Publications by authors named "Arantxa Gutierrez"

25 Publications

  • Page 1 of 1

The Polycomb-associated factor PHF19 controls hematopoietic stem cell state and differentiation.

Sci Adv 2020 Aug 5;6(32):eabb2745. Epub 2020 Aug 5.

Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Dr. Aiguader 88, Barcelona 08003, Spain.

Adult hematopoietic stem cells (HSCs) are rare multipotent cells in bone marrow that are responsible for generating all blood cell types. HSCs are a heterogeneous group of cells with high plasticity, in part, conferred by epigenetic mechanisms. PHF19, a subunit of the Polycomb repressive complex 2 (PRC2), is preferentially expressed in mouse hematopoietic precursors. Here, we now show that, in stark contrast to results published for other PRC2 subunits, genetic depletion of increases HSC identity and quiescence. While proliferation of HSCs is normally triggered by forced mobilization, defects in differentiation impede long-term correct blood production, eventually leading to aberrant hematopoiesis. At molecular level, PHF19 deletion triggers a redistribution of the histone repressive mark H3K27me3, which notably accumulates at blood lineage-specific genes. Our results provide novel insights into how epigenetic mechanisms determine HSC identity, control differentiation, and are key for proper hematopoiesis.
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http://dx.doi.org/10.1126/sciadv.abb2745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406347PMC
August 2020

Hydrophilic intraocular lens opacification after repeated intracameral gas injection for Descemet membrane detachment.

Oman J Ophthalmol 2019 Jan-Apr;12(1):46-49

Department of Internal Medicine, Hospital General de Castellón, Castellón De La Plana, Castellón, Spain.

Descemet membrane detachment (DMD) is a complication of a variety of eye procedures that can result in severe visual loss. We report a new case of the condition, in a highly myopic patient that had undergone cataract surgery, and presented a macular hemorrhage during the intervention. DMD was successfully treated with a combined technique of intracameral gas injection and transcorneal suturing. Following resolution of this complication, intraocular lens opacification was observed.
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http://dx.doi.org/10.4103/ojo.OJO_173_2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380154PMC
February 2019

EPOP Functionally Links Elongin and Polycomb in Pluripotent Stem Cells.

Mol Cell 2016 11;64(4):645-658

Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Dr. Aiguader 88, Barcelona 08003, Spain; Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain; ICREA, Pg. Lluis Companys 23, 08010 Barcelona, Spain. Electronic address:

The cellular plasticity of pluripotent stem cells is thought to be sustained by genomic regions that display both active and repressive chromatin properties. These regions exhibit low levels of gene expression, yet the mechanisms controlling these levels remain unknown. Here, we describe Elongin BC as a binding factor at the promoters of bivalent sites. Biochemical and genome-wide analyses show that Elongin BC is associated with Polycomb Repressive Complex 2 (PRC2) in pluripotent stem cells. Elongin BC is recruited to chromatin by the PRC2-associated factor EPOP (Elongin BC and Polycomb Repressive Complex 2 Associated Protein, also termed C17orf96, esPRC2p48, E130012A19Rik), a protein expressed in the inner cell mass of the mouse blastocyst. Both EPOP and Elongin BC are required to maintain low levels of expression at PRC2 genomic targets. Our results indicate that keeping the balance between activating and repressive cues is a more general feature of chromatin in pluripotent stem cells than previously appreciated.
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http://dx.doi.org/10.1016/j.molcel.2016.10.018DOI Listing
November 2016

Direct interaction between Id1 and Zrf1 controls neural differentiation of embryonic stem cells.

EMBO Rep 2015 Jan 31;16(1):63-70. Epub 2014 Oct 31.

Centre for Genomic Regulation (CRG), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Id proteins are dominant-negative regulators within the HLH family of proteins. In embryonic stem cells (ESCs), Id1 and Id3 maintain the pluripotent state by preventing neural differentiation. The Id1-interacting protein Zrf1 plays a crucial role as a chromatin-bound factor in specification of the neural fate from ESCs. Here, we show that Id1 blocks Zrf1 recruitment to chromatin, thus preventing the activation of neural genes in ESCs. Upon differentiation, Id1 expression decreases thus inducing Zrf1 binding to neural genes. Importantly, depletion of Zrf1 rescues the expression of Polycomb targets involved in neural specification which are up-regulated in Id1 knock-out ESCs. We therefore identified Zrf1 as transcriptional regulator of neural fate downstream of Id1 in ESCs.
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http://dx.doi.org/10.15252/embr.201439560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4304729PMC
January 2015

Role of UTX in retinoic acid receptor-mediated gene regulation in leukemia.

Mol Cell Biol 2014 Oct 28;34(19):3765-75. Epub 2014 Jul 28.

Center for Genomic Regulation, Barcelona, Spain Universitat Pompeu Fabra, Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain

Human UTX, a member of the Jumonji C family of proteins, associates with mixed-lineage leukemia 3/4 complexes. Stimulation with retinoic acid leads to the recruitment of UTX-containing complexes to HOX genes, which results in demethylation of histone H3 lysine 27 and concomitant methylation of histone H3 lysine 4. Here, we show that UTX interacts with the retinoic acid receptor α (RARα) and that this interaction is essential for proper differentiation of leukemic U937 cells in response to retinoic acid. UTX occupies the promoters of several RAR target genes and regulates their transcriptional output by modulating ASH2L complex recruitment. Overexpression of UTX in promyelocytic NB4 cells results in enhanced cellular differentiation upon retinoic acid treatment. Our results show that UTX is important for RAR-mediated transcription and provide insight into the critical role of cross talk between histone H3 lysine 4 methylation and histone H3 lysine 27 demethylation during cellular differentiation.
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http://dx.doi.org/10.1128/MCB.00839-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187727PMC
October 2014

Zrf1 is required to establish and maintain neural progenitor identity.

Genes Dev 2014 Jan;28(2):182-97

Centre for Genomic Regulation (CRG), Universitat Pompeu Fabra, 08003 Barcelona, Spain;

The molecular mechanisms underlying specification from embryonic stem cells (ESCs) and maintenance of neural progenitor cells (NPCs) are largely unknown. Recently, we reported that the Zuotin-related factor 1 (Zrf1) is necessary for chromatin displacement of the Polycomb-repressive complex 1 (PRC1). We found that Zrf1 is required for NPC specification from ESCs and that it promotes the expression of NPC markers, including the key regulator Pax6. Moreover, Zrf1 is essential to establish and maintain Wnt ligand expression levels, which are necessary for NPC self-renewal. Reactivation of proper Wnt signaling in Zrf1-depleted NPCs restores Pax6 expression and the self-renewal capacity. ESC-derived NPCs in vitro resemble most of the characteristics of the self-renewing NPCs located in the developing embryonic cortex, which are termed radial glial cells (RGCs). Depletion of Zrf1 in vivo impairs the expression of key self-renewal regulators and Wnt ligand genes in RGCs. Thus, we demonstrate that Zrf1 plays an essential role in NPC generation and maintenance.
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http://dx.doi.org/10.1101/gad.228510.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3909791PMC
January 2014

DPY30 regulates pathways in cellular senescence through ID protein expression.

EMBO J 2013 Aug 19;32(16):2217-30. Epub 2013 Jul 19.

Centre for Genomic Regulation (CRG) and UPF, Department of Gene Regulation, Stem Cells and Cancer, Barcelona, Spain.

Cellular senescence is an intrinsic defense mechanism to various cellular stresses: while still metabolically active, senescent cells stop dividing and enter a proliferation arrest. Here, we identify DPY30, a member of all mammalian histone H3K4 histone methyltransferases (HMTases), as a key regulator of the proliferation potential of human primary cells. Following depletion of DPY30, cells show a severe proliferation defect and display a senescent phenotype, including a flattened and enlarged morphology, elevated level of reactive oxygen species (ROS), increased SA-β-galactosidase activity, and formation of senescence-associated heterochromatin foci (SAHFs). While DPY30 depletion leads to a reduced level of H3K4me3-marked active chromatin, we observed a concomitant activation of CDK inhibitors, including p16INK4a, independent of H3K4me3. ChIP experiments show that key regulators of cell-cycle progression, including ID proteins, are under direct control of DPY30. Because ID proteins are negative regulators of the transcription factors ETS1/2, depletion of DPY30 leads to the transcriptional activation of p16INK4a by ETS1/2 and thus to a senescent-like phenotype. Ectoptic re-introduction of ID protein expression can partially rescue the senescence-like phenotype induced by DPY30 depletion. Thus, our data indicate that DPY30 controls proliferation by regulating ID proteins expression, which in turn lead to senescence bypass.
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http://dx.doi.org/10.1038/emboj.2013.159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3746196PMC
August 2013

The DNA demethylating agent decitabine activates the TRAIL pathway and induces apoptosis in acute myeloid leukemia.

Biochim Biophys Acta 2013 Jan 6;1832(1):114-20. Epub 2012 Oct 6.

Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.

Although epigenetic drugs have been approved for use in selected malignancies, there is significant need for a better understanding of their mechanism of action. Here, we study the action of a clinically approved DNA-methyltransferase inhibitor - decitabine (DAC) - in acute myeloid leukemia (AML) cells. At low doses, DAC treatment induced apoptosis of NB4 Acute Promyelocytic Leukemia (APL) cells, which was associated with the activation of the extrinsic apoptotic pathway. Expression studies of the members of the Death Receptor family demonstrated that DAC induces the expression of TNF-related apoptosis-inducing ligand (TRAIL). Upregulation of TRAIL, upon DAC treatment, was associated with specific epigenetic modifications induced by DAC in the proximity of the TRAIL promoter, as demonstrated by DNA demethylation, increased DNaseI sensitivity and histone acetylation of a non-CpG island, CpG-rich region located 2kb upstream to the transcription start site. Luciferase assay experiments showed that this region behave as a DNA methylation sensitive transcriptional regulatory element. The CpG regulatory element was also found methylated in samples derived from APL patients. These findings have been confirmed in the non-APL, AML Kasumi cell line, suggesting that this regulatory mechanism may be extended to other AMLs. Our study suggests that DNA methylation is a regulatory mechanism relevant for silencing of the TRAIL apoptotic pathway in leukemic cells, and further elucidates the mechanism by which epigenetic drugs mediate their anti-leukemic effects.
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http://dx.doi.org/10.1016/j.bbadis.2012.10.001DOI Listing
January 2013

DNA methylation of the gonadal aromatase (cyp19a) promoter is involved in temperature-dependent sex ratio shifts in the European sea bass.

PLoS Genet 2011 Dec 29;7(12):e1002447. Epub 2011 Dec 29.

Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain.

Sex ratio shifts in response to temperature are common in fish and reptiles. However, the mechanism linking temperature during early development and sex ratios has remained elusive. We show in the European sea bass (sb), a fish in which temperature effects on sex ratios are maximal before the gonads form, that juvenile males have double the DNA methylation levels of females in the promoter of gonadal aromatase (cyp19a), the enzyme that converts androgens into estrogens. Exposure to high temperature increased the cyp19a promoter methylation levels of females, indicating that induced-masculinization involves DNA methylation-mediated control of aromatase gene expression, with an observed inverse relationship between methylation levels and expression. Although different CpGs within the sb cyp19a promoter exhibited different sensitivity to temperature, we show that the increased methylation of the sb cyp19a promoter, which occurs in the gonads but not in the brain, is not a generalized effect of temperature. Importantly, these effects were also observed in sexually undifferentiated fish and were not altered by estrogen treatment. Thus, methylation of the sb cyp19a promoter is the cause of the lower expression of cyp19a in temperature-masculinized fish. In vitro, induced methylation of the sb cyp19a promoter suppressed the ability of SF-1 and Foxl2 to stimulate transcription. Finally, a CpG differentially methylated by temperature and adjacent to a Sox transcription factor binding site is conserved across species. Thus, DNA methylation of the aromatase promoter may be an essential component of the long-sought-after mechanism connecting environmental temperature and sex ratios in vertebrate species with temperature-dependent sex determination.
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http://dx.doi.org/10.1371/journal.pgen.1002447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3248465PMC
December 2011

E-box-independent regulation of transcription and differentiation by MYC.

Nat Cell Biol 2011 Oct 23;13(12):1443-9. Epub 2011 Oct 23.

Centre de Regulació Genòmica and UPF, Barcelona 08003, Spain.

MYC proto-oncogene is a key player in cell homeostasis that is commonly deregulated in human carcinogenesis(1). MYC can either activate or repress target genes by forming a complex with MAX (ref. 2). MYC also exerts MAX-independent functions that are not yet fully characterized(3). Cells possess an intrinsic pathway that can abrogate MYC-MAX dimerization and E-box interaction, by inducing phosphorylation of MYC in a PAK2-dependent manner at three residues located in its helix-loop-helix domain(4). Here we show that these carboxy-terminal phosphorylation events switch MYC from an oncogenic to a tumour-suppressive function. In undifferentiated cells, MYC-MAX is targeted to the promoters of retinoic-acid-responsive genes by its direct interaction with the retinoic acid receptor-α (RARα). MYC-MAX cooperates with RARα to repress genes required for differentiation, in an E-box-independent manner. Conversely, on C-terminal phosphorylation of MYC during differentiation, the complex switches from a repressive to an activating function, by releasing MAX and recruiting transcriptional co-activators. Phospho-MYC synergizes with retinoic acid to eliminate circulating leukaemic cells and to decrease the level of tumour invasion. Our results identify an E-box-independent mechanism for transcriptional regulation by MYC that unveils previously unknown functions for MYC in differentiation. These may be exploited to develop alternative targeted therapies.
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http://dx.doi.org/10.1038/ncb2355DOI Listing
October 2011

The histone variant macroH2A is an epigenetic regulator of key developmental genes.

Nat Struct Mol Biol 2009 Oct 6;16(10):1074-9. Epub 2009 Sep 6.

Centre de Regulació Genòmica (CRG), PRBB, Barcelona, Spain.

The histone variants macroH2A1 and macroH2A2 are associated with X chromosome inactivation in female mammals. However, the physiological function of macroH2A proteins on autosomes is poorly understood. Microarray-based analysis in human male pluripotent cells uncovered occupancy of both macroH2A variants at many genes encoding key regulators of development and cell fate decisions. On these genes, the presence of macroH2A1+2 is a repressive mark that overlaps locally and functionally with Polycomb repressive complex 2. We demonstrate that macroH2A1+2 contribute to the fine-tuning of temporal activation of HOXA cluster genes during neuronal differentiation. Furthermore, elimination of macroH2A2 function in zebrafish embryos produced severe but specific phenotypes. Taken together, our data demonstrate that macroH2A variants constitute an important epigenetic mark involved in the concerted regulation of gene expression programs during cellular differentiation and vertebrate development.
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http://dx.doi.org/10.1038/nsmb.1665DOI Listing
October 2009

ERalpha as ligand-independent activator of CDH-1 regulates determination and maintenance of epithelial morphology in breast cancer cells.

Proc Natl Acad Sci U S A 2009 May 21;106(18):7420-5. Epub 2009 Apr 21.

Department of Medicine, Howard Hughes Medical Institute, University of California at San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0651, USA.

Estrogen receptor alpha (ERalpha) and E-cadherin are primary markers of luminal epithelial breast cancer cells with E-cadherin being a main caretaker of the epithelial phenotype. E-cadherin repression is needed for cancer cells to acquire motile and invasive properties, and it is known that in ER-positive breast cancer cells, estrogen down-regulate E-cadherin gene transcription. We report here that ERalpha is bound to the E-cadherin promoter in both the presence and the complete absence of estrogen, suggesting an unexpected role for unliganded ERalpha in E-cadherin transcription. Indeed, our data reveal that activation by unliganded ERalpha and repression by estrogen-activated ERalpha require direct binding to a half-estrogen response element within the E-cadherin promoter and exchange from associated coactivators to corepressors. Therefore, these results suggest a pivotal role for unliganded ERalpha in controlling a fundamental caretaker of the epithelial phenotype in breast cancer cells. Here, we show that ERalpha-positive breast cancer T47D cells transduced with the sfRON kinase undergo a full epithelial-mesenchymal conversion and lose E-cadherin and ERalpha expression. Our data show that, although the E-cadherin gene becomes hypermethylated and heterochromatic, kinase inhibitors can restore E-cadherin expression, together with an epithelial morphology in an ERalpha-dependent fashion. Similarly, transfection of ERalpha, in the absence of ligands, was sufficient to restore E-cadherin transcription in both sfRON-T47D and other ERalpha-, E-cadherin-negative cells. Therefore, our results suggest a novel role for the ERalpha that plays the dual role of ligand-independent activator and ligand-dependent repressor of E-cadherin in breast cancer cells.
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http://dx.doi.org/10.1073/pnas.0903033106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671327PMC
May 2009

MBD3, a component of the NuRD complex, facilitates chromatin alteration and deposition of epigenetic marks.

Mol Cell Biol 2008 Oct 21;28(19):5912-23. Epub 2008 Jul 21.

Centre de Regulacio Genomica, Universitat Pompeu Fabra, Barcelona, Spain.

In plants, as in mammals, mutations in SNF2-like DNA helicases/ATPases were shown to affect not only chromatin structure but also global methylation patterns, suggesting a potential functional link between chromatin structure and epigenetic marks. The SNF2-like ATPase containing nucleosome remodeling and deacetylase corepressor complex (NuRD) is involved in gene transcriptional repression and chromatin remodeling. We have previously shown that the leukemogenic protein PML-RARa represses target genes through recruitment of DNA methytransferases and Polycomb complex. Here, we demonstrate a direct role of the NuRD complex in aberrant gene repression and transmission of epigenetic repressive marks in acute promyelocytic leukemia (APL). We show that PML-RARa binds and recruits NuRD to target genes, including to the tumor-suppressor gene RARbeta2. In turn, the NuRD complex facilitates Polycomb binding and histone methylation at lysine 27. Retinoic acid treatment, which is often used for patients at the early phase of the disease, reduced the promoter occupancy of the NuRD complex. Knockdown of the NuRD complex in leukemic cells not only prevented histone deacetylation and chromatin compaction but also impaired DNA and histone methylation, as well as stable silencing, thus favoring cellular differentiation. These results unveil an important role for NuRD in the establishment of altered epigenetic marks in APL, demonstrating an essential link between chromatin structure and epigenetics in leukemogenesis that could be exploited for therapeutic intervention.
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http://dx.doi.org/10.1128/MCB.00467-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2546998PMC
October 2008

Polycomb complex 2 is required for E-cadherin repression by the Snail1 transcription factor.

Mol Cell Biol 2008 Aug 2;28(15):4772-81. Epub 2008 Jun 2.

Programa de Recerca en Càncer, IMIM-Hospital del Mar, Barcelona, Spain.

The transcriptional factor Snail1 is a repressor of E-cadherin (CDH1) gene expression essential for triggering epithelial-mesenchymal transition. Snail1 represses CDH1, directly binding its promoter and inducing the synthesis of the Zeb1 repressor. In this article, we show that repression of CDH1 by Snail1, but not by Zeb1, is dependent on the activity of Polycomb repressive complex 2 (PRC2). Embryonic stem (ES) cells null for Suz12, one of the components of PRC2, show higher levels of Cdh1 mRNA than control ES cells. In tumor cells, interference of PRC2 activity prevents the ability of Snail1 to downregulate CDH1 and partially derepresses CDH1. Chromatin immunoprecipitation assays demonstrated that Snail1 increases the binding of Suz12 to the CDH1 promoter and the trimethylation of lysine 27 in histone H3. Moreover, Snail1 interacts with Suz12 and Ezh2, as shown by coimmunoprecipitation experiments. In conclusion, these results demonstrate that Snail1 recruits PRC2 to the CDH1 promoter and requires the activity of this complex to repress E-cadherin expression.
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http://dx.doi.org/10.1128/MCB.00323-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493371PMC
August 2008

Role of the polycomb repressive complex 2 in acute promyelocytic leukemia.

Cancer Cell 2007 Jun;11(6):513-25

Centre de Regulacio Genomica, c/ Dr. Aiguader 88, 08003 Barcelona, Spain.

Epigenetic changes are common alterations in cancer cells. Here, we have investigated the role of Polycomb group proteins in the establishment and maintenance of the aberrant silencing of tumor suppressor genes during transformation induced by the leukemia-associated PML-RARalpha fusion protein. We show that in leukemic cells knockdown of SUZ12, a key component of Polycomb repressive complex 2 (PRC2), reverts not only histone modification but also induces DNA demethylation of PML-RARalpha target genes. This results in promoter reactivation and granulocytic differentiation. Importantly, the epigenetic alterations caused by PML-RARalpha can be reverted by retinoic acid treatment of primary blasts from leukemic patients. Our results demonstrate that the direct targeting of Polycomb group proteins by an oncogene plays a key role during carcinogenesis.
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http://dx.doi.org/10.1016/j.ccr.2007.04.009DOI Listing
June 2007

The methyl-CpG binding protein MBD1 is required for PML-RARalpha function.

Proc Natl Acad Sci U S A 2006 Jan 23;103(5):1400-5. Epub 2006 Jan 23.

Centre de Regulacio Genomica, Universitat Pompeu Fabra, Passeig Maritim 37-49, 08003 Barcelona, Spain.

PML-RARalpha induces a block of hematopoietic differentiation and acute promyelocytic leukemia. This block is based on its capacity to inactivate target genes by recruiting histone deacetylase (HDAC) and DNA methyltransferase activities. Here we report that MBD1, a member of a conserved family of proteins able to bind methylated DNA, cooperates with PML-RARalpha in transcriptional repression and cellular transformation. PML-RARalpha recruits MBD1 to its target promoter through an HDAC3-mediated mechanism. Binding of HDAC3 and MBD1 is not confined to the promoter region but instead is spread over the locus. Knock-down of HDAC3 expression by RNA interference in acute promyelocytic leukemia cells alleviates PML-RAR-induced promoter silencing. We further demonstrate that retroviral expression of dominant-negative mutants of MBD1 in hematopoietic precursors compromises the ability of PML-RARalpha to block their differentiation and thus restored cell differentiation. Our results demonstrate that PML-RARalpha functions by recruiting an HDAC3-MBD1 complex that contributes to the establishment and maintenance of the silenced chromatin state.
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http://dx.doi.org/10.1073/pnas.0509343103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1360559PMC
January 2006

Myc represses transcription through recruitment of DNA methyltransferase corepressor.

EMBO J 2005 Jan 16;24(2):336-46. Epub 2004 Dec 16.

Free University of Brussels, Faculty of Medicine, Laboratory of Molecular Virology, Brussels, Belgium.

The Myc transcription factor is an essential mediator of cell growth and proliferation through its ability to both positively and negatively regulate transcription. The mechanisms by which Myc silences gene expression are not well understood. The current model is that Myc represses transcription through functional interference with transcriptional activators. Here we show that Myc binds the corepressor Dnmt3a and associates with DNA methyltransferase activity in vivo. In cells with reduced Dnmt3a levels, we observe specific reactivation of the Myc-repressed p21Cip1 gene, whereas the expression of Myc-activated E-boxes genes is unchanged. In addition, we find that Myc can target Dnmt3a selectively to the promoter of p21Cip1. Myc is known to be recruited to the p21Cip1 promoter by the DNA-binding factor Miz-1. Consistent with this, we observe that Myc and Dnmt3a form a ternary complex with Miz-1 and that this complex can corepress the p21Cip1 promoter. Finally, we show that DNA methylation is required for Myc-mediated repression of p21Cip1. Our data identify a new mechanism by which Myc can silence gene expression not only by passive functional interference but also by active recruitment of corepressor proteins. Furthermore, these findings suggest that targeting of DNA methyltransferases by transcription factors is a wide and general mechanism for the generation of specific DNA methylation patterns within a cell.
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http://dx.doi.org/10.1038/sj.emboj.7600509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC545804PMC
January 2005

HIV-1 resistance to the gp41-dependent fusion inhibitor C-34.

Antiviral Res 2003 Jul;59(2):137-42

Hospital Germans Trias i Pujol, Universitat Autónoma de Bercelona, 08916 Badalona, Spain.

The gp41 subunit of HIV-1 has been recently recognized as a target for antiviral therapy. C-34 is a peptide that mimics the heptad repeat 2 in the ectodomain of gp41. Here, we describe two HIV-1 strains selected after 5 and 17 passages in culture with increasing concentrations of C-34 (breakthrough concentration of 10 microg/ml). The HXB2-derived strain was more than 1000-fold resistant and contained a V38E mutation in the gp41 coding DNA sequence. The NL4-3-derived strain was more than 500-fold resistant and contained a L33S mutation in gp41. No cross-resistance to the RT inhibitor AZT, the HIV binding inhibitor dextran sulfate (DS), or the chemokine receptor antagonist ALX-40-4C was detected. These data indicate that HIV-1 can overcome C-34 inhibition through mutations at residues not involved in the formation of the hydrophobic cavity of gp41.
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http://dx.doi.org/10.1016/s0166-3542(03)00071-8DOI Listing
July 2003

Role of the human immunodeficiency virus type 1 envelope gene in viral fitness.

J Virol 2003 Aug;77(16):9069-73

Department of Virology, Lerner Research Institute, Cleveland Clinic Foundation, OH 44195, USA.

A human host offers a variety of microenvironments to the infecting human immunodeficiency virus type 1 (HIV-1), resulting in various selective pressures, most of them directed against the envelope (env) gene. Therefore, it seems evident that the replicative capacity of the virus is largely related to viral entry. In this study we have used growth competition experiments and TaqMan real-time PCR detection to measure the fitness of subtype B HIV-1 primary isolates and autologous env-recombinant viruses in order to analyze the contribution of wild-type env sequences to overall HIV-1 fitness. A significant correlation was observed between fitness values obtained for wild-type HIV-1 isolates and those for the corresponding env-recombinant viruses (r = 0.93; P = 0.002). Our results suggest that the env gene, which is linked to a myriad of viral characteristics (e.g., entry into the host cell, transmission, coreceptor usage, and tropism), plays a major role in fitness of wild-type HIV-1. In addition, this new recombinant assay may be useful for measuring the contribution of HIV-1 env to fitness in viruses resistant to novel antiretroviral entry inhibitors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC167250PMC
http://dx.doi.org/10.1128/jvi.77.16.9069-9073.2003DOI Listing
August 2003

Anti-HIV-1 activity of enfuvirtide (T-20) by inhibition of bystander cell death.

Antivir Ther 2003 Apr;8(2):155-61

Retrovirology Laboratory irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain.

Infection by human immunodeficiency virus type 1 (HIV-1) has been associated with increased cell death of both infected and bystander cells. The envelope glycoprotein complex appears to play an active role in HIV-induced death of bystander cells. We quantified cell-to-cell fusion, single cell death and membrane lipid mixing in cocultures of effector, HIV-1 envelope-expressing cells with peripheral blood mononuclear cells or purified CD4 T lymphocytes from HIV-negative donors, in the presence or the absence of the fusion inhibitor enfuvirtide (T-20, pentafuside, Fuzeon). T-20, which blocks gp41-dependent virus-cell fusion, showed a complete and dose-dependent inhibition of syncytium formation in cocultures of envelope-expressing cells with uninfected cells. Similarly, T-20 totally abrogated death of single bystander CD4 T cells with an IC50 of 0.04 microg/ml. Membrane lipid mixing, as a measure of interaction between envelope-expressing cells and CD4 cells, was also dose-dependently inhibited by T-20. Moreover, effector cells chronically infected with a T-20-resistant virus recovered the ability to induce bystander cell death in the presence of the drug, supporting the role of gp41 in single cell death. In conclusion, T-20 is able to protect CD4 T cells from envelope presentation with a dual effect: inhibition of virus replication and blockade of HIV-1 envelope-induced cell death of bystander CD4 T cells. Protection of cells prior to infection from HIV envelope-dependent bystander effect could lead to a better immune restoration of HIV-1-infected patients that are treated with T-20.
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April 2003

Interleukin-7-dependent production of RANTES that correlates with human immunodeficiency virus disease progression.

J Virol 2003 Apr;77(7):4389-95

Retrovirology Laboratory irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Spain.

There is a relationship between CD4-T-cell number and circulating interleukin 7 (IL-7) levels in human immunodeficiency virus (HIV)-positive individuals. Here, we show that IL-7 induced a dose-dependent production of CCL3 (MIP-1alpha), CCL4 (MIP-1beta), and CCL5 (RANTES) in peripheral blood mononuclear cells (PBMC), ex vivo tonsil lymphoid tissue of HIV(-) individuals, and PBMC from HIV(+) individuals, suggesting that IL-7 may regulate beta-chemokine production in vivo. In a cross-sectional study of HIV(+) individuals (n = 130), a weak but significant correlation between IL-7 and RANTES was noted (r = 0.379; P < 0.001). Remarkably, the correlation between IL-7 and RANTES increased to an r value of 0.798 (P < 0.001) if individuals with low CD4 cell counts (<200 cells/ micro l) were excluded from the analysis. Our results suggest that there is a relationship between IL-7 and the production of RANTES both in vitro and in vivo that is lost in immune-compromised patients (CD4 count of <200 cells/ micro l) but that could be restored by antiretroviral therapy. Unlike the case for IL-7, high levels of RANTES suggest an intermediate stage of HIV disease progression.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150631PMC
http://dx.doi.org/10.1128/jvi.77.7.4389-4395.2003DOI Listing
April 2003

Cell-surface-expressed HIV-1 envelope induces the death of CD4 T cells during GP41-mediated hemifusion-like events.

Virology 2003 Jan;305(2):318-29

Laboratori de Retrovirologia, Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Catalonia, Spain.

Cells expressing the HIV-1 envelope glycoprotein complex (gp120/gp41, Env) induce the death of target cells either after cell-to-cell fusion or after cell-to-cell contact in a fusion-independent fashion. Here, we demonstrate that Env-induced death of single cells (including primary CD4 T cells) required gp120 and gp41 function. The gp41 peptide C34, which blocked syncytium formation, completely inhibited the death of single target cells by specifically acting on gp41 function. Moreover, Env-induced single cell death was exclusively observed in CD4 cells and was associated with specific gp41-mediated transfer of lipids from the membrane of Env-expressing cells to the target cell but not with detectable cytoplasm mixing (complete fusion). We conclude that after gp120 function, gp41 mediates close cell-to-cell contacts, thereby triggering cell death in single uninfected cells in the absence of detectable cell-to-cell fusion.
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http://dx.doi.org/10.1006/viro.2002.1764DOI Listing
January 2003

Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication.

AIDS 2002 Dec;16(18):2385-90

Retrovirology Laboratory IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916 Badalona, Spain.

Objectives: Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1.

Methods: A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively.

Results: siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed.

Conclusion: Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.
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http://dx.doi.org/10.1097/00002030-200212060-00002DOI Listing
December 2002

Preferential attachment of HIV particles to activated and CD45RO+CD4+ T cells.

AIDS Res Hum Retroviruses 2002 Jan;18(1):27-38

Laboratori de Retrovirologia, Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona 08916, Catalonia, Spain.

We have studied the binding of biotinylated HIV particles to various cell lines and peripheral blood mononuclear cells (PBMCs). Viruses were harvested from cultures of cell surface-biotinylated cells productively infected with HIV-IIIB. Labeled HIV particles bound to and infected CD4(+) cell lines and PBMCs. The interaction between gp120 and CD4 contributed in part to HIV binding to CD4(+) cells. However, HIV binding was for the most part independent of CD4 expression and sensitive to polyanion inhibition. Polyanion-sensitive interactions involved heparan sulfate in cell lines but not in primary T cells. Interestingly, HIV binding to primary cells was heterogeneous and targeted discrete subsets of CD4(+) and CD4(-) cells. The CD4(+) T cell subset that displayed high HIV-binding capacity contained mostly CD4(+)CD45RO(+) cells, whereas the subset showing undetectable HIV binding contained higher proportions of CD4(+)CD45RO(-) cells. Consistently, purified CD4(+)CD45RO(-) cells or purified CD4(+) T cells with low virus-binding capacity showed lower HIV entry and delayed HIV replication when compared with purified CD4(+)CD45RO(+) or purified CD4(+) T cells with high virus-binding capacity, respectively. Our data suggest that the binding of HIV to cell surface-expressed CD4 might be inefficient in a subset of CD4(+) T cells and that increased binding of HIV to activated and CD4(+)CD45RO(+) cells may contribute to the higher susceptibility of these cells to HIV infection.
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http://dx.doi.org/10.1089/088922202753394691DOI Listing
January 2002

Anti-HIV activity of a novel aminoglycoside-arginine conjugate.

Antiviral Res 2002 Jan;53(1):1-8

Retrovirology Laboratory, Fundació irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autonoma de Barcelona, 08916, Badalona, Spain.

We have previously described conjugates of L-arginine with aminoglycosides (AAC) that have shown anti-human immunodeficiency virus type 1 (HIV-1) activity in in vitro cell culture systems. Here, we extend our report to a novel neomycin B-arginine conjugate (NeoR) that has shown up to 30-fold increased potency over previous AAC compounds. NeoR inhibited the replication of both R5 and X4 strains of HIV-1 in cells expressing the appropriate coreceptor or peripheral blood mononuclear cells. In lymphoid tissue ex vivo, NeoR blocked the replication of the dualtropic strain 89.6 suggesting anti-HIV activity of AAC on the site of in vivo virus replication. NeoR blocked the binding of HIV particles to lymphoid cells and was also able to antagonize the activity of the CXCR4 receptor so it may prevent the emergence of X4 HIV-1 strains. Nevertheless, in a cellular assay, we were unable to detect anti-Tat dependent transactivation activity as previously suggested for this family of compounds.
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http://dx.doi.org/10.1016/s0166-3542(01)00188-7DOI Listing
January 2002