading to DNA breaks. Given the transcriptional promiscuity of the genome, which leads to large amounts of RNAs from mRNAs to different types of ncRNAs, these can become a major threat to genome integrity if they form R-loops. Consequently, cells have evolved nuclear factors to prevent this phenomenon that includes THO, a conserved eukaryotic complex acting in transcription elongation and RNA processing and export that upon inactivation causes genome instability linked to R-loop accumulation. We revise and discuss here the biological relevance of THO and a number of RNA helicases, including the THO partner UAP56/DDX39B, as a paradigm of the cellular mechanisms of cotranscriptional R-loop prevention.
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http://dx.doi.org/10.1101/sqb.2019.84.039594DOI Listing
June 2020

Depletion of the MFAP1/SPP381 Splicing Factor Causes R-Loop-Independent Genome Instability.

Cell Rep 2019 08;28(6):1551-1563.e7

Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Seville 41092, Spain. Electronic address:

THO/TREX is a conserved complex with a role in messenger ribonucleoprotein biogenesis that links gene expression and genome instability. Here, we show that human THO interacts with MFAP1 (microfibrillar-associated protein 1), a spliceosome-associated factor. Interestingly, MFAP1 depletion impairs cell proliferation and genome integrity, increasing γH2AX foci and DNA breaks. This phenotype is not dependent on either transcription or RNA-DNA hybrids. Mutations in the yeast orthologous gene SPP381 cause similar transcription-independent genome instability, supporting a conserved role. MFAP1 depletion has a wide effect on splicing and gene expression in human cells, determined by transcriptome analyses. MFAP1 depletion affects a number of DNA damage response (DDR) genes, which supports an indirect role of MFAP1 on genome integrity. Our work defines a functional interaction between THO and RNA processing and argues that splicing factors may contribute to genome integrity indirectly by regulating the expression of DDR genes rather than by a direct role.
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http://dx.doi.org/10.1016/j.celrep.2019.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693559PMC
August 2019

Human THO-Sin3A interaction reveals new mechanisms to prevent R-loops that cause genome instability.

EMBO J 2017 12 26;36(23):3532-3547. Epub 2017 Oct 26.

Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Consejo Superior de Investigaciones Científicas-Universidad Pablo de Olavide-Universidad de Sevilla, Seville, Spain

R-loops, formed by co-transcriptional DNA-RNA hybrids and a displaced DNA single strand (ssDNA), fulfill certain positive regulatory roles but are also a source of genomic instability. One key cellular mechanism to prevent R-loop accumulation centers on the conserved THO/TREX complex, an RNA-binding factor involved in transcription elongation and RNA export that contributes to messenger ribonucleoprotein (mRNP) assembly, but whose precise function is still unclear. To understand how THO restrains harmful R-loops, we searched for new THO-interacting factors. We found that human THO interacts with the Sin3A histone deacetylase complex to suppress co-transcriptional R-loops, DNA damage, and replication impairment. Functional analyses show that histone hypo-acetylation prevents accumulation of harmful R-loops and RNA-mediated genomic instability. Diminished histone deacetylase activity in THO- and Sin3A-depleted cell lines correlates with increased R-loop formation, genomic instability, and replication fork stalling. Our study thus uncovers physical and functional crosstalk between RNA-binding factors and chromatin modifiers with a major role in preventing R-loop formation and RNA-mediated genome instability.
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http://dx.doi.org/10.15252/embj.201797208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709763PMC
December 2017

Excess of Yra1 RNA-Binding Factor Causes Transcription-Dependent Genome Instability, Replication Impairment and Telomere Shortening.

PLoS Genet 2016 Apr 1;12(4):e1005966. Epub 2016 Apr 1.

Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Seville, Spain.

Yra1 is an essential nuclear factor of the evolutionarily conserved family of hnRNP-like export factors that when overexpressed impairs mRNA export and cell growth. To investigate further the relevance of proper Yra1 stoichiometry in the cell, we overexpressed Yra1 by transforming yeast cells with YRA1 intron-less constructs and analyzed its effect on gene expression and genome integrity. We found that YRA1 overexpression induces DNA damage and leads to a transcription-associated hyperrecombination phenotype that is mediated by RNA:DNA hybrids. In addition, it confers a genome-wide replication retardation as seen by reduced BrdU incorporation and accumulation of the Rrm3 helicase. In addition, YRA1 overexpression causes a cell senescence-like phenotype and telomere shortening. ChIP-chip analysis shows that overexpressed Yra1 is loaded to transcribed chromatin along the genome and to Y' telomeric regions, where Rrm3 is also accumulated, suggesting an impairment of telomere replication. Our work not only demonstrates that a proper stoichiometry of the Yra1 mRNA binding and export factor is required to maintain genome integrity and telomere homeostasis, but suggests that the cellular imbalance between transcribed RNA and specific RNA-binding factors may become a major cause of genome instability mediated by co-transcriptional replication impairment.
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http://dx.doi.org/10.1371/journal.pgen.1005966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4818039PMC
April 2016

A genome-wide function of THSC/TREX-2 at active genes prevents transcription-replication collisions.

Nucleic Acids Res 2014 Oct 7;42(19):12000-14. Epub 2014 Oct 7.

Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, 41092 Seville, Spain

The THSC/TREX-2 complex of Saccharomyces cerevisiae mediates the anchoring of transcribed genes to the nuclear pore, linking transcription elongation with mRNA export and genome stability, as shown for specific reporters. However, it is still unknown whether the function of TREX-2 is global and the reason for its relevant role in genome integrity. Here, by studying two TREX-2 representative subunits, Thp1 and Sac3, we show that TREX-2 has a genome-wide role in gene expression. Both proteins show similar distributions along the genome, with a gradient disposition at active genes that increases towards the 3' end. Thp1 and Sac3 have a relevant impact on the expression of long, G+C-rich and highly transcribed genes. Interestingly, replication impairment detected by the genome-wide accumulation of the replicative Rrm3 helicase is increased preferentially at highly expressed genes in the thp1Δ and sac3Δ mutants analyzed. Therefore, our work provides evidence of a function of TREX-2 at the genome-wide level and suggests a role for TREX-2 in preventing transcription-replication conflicts, as a source of genome instability derived from a defective messenger ribonucleoprotein particle (mRNP) biogenesis.
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http://dx.doi.org/10.1093/nar/gku906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231764PMC
October 2014

Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation.

Proc Natl Acad Sci U S A 2014 Aug 4;111(33):12163-8. Epub 2014 Aug 4.

Janssen Research and Development, Spring House, PA 19002.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4(+) Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7β, 27-dihydroxycholesterol (7β, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ- or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7β, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7β, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4(+) and γδ(+) T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.
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http://dx.doi.org/10.1073/pnas.1322807111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143045PMC
August 2014

R-loop mediated transcription-associated recombination in trf4Δ mutants reveals new links between RNA surveillance and genome integrity.

PLoS One 2013 7;8(6):e65541. Epub 2013 Jun 7.

Departamento de Biología Molecular, Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Seville, Spain.

To get further insight into the factors involved in the maintenance of genome integrity we performed a screening of Saccharomyces cerevisiae deletion strains inducing hyperrecombination. We have identified trf4, a gene encoding a non-canonical polyA-polymerase involved in RNA surveillance, as a factor that prevents recombination between DNA repeats. We show that trf4Δ confers a transcription-associated recombination phenotype that is mediated by the nascent mRNA. In addition, trf4Δ also leads to an increase in the mutation frequency. Both genetic instability phenotypes can be suppressed by overexpression of RNase H and are exacerbated by overexpression of the human cytidine deaminase AID. These results suggest that in the absence of Trf4 R-loops accumulate co-transcriptionally increasing the recombination and mutation frequencies. Altogether our data indicate that Trf4 is necessary for both mRNA surveillance and maintenance of genome integrity, serving as a link between RNA and DNA metabolism in S. cerevisiae.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065541PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3676323PMC
January 2014

Identification of benzofuran central cores for the inhibition of leukotriene A(4) hydrolase.

Bioorg Med Chem Lett 2013 Feb 5;23(3):811-5. Epub 2012 Dec 5.

Janssen Research & Development, LLC, 3210 Merryfield Row, San Diego, CA 92121, USA.

Leukotrienes (LT's) are known to play a physiological role in inflammatory immune response. Leukotriene A(4) hydrolase (LTA(4)H) is a cystolic enzyme that stereospecifically catalyzes the transformation of LTA(4) to LTB(4). LTB(4) is a known pro-inflammatory mediator. This paper describes the identification and synthesis of substituted benzofurans as LTH(4)H inhibitors. The benzofuran series demonstrated reduced mouse and human whole blood LTB(4) levels in vitro and led to the identification one analog for advanced profiling. Benzofuran 28 showed dose responsive target engagement and provides a useful tool to explore a LTA(4)H inhibitor for the treatment of inflammatory diseases, such as asthma and inflammatory bowel disease (IBD).
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http://dx.doi.org/10.1016/j.bmcl.2012.11.074DOI Listing
February 2013

New clues to understand the role of THO and other functionally related factors in mRNP biogenesis.

Biochim Biophys Acta 2012 Jun 20;1819(6):514-20. Epub 2011 Dec 20.

Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Av Américo Vespucio s/n, 41092 Sevilla, Spain. [email protected]/es

Coupling of transcription with mRNA processing and export has been shown to be relevant to efficient gene expression. A number of studies have determined that THO/TREX, a nuclear protein complex conserved from yeast to humans, plays an important role in mRNP biogenesis connecting transcription elongation, mRNA export and preventing genetic instability. Recent data indicates that THO could be relevant to different mRNA processing steps, including the 3'-end formation, transcript release and export. Novel connections of THO to proteins related to the splicing machinery, provide new views about possible functions of THO in mRNP biogenesis. In this review, we summarize the previous and new results concerning the impact of THO in transcription and its biological implications, with a special emphasis on the relationship with THSC/TREX-2 and other functionally related factors involved in mRNA biogenesis and export. The emerging picture presents THO as a dynamic complex interacting with the nascent RNA and with different factors connecting nuclear functions necessary for mRNP biogenesis with genome integrity, cellular homeostasis and development. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.
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http://dx.doi.org/10.1016/j.bbagrm.2011.11.012DOI Listing
June 2012

Mechanisms establishing TLR4-responsive activation states of inflammatory response genes.

PLoS Genet 2011 Dec 8;7(12):e1002401. Epub 2011 Dec 8.

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California, United States of America.

Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Comprehensive genome-wide analysis of the epigenetic and transcription status of the TLR4-induced transcriptional program in macrophages suggests that Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early- (I/E) and late-response genes results from a sequential process in which signal-independent factors initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by encoding a distinct set of signal-dependent transcription factor elements, including TATA boxes, which lead to preferential binding of TBP and basal enrichment for RNA polymerase II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases the overall rates of both transcriptional initiation and the efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. Collectively, these findings reveal broadly utilized mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses.
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http://dx.doi.org/10.1371/journal.pgen.1002401DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234212PMC
December 2011

Genome instability and transcription elongation impairment in human cells depleted of THO/TREX.

PLoS Genet 2011 Dec 1;7(12):e1002386. Epub 2011 Dec 1.

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla - Consejo Superior de Investigaciones Científicas (CSIC), Sevilla, Spain.

THO/TREX connects transcription with genome integrity in yeast, but a role of mammalian THO in these processes is uncertain, which suggests a differential implication of mRNP biogenesis factors in genome integrity in yeast and humans. We show that human THO depletion impairs transcription elongation and mRNA export and increases instability associated with DNA breaks, leading to hyper-recombination and γH2AX and 53BP1 foci accumulation. This is accompanied by replication alteration as determined by DNA combing. Genome instability is R-loop-dependent, as deduced from the ability of the AID enzyme to increase DNA damage and of RNaseH to reduce it, or from the enhancement of R-loop-dependent class-switching caused by THOC1-depletion in CH12 murine cells. Therefore, mammalian THO prevents R-loop formation and has a role in genome dynamics and function consistent with an evolutionary conservation of the functional connection between these mRNP biogenesis factors and genome integrity that had not been anticipated.
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http://dx.doi.org/10.1371/journal.pgen.1002386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3228816PMC
December 2011

Nab2 functions in the metabolism of RNA driven by polymerases II and III.

Mol Biol Cell 2011 Aug 16;22(15):2729-40. Epub 2011 Jun 16.

Centro Andaluz de Biología Molecular y Medicina Regenerativa, Universidad de Sevilla-Consejo Superior de Investigaciones Científicas, 41092 Seville, Spain.

Gene expression in eukaryotes is an essential process that includes transcription, RNA processing, and export. One important player in this interface is the poly(A)(+)-RNA-binding protein Nab2, which regulates the mRNA poly(A)(+)-tail length and export. Here we show that Nab2 has additional roles during mRNA transcription, tRNA metabolism, and ribosomal subunit export. Nab2 is associated with the entire open reading frame of actively transcribed RNA polymerase (RNAP) II and III genes. As a consequence, nab2 mutations confer translation defects that are detected by polysome profiling. Genome-wide analysis of expression of a conditional degron nab2 mutant shows that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is direct. Taken together, our results identify novel functions for Nab2 in transcription and metabolism of most types of RNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, and that it is a central player in the general and coordinated control of gene expression from transcription to translation.
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http://dx.doi.org/10.1091/mbc.E11-01-0055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145548PMC
August 2011

A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors.

EMBO J 2011 May 8;30(10):1953-64. Epub 2011 Apr 8.

Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Sevilla, Spain.

To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G-less cassettes separated by either a long and GC-rich or a short and GC-poor DNA sequence (G-less-based run-on (GLRO) assay). We demonstrate that PAF, THSC/TREX-2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo, this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre-mRNA processing and nuclear export with transcription elongation.
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http://dx.doi.org/10.1038/emboj.2011.109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3098481PMC
May 2011

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers.

BMC Cancer 2011 Feb 17;11:77. Epub 2011 Feb 17.

Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Av, Américo Vespucio s/n, 41092 Sevilla, Spain.

Background: One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples.

Methods: The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis.

Results: We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers.

Conclusions: These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development.
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http://dx.doi.org/10.1186/1471-2407-11-77DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3050854PMC
February 2011

Female-biased incidence of experimental autoimmune encephalomyelitis reflects sexually dimorphic expression of surface CTLA-4 (CD152) on T lymphocytes.

Gend Med 2010 Aug;7(4):296-308

Department of Experimental Pediatrics, Otto-von-Guericke University, Magdeburg, Germany.

Background: Autoimmune reactions occur naturally and in most cases are controlled by regulatory mechanisms. However, unwanted autoimmune responses still appear in 5% to 7% of the population, in strikingly greater frequencies in women compared with men. The chronic inflammation characteristic of autoimmune diseases is mainly initiated and maintained by autoreactive CD4(+) T lymphocytes. Costimu-lation is required for an optimal response of T lymphocytes: CD28 is a T-cell activator, whereas CTLA-4 (cytotoxic T-lymphocyte antigen 4, also known as CD152) downregulates T-cell activity. Together these costimulatory molecules provide a balance in T-cell immune response.

Objective: The aim of this study was to elucidate the role of the inhibitory receptor CTLA-4 in the quality of sex-specific immune responses.

Methods: At the German Rheumatism Research Center (DRFZ), Berlin, Germany, between 2006 and 2010, we tested mouse strains commonly used for the development of experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis (MS). The SJL mouse strain not only mimics MS pathogenesis, but also exhibits the female predominance that occurs in patients with MS.

Results: Cells derived from SJL females revealed increased proliferation and a doubled frequency of T-helper (Th)1- and Th2-like cytokines, compared with their male counterparts. Moreover, activated Th cells from male mice express significantly higher frequencies (61%) of CTLA-4 expressed at the cell surface in comparison with those of females (46%). Accordingly, close to 50% reduction of CTLA-4 expression occurred in cells of both sexes after the addition of estrogen. We observed that interferon (IFN)-γ(high) production in females occurred in a higher frequency in CD4(+) T cells cultured under neutral conditions (24.6% in females, 15.9% in males). Moreover, we observed that the IFN-yhigh producers were mainly present in females (4.5% vs 0.4% in males).

Conclusion: Our results suggest that induction of CTLA-4 expression could serve as a target for an immunomodulatory strategy to downregulate immune responses in sexually dimorphic autoimmune diseases.
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http://dx.doi.org/10.1016/j.genm.2010.08.005DOI Listing
August 2010

The SWI/SNF protein BAF60b is ubiquitinated through a signalling process involving Rac GTPase and the RING finger protein Unkempt.

FEBS J 2010 Mar 8;277(6):1453-64. Epub 2010 Feb 8.

Institut Cochin, Université Paris Descartes, CNRS (UMR8104), Paris, France.

The SWI/SNF chromatin remodelling complexes are important regulators of transcription; they consist of large multisubunit assemblies containing either Brm or Brg1 as the catalytic ATPase subunit and a variable subset of approximately 10 Brg/Brm-associated factors (BAF). Among these factors, BAF60 proteins (BAF60a, BAF60b or BAF60c), which are found in most complexes, are thought to bridge interactions between transcription factors and SWI/SNF complexes. We report here on a Rac-dependent process leading to BAF60b ubiquitination. Using two-hybrid cloning procedures, we identified a mammalian RING finger protein homologous to drosophila Unkempt as a new partner of the activated form of RacGTPases and demonstrated that mammalian Unkempt specifically binds to BAF60b and promotes its ubiquitination in a Rac1-dependent manner. Immunofluorescence studies demonstrated that Unkempt is primarily localized in the cytoplasmic compartment, but has the ability to shuttle between the nucleus and the cytoplasm, suggesting that the Rac- and Unkempt-dependent process leading to BAF60b ubiquitination takes place in the nuclear compartment. Ubiquitinated forms of BAF60b were found to accumulate upon treatment with the proteasome inhibitor MG132, indicating that BAF60b ubiquitination is of the degradative type and could regulate the level of BAF60b in SWI/SNF complexes. Our observations support the new idea of a direct connection between Rac signalling and chromatin remodelling.
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http://dx.doi.org/10.1111/j.1742-4658.2010.07575.xDOI Listing
March 2010

Transcription at the proximity of the nuclear pore: a role for the THP1-SAC3-SUS1-CDC31 (THSC) complex.

RNA Biol 2009 Apr-Jun;6(2):145-8. Epub 2009 Apr 8.

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC, Seville, Spain.

A key aspect of eukaryotic gene expression is the coupling of transcription with RNA processing, polyadenylation and export. The use of new techniques based on tandem affinity purification (TAP) and chromatin immunoprecipitation (ChIP), and of genetic and cell biology approaches has contributed to the beginning of deciphering the network of protein-mRNA interactions accompanying this coupling. Although an extensive amount of work has shed light on this matter, the order of participation and precise role of the different proteins remain to be deciphered. It seems that different and sequential protein interactions must converge to finally promote the anchoring of genes to the nuclear periphery. Here we discuss the new data on the coupling of gene expression and RNA export, with emphasis on the THP1-SAC3-SUS1-CDC31 complex and the possible implications of these results on transcription at the nuclear pore.
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http://dx.doi.org/10.4161/rna.6.2.7803DOI Listing
February 2010

A reduction in RNA polymerase II initiation rate suppresses hyper-recombination and transcription-elongation impairment of THO mutants.

Mol Genet Genomics 2008 Oct 6;280(4):327-36. Epub 2008 Aug 6.

Centro Andaluz de Biologia Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Av. Américo Vespucio s/n, 41092 Sevilla, Spain.

Hrs1/Med3, a component of the Mediator involved in transcription initiation, was previously isolated as a suppressor of hpr1Delta hyper-recombination linked to transcription elongation. Here we show that hrs1Delta-mediated suppression is specific of transcription-associated hyper-recombination (TAR). The decrease in recombination associated with hrs1Delta, either in wild-type or hpr1Delta cells is only observed in DNA repeats constructs in which transcription is Hrs1-dependent. We propose that the suppression of THO mutants by hrs1Delta is due to the specific effect of hrs1Delta on transcription initiation of the recombination system. In parallel we show that the higher the transcription of a gene the more important becomes the THO complex for its expression, implying that the in vivo relevance of this complex is dependent on the frequency of RNAPII transcription initiation. This study furthers the understanding of the importance of THO in transcription and the maintenance of genome stability.
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http://dx.doi.org/10.1007/s00438-008-0368-8DOI Listing
October 2008

The THP1-SAC3-SUS1-CDC31 complex works in transcription elongation-mRNA export preventing RNA-mediated genome instability.

Mol Biol Cell 2008 Oct 30;19(10):4310-8. Epub 2008 Jul 30.

Centro Andaluz de Biologia Molecular y Medicina Regenerativa, Universidad de Sevilla-CSIC, 41092 Sevilla, Spain.

The eukaryotic THO/TREX complex, involved in mRNP biogenesis, plays a key role in the maintenance of genome integrity in yeast. mRNA export factors such as Thp1-Sac3 also affect genome integrity, but their mutations have other phenotypes different from those of THO/TREX. Sus1 is a novel component of SAGA transcription factor that also associates with Thp1-Sac3, but little is known about its effect on genome instability and transcription. Here we show that Thp1, Sac3, and Sus1 form a functional unit with a role in mRNP biogenesis and maintenance of genome integrity that is independent of SAGA. Importantly, the effects of ribozyme-containing transcription units, RNase H, and the action of human activation-induced cytidine deaminase on transcription and genome instability are consistent with the possibility that R-loops are formed in Thp1-Sac3-Sus1-Cdc31 as in THO mutants. Our data reveal that Thp1-Sac3-Sus1-Cdc31, together with THO/TREX, define a specific pathway connecting transcription elongation with export via an RNA-dependent dynamic process that provides a feedback mechanism for the control of transcription and the preservation of genetic integrity of transcribed DNA regions.
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http://dx.doi.org/10.1091/mbc.e08-04-0355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2555943PMC
October 2008

Biogenesis of mRNPs: integrating different processes in the eukaryotic nucleus.

Chromosoma 2008 Aug 22;117(4):319-31. Epub 2008 Apr 22.

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-CSIC, Seville, Spain.

Transcription is a central function occurring in the nucleus of eukaryotic cells in coordination with other nuclear processes. During transcription, the nascent pre-mRNA associates with mRNA-binding proteins and undergoes a series of processing steps, resulting in export-competent mRNA ribonucleoprotein complexes (mRNPs) that are transported into the cytoplasm. Experimental evidence increasingly indicates that the different processing steps (5'-end capping, splicing, 3'-end cleavage) and mRNP export are connected to each other as well as to transcription, both functionally and physically. Here, we review the overall process of mRNP biogenesis with particular emphasis on the functional coupling of transcription with mRNP biogenesis and export and its relationship to nuclear organization.
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http://dx.doi.org/10.1007/s00412-008-0158-4DOI Listing
August 2008

Different physiological relevance of yeast THO/TREX subunits in gene expression and genome integrity.

Mol Genet Genomics 2008 Feb 25;279(2):123-32. Epub 2007 Oct 25.

Departamento de Biología Molecular, CABIMER, CSIC, Universidad de Sevilla, Av. Américo Vespucio s/n, Seville, Spain.

THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and plays a role in preventing the transcription-associated genetic instability. THO is composed of Tho2, Hpr1, Mft1 and Thp2 subunits, which associate with the Sub2-Yra1 export factors and Tex1 to form the TREX complex. To compare the functional relevance of the different THO/TREX subunits, we determined the effect of their null mutations on mRNA accumulation and recombination. Unexpectedly, we noticed that a full deletion of HPR1, hpr1DeltaK, conferred stronger hyper-recombination phenotype and gene expression defects than did hpr1DeltaH, the allele encoding a C-terminal truncated protein which was used in most previous studies. We show that tho2Delta and, to a lesser extent, hpr1DeltaK are the THO mutations with the highest impact on all phenotypes, and that sub2Delta shows a similar transcription-dependent hyper-recombination phenotype and in vivo transcription impairment as hpr1DeltaK and tho2Delta. Recombination and transcription analyses indicate that THO/TREX mutants share a moderate but significant effect on gene conversion and ectopic recombination, as well as transcription impairment of even short and low GC-content genes. Our data provide new information on the relevance of these proteins in mRNP biogenesis and in the maintenance of genomic integrity.
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http://dx.doi.org/10.1007/s00438-007-0301-6DOI Listing
February 2008

Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project.

Nature 2007 Jun;447(7146):799-816

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2212820PMC
http://dx.doi.org/10.1038/nature05874DOI Listing
June 2007

Differential repression of c-myc and cdc2 gene expression by ERF and PE-1/METS.

Cell Cycle 2007 Jul 20;6(13):1594-604. Epub 2007 Apr 20.

Biomedical Sciences Graduate Program, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California 92093, USA.

The molecular mechanisms that control the proliferation and differentiation of specific cell types remain poorly understood. Positive ETS factors play important roles in mediating proliferative responses to Ras/MAPK signaling in many cell types following mitogenic stimulation. PE-1/METS, a member of the ETS-domain family transcription factors that functions as a transcriptional repressor, can block mitogenic responses mediated by positively acting Ets factors. The anti-proliferative functions of PE-1/METS require its interaction with DP103, a multifunctional DEAD-box protein that mediates interactions with corepressor proteins and acts in a cooperative manner with Rb family members and to repress cell cycle control genes. ETS-2 repressor factor (ERF) is structurally related to and also functions as a transcriptional repressor, but endogenous target genes and mechanisms of repression remain unknown. Here, we demonstrate that like PE-1/METS, ERF-mediated repression also requires DP103, and that ERF negatively regulates the c-myc and cdc2 genes. In contrast to PE-1/METS, however, ERF-mediated repression of these genes is inactivated by MAPK signaling through phosphorylation sites that are ERF-specific. Furthermore, constitutive activation of the Ras/MAPK pathway in RAW 264.7 cells transformed by the v-Abelson leukemia virus is associated with constitutive inactivation of ERF in this cell type. We propose that ERF and PE-1/METS function to impose 'repression checkpoints' on a subset of cell cycle control genes that are differentially regulated by growth factor signaling pathways that control proliferation and differentiation and that ERF is targeted for inactivation by transforming oncogenes such as vAbl.
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http://dx.doi.org/10.4161/cc.6.13.4336DOI Listing
July 2007

An hpr1 point mutation that impairs transcription and mRNP biogenesis without increasing recombination.

Mol Cell Biol 2006 Oct 14;26(20):7451-65. Epub 2006 Aug 14.

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avd. Reina Mercedes 6, 41012 Sevilla, Spain.

THO/TREX, a conserved eukaryotic protein complex, is a key player at the interface between transcription and mRNP metabolism. The lack of a functional THO complex impairs transcription, leads to transcription-dependent hyperrecombination, causes mRNA export defects and fast mRNA decay, and retards replication fork progression in a transcription-dependent manner. To get more insight into the interconnection between mRNP biogenesis and genomic instability, we searched for HPR1 mutations that differentially affect gene expression and recombination. We isolated mutants that were barely affected in gene expression but exhibited a hyperrecombination phenotype. In addition, we isolated a mutant, hpr1-101, with a strong defect in transcription, as observed for lacZ, and a general defect in mRNA export that did not display a relevant hyperrecombination phenotype. In THO single-null mutants, but not in the hpr1 point mutants studied, THO and its subunits were unstable. Interestingly, in contrast to hyperrecombinant null mutants, hpr1-101 did not cause retardation of replication fork progression. Transcription and mRNP biogenesis can therefore be impaired by THO/TREX dysfunction without increasing recombination, suggesting that it is possible to separate the mechanism(s) responsible for mRNA biogenesis defects from the further step of triggering transcription-dependent recombination.
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http://dx.doi.org/10.1128/MCB.00684-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1636866PMC
October 2006

Tho1, a novel hnRNP, and Sub2 provide alternative pathways for mRNP biogenesis in yeast THO mutants.

Mol Cell Biol 2006 Jun;26(12):4387-98

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avd. Reina Mercedes 6, 41012 Sevilla, Spain.

THO is a protein complex that functions in cotranscriptional mRNP formation. Yeast THO1 and SUB2 (Saccharomyces cerevisiae) were identified as multicopy suppressors of the expression defects of the hpr1Delta mutant of THO. Here we show that multicopy THO1 suppresses the mRNA accumulation and export defects and the hyperrecombination phenotype of THO mutants but not those of sub2Delta, thp1Delta, or spt4Delta. Similarly, Sub2 overexpression suppresses the RNA export defect of hpr1Delta. Tho1 is a conserved RNA binding nuclear protein that specifically binds to transcribed chromatin in a THO- and RNA-dependent manner and genetically interacts with the shuttling hnRNP Nab2. The ability of Tho1 to suppress hpr1Delta resides in its C-terminal half, which contains the RNA binding activity and is located after a SAP/SAF (scaffold-associated protein/scaffold-associated factor) domain. Altogether, these results suggest that Tho1 is an hnRNP that, similarly to Sub2, assembles onto the nascent mRNA during transcription and participates in mRNP biogenesis and export. Overexpression of Tho1 or Sub2 may provide alternative ways for mRNP formation and export in the absence of a functional THO complex.
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http://dx.doi.org/10.1128/MCB.00234-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1489133PMC
June 2006

Interdependence between transcription and mRNP processing and export, and its impact on genetic stability.

Mol Cell 2005 Jun;18(6):711-22

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Spain.

The conserved eukaryotic THO-TREX complex acts at the interface between transcription and mRNA export and affects transcription-associated recombination. To investigate the interdependence of nuclear mRNA processes and their impact on genomic integrity, we analyzed transcript accumulation and recombination of 40 selected mutants covering representative steps of the biogenesis and export of the messenger ribonucleoprotein particle (mRNP). None of the mutants analyzed shared the strong transcript-accumulation defect and hyperrecombination of THO mutants. Nevertheless, mutants in 3' end cleavage/polyadenylation, nuclear exosome, and mRNA export showed a weak but significant effect on recombination and transcript accumulation. Mutants of the nuclear exosome (rrp6) and 3' end processing factors (rna14 and rna15) showed inefficient transcription elongation and genetic interactions with THO. The results suggest a tight interdependence among mRNP biogenesis steps and transcription and an unexpected effect of the nuclear exosome and the cleavage/polyadenylation factors on transcription elongation and genetic integrity.
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http://dx.doi.org/10.1016/j.molcel.2005.05.001DOI Listing
June 2005

Direct isolation and identification of promoters in the human genome.

Genome Res 2005 Jun 17;15(6):830-9. Epub 2005 May 17.

Ludwig Institute for Cancer Research, University of California, San Diego, La Jolla, California 92093, USA.

Transcriptional regulatory elements play essential roles in gene expression during animal development and cellular response to environmental signals, but our knowledge of these regions in the human genome is limited despite the availability of the complete genome sequence. Promoters mark the start of every transcript and are an important class of regulatory elements. A large, complex protein structure known as the pre-initiation complex (PIC) is assembled on all active promoters, and the presence of these proteins distinguishes promoters from other sequences in the genome. Using components of the PIC as tags, we isolated promoters directly from human cells as protein-DNA complexes and identified the resulting DNA sequences using genomic tiling microarrays. Our experiments in four human cell lines uncovered 252 PIC-binding sites in 44 semirandomly selected human genomic regions comprising 1% (30 megabase pairs) of the human genome. Nearly 72% of the identified fragments overlap or immediately flank 5' ends of known cDNA sequences, while the remainder is found in other genomic regions that likely harbor putative promoters of unannotated transcripts. Indeed, molecular analysis of the RNA isolated from one cell line uncovered transcripts initiated from over half of the putative promoter fragments, and transient transfection assays revealed promoter activity for a significant proportion of fragments when they were fused to a luciferase reporter gene. These results demonstrate the specificity of a genome-wide analysis method for mapping transcriptional regulatory elements and also indicate that a small, yet significant number of human genes remains to be discovered.
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http://dx.doi.org/10.1101/gr.3430605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1142473PMC
June 2005

Evidence for linkage of nonsyndromic cleft lip with or without cleft palate to a region on chromosome 2.

Eur J Hum Genet 2003 Nov;11(11):835-9

Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD, USA.

Results from a genome-wide screen of 10 multiplex families ascertained through probands with nonsyndromic cleft lip with or without cleft palate (CL/P) in Mexico, Argentina, and the United States yielded suggestive evidence of linkage to chromosomes 2, 6, 17 and 18. Fine mapping excluded all regions except chromosome 2. Subsequent analysis was performed on the original 10 families plus an additional 16 families using 31 markers on chromosome 2. This analysis showed intriguing evidence of linkage to 2q (Zlr=2.26, empirical P-value=0.028 in a chromosome-wide analysis). Transmission disequilibrium tests also revealed evidence of linkage and disequilibrium for two markers in this region (D2S168 and D2S1400 with P-values=0.022 and 0.006, respectively). A subset of these 26 families provided additional evidence for a susceptibility gene for CL/P on 2q, suggesting that further studies of genes in this region are warranted.
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http://dx.doi.org/10.1038/sj.ejhg.5201052DOI Listing
November 2003

Nab2p and the Thp1p-Sac3p complex functionally interact at the interface between transcription and mRNA metabolism.

J Biol Chem 2003 Jun 17;278(26):24225-32. Epub 2003 Apr 17.

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes 6, 41012 Sevilla, Spain.

THP1 is a conserved eukaryotic gene whose null mutations confer, in yeast, transcription and genetic instability phenotypes and RNA export defects similar to those of the THO/TREX complex null mutations. In a search for multicopy suppressors of the transcription defect of thp1Delta cells, we identified the poly(A)+ RNA-binding heterogeneous nuclear ribonucleoprotein Nab2p. Multicopy NAB2 also suppressed the RNA export defect of thp1Delta cells. This result suggests a functional relationship between Thp1p and Nab2p. Consistently, the leaky mutation nab2-1 conferred a transcription defect and hyper-recombination phenotype similar to those of thp1Delta, although to a minor degree. Reciprocally, a purified His6-tagged Thp1p fusion bound RNA in vitro. In a different approach, we show by Western analyses that a highly purified Thp1p-Sac3p complex does not contain components of THO/TREX and that sac3Delta confers a transcription defect and hyper-recombination phenotype identical to those of thp1Delta. mRNA degradation was not affected in thp1Delta mutants, implying that their expression defects are not due to mRNA decay. This indicates that Thp1p-Sac3p is a structural and functional unit. Altogether, our results suggest that Thp1p-Sac3p and Nab2p are functionally related heterogeneous nuclear ribonucleoproteins that define a further link between mRNA metabolism and transcription.
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http://dx.doi.org/10.1074/jbc.M302900200DOI Listing
June 2003
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