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1.
Cell ; 138(2): 300-13, 2009 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-19632180

RESUMEN

While hematopoietic stem cell (HSC) self-renewal is well studied, it remains unknown whether distinct control mechanisms enable HSC divisions that generate progeny cells with specific lineage bias. Here, we report that the monocytic transcription factor MafB specifically restricts the ability of M-CSF to instruct myeloid commitment divisions in HSCs. MafB deficiency specifically enhanced sensitivity to M-CSF and caused activation of the myeloid master-regulator PU.1 in HSCs in vivo. Single-cell analysis revealed that reduced MafB levels enabled M-CSF to instruct divisions producing asymmetric daughter pairs with one PU.1(+) cell. As a consequence, MafB(-/-) HSCs showed a PU.1 and M-CSF receptor-dependent competitive repopulation advantage specifically in the myelomonocytic, but not T lymphoid or erythroid, compartment. Lineage-biased repopulation advantage was progressive, maintained long term, and serially transplantable. Together, this indicates that an integrated transcription factor/cytokine circuit can control the rate of specific HSC commitment divisions without compromising other lineages or self-renewal.


Asunto(s)
Linaje de la Célula , Células Madre Hematopoyéticas/citología , Factor Estimulante de Colonias de Macrófagos/metabolismo , Factor de Transcripción MafB/metabolismo , Células Mieloides/citología , Animales , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Transactivadores/metabolismo
2.
EMBO J ; 34(15): 2042-58, 2015 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-26139534

RESUMEN

T cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T cells, often termed "exhausted" T cells. We compared the transcriptome of "exhausted" CD8 T cells infiltrating autochthonous melanomas to those of naïve and acutely stimulated CD8 T cells. Despite strong similarities between transcriptional signatures of tumor- and virus-induced exhausted CD8 T cells, notable differences appeared. Among transcriptional regulators, Nr4a2 and Maf were highly overexpressed in tumor-exhausted T cells and significantly upregulated in CD8 T cells from human melanoma metastases. Transduction of murine tumor-specific CD8 T cells to express Maf partially reproduced the transcriptional program associated with tumor-induced exhaustion. Upon adoptive transfer, the transduced cells showed normal homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor effector responses. We further identified TGFß and IL-6 as main inducers of Maf expression in CD8 T cells and showed that Maf-deleted tumor-specific CD8 T cells were much more potent to restrain tumor growth in vivo. Therefore, the melanoma microenvironment contributes to skewing of CD8 T cell differentiation programs, in part by TGFß/IL-6-mediated induction of Maf.


Asunto(s)
Linfocitos T CD8-positivos/citología , Diferenciación Celular/inmunología , Melanoma/metabolismo , Proteínas Proto-Oncogénicas c-maf/metabolismo , Microambiente Tumoral/fisiología , Animales , Linfocitos T CD8-positivos/metabolismo , Cartilla de ADN/genética , Citometría de Flujo , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Interleucina-6/metabolismo , Luciferasas , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-maf/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta/metabolismo
3.
Nucleic Acids Res ; 44(8): 3567-85, 2016 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-26673693

RESUMEN

Ets1 is a sequence-specific transcription factor that plays an important role during hematopoiesis, and is essential for the transition of CD4(-)/CD8(-) double negative (DN) to CD4(+)/CD8(+) double positive (DP) thymocytes. Using genome-wide and functional approaches, we investigated the binding properties, transcriptional role and chromatin environment of Ets1 during this transition. We found that while Ets1 binding at distal sites was associated with active genes at both DN and DP stages, its enhancer activity was attained at the DP stage, as reflected by levels of the core transcriptional hallmarks H3K4me1/3, RNA Polymerase II and eRNA. This dual, stage-specific ability reflected a switch from non-T hematopoietic toward T-cell specific gene expression programs during the DN-to-DP transition, as indicated by transcriptome analyses of Ets1(-/-) thymic cells. Coincidentally, Ets1 associates more specifically with Runx1 in DN and with TCF1 in DP cells. We also provide evidence that Ets1 predominantly binds distal nucleosome-occupied regions in DN and nucleosome-depleted regions in DP. Finally and importantly, we demonstrate that Ets1 induces chromatin remodeling by displacing H3K4me1-marked nucleosomes. Our results thus provide an original model whereby the ability of a transcription factor to bind nucleosomal DNA changes during differentiation with consequences on its cognate enhancer activity.


Asunto(s)
Diferenciación Celular/genética , Elementos de Facilitación Genéticos/genética , Nucleosomas/genética , Proteína Proto-Oncogénica c-ets-1/metabolismo , Linfocitos T/citología , Animales , Secuencia de Bases , Sitios de Unión/genética , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Línea Celular , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/genética , Hematopoyesis/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleosomas/metabolismo , Proteína Proto-Oncogénica c-ets-1/genética , ARN Polimerasa II/metabolismo , Análisis de Secuencia de ADN
4.
J Immunol ; 194(7): 3432-43, 2015 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-25732733

RESUMEN

V(D)J recombination assembles Ag receptor genes during lymphocyte development. Enhancers at AR loci are known to control V(D)J recombination at associated alleles, in part by increasing chromatin accessibility of the locus, to allow the recombination machinery to gain access to its chromosomal substrates. However, whether there is a specific mechanism to induce chromatin accessibility at AR loci is still unclear. In this article, we highlight a specialized epigenetic marking characterized by high and extended H3K4me3 levels throughout the Dß-Jß-Cß gene segments. We show that extended H3K4 trimethylation at the Tcrb locus depends on RNA polymerase II (Pol II)-mediated transcription. Furthermore, we found that the genomic regions encompassing the two DJCß clusters are highly enriched for Ser(5)-phosphorylated Pol II and short-RNA transcripts, two hallmarks of transcription initiation and early transcription. Of interest, these features are shared with few other tissue-specific genes. We propose that the entire DJCß regions behave as transcription "initiation" platforms, therefore linking a specialized mechanism of Pol II transcription with extended H3K4 trimethylation and highly accessible Dß and Jß gene segments.


Asunto(s)
Cromatina/genética , Sitios Genéticos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Transcripción Genética , Animales , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Inmunoprecipitación de Cromatina , Metilación de ADN , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , ARN Polimerasa II/metabolismo , Recombinación V(D)J
5.
BMC Genomics ; 14: 914, 2013 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-24365181

RESUMEN

BACKGROUND: Divergent transcription is a wide-spread phenomenon in mammals. For instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the RNA degradation machinery is inhibited. Moreover, many described long non-coding RNAs (lncRNAs) are transcribed antisense from coding gene promoters. However, the general significance of divergent lncRNA/mRNA gene pair transcription is still poorly understood. Here, we used strand-specific RNA-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues. RESULTS: We found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding RNA (lncRNA)/mRNA gene pairs. Strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. Their promoters share several characteristics with those of transcriptional developmental genes, including very large CpG islands, high degree of conservation and epigenetic regulation in ES cells. In-depth analysis revealed a unique GC skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. Finally, genes associated with antisense transcription harbor specific H3K79me2 epigenetic marking and RNA polymerase II enrichment profiles linked to an intensified rate of early transcriptional elongation. CONCLUSIONS: We concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription.


Asunto(s)
Elementos sin Sentido (Genética) , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , ARN Mensajero/genética , Transcripción Genética , Animales , Composición de Base , Cromatina/genética , Islas de CpG , Epigénesis Genética , Exones , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia de ARN , Timocitos
6.
EMBO J ; 27(14): 2006-17, 2008 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-18566588

RESUMEN

The function of the Ets-1 transcription factor is regulated by two regions that flank its DNA-binding domain. A previously established mechanism for auto-inhibition of monomeric Ets-1 on DNA response elements with a single ETS-binding site, however, has not been observed for the stromelysin-1 promoter containing two palindromic ETS-binding sites. We present the structure of Ets-1 on this promoter element, revealing a ternary complex in which protein homo-dimerization is mediated by the specific arrangement of the two ETS-binding sites. In this complex, the N-terminal-flanking region is required for ternary protein-DNA assembly. Ets-1 variants, in which residues from this region are mutated, loose the ability for DNA-mediated dimerization and stromelysin-1 promoter transactivation. Thus, our data unravel the molecular basis for relief of auto-inhibition and the ability of Ets-1 to function as a facultative dimeric transcription factor on this site. Our findings may also explain previous data of Ets-1 function in the context of heterologous transcription factors, thus providing a molecular model that could also be valid for Ets-1 regulation by hetero-oligomeric assembly.


Asunto(s)
ADN/metabolismo , Metaloproteinasa 3 de la Matriz/genética , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-ets-1/química , Proteína Proto-Oncogénica c-ets-1/metabolismo , Línea Celular , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Elementos Reguladores de la Transcripción , Activación Transcripcional
7.
Mol Cell Biol ; 27(15): 5554-64, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17548468

RESUMEN

During the execution of differentiation programs, lineage-specific transcription factors are in competition with antagonistic factors that drive progenitor proliferation. Thus, the myeloid transcription factor MafB promotes macrophage differentiation of myeloid progenitors, but a constitutively active Myb transcription factor (v-Myb) can maintain proliferation and block differentiation. Little is known, however, about the regulatory mechanisms that control such competing activities. Here we report that the small ubiquitin-like protein SUMO-1 can modify MafB in vitro and in vivo on lysines 32 and 297. The absence of MafB SUMO modification increased MafB-driven transactivation and macrophage differentiation potential but inhibited cell cycle progression and myeloid progenitor growth. Furthermore, we observed that direct repression of MafB transactivation by v-Myb was strictly dependent on MafB SUMO modification. Consequently, a SUMOylation-deficient MafB K32R K297R (K32,297R) mutant could specify macrophage fate even after activation of inducible Myb alleles and resist their differentiation-inhibiting activity. Our findings suggest that SUMO modification of MafB affects the balance between myeloid progenitor expansion and terminal macrophage differentiation by controlling MafB transactivation capacity and susceptibility to Myb repression. SUMO modification of lineage-specific transcription factors may thus modulate transcription factor antagonism to control tissue homeostasis in the hematopoietic system.


Asunto(s)
Diferenciación Celular , Macrófagos/citología , Factor de Transcripción MafB/metabolismo , Proteínas Oncogénicas v-myb/metabolismo , Proteínas Represoras/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Transcripción Genética , Animales , Línea Celular , Proliferación Celular , Pollos , Humanos , Ratones , Modelos Biológicos , Células Mieloides/citología , Unión Proteica , Células Madre/citología , Activación Transcripcional/genética
9.
Mol Cell Biol ; 26(18): 6808-18, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16943423

RESUMEN

In the hematopoietic system the bZip transcription factor MafB is selectively expressed at high levels in monocytes and macrophages and promotes macrophage differentiation in myeloid progenitors, whereas a dominant-negative allele can inhibit this process. To analyze the requirement of MafB for macrophage development, we generated MafB-deficient mice and, due to their neonatal lethal phenotype, analyzed macrophage differentiation in vitro, in the embryo, and in reconstituted mice. Surprisingly we observed in vitro differentiation of macrophages from E14.5 fetal liver (FL) cells and E18.5 splenocytes. Furthermore we found normal numbers of F4/80(+)/Mac-1(+) macrophages and monocytes in fetal liver, spleen, and blood as well as in bone marrow, spleen, and peritoneum of adult MafB(-/-) FL reconstituted mice. MafB(-/-) macrophages showed intact basic macrophage functions such as phagocytosis of latex beads or Listeria monocytogenes and nitric oxide production in response to lipopolysaccharide. By contrast, MafB(-/-) macrophages expressed increased levels of multiple genes involved in actin organization. Consistent with this, phalloidin staining revealed an altered morphology involving increased numbers of branched protrusions of MafB(-/-) macrophages in response to macrophage colony-stimulating factor. Together these data point to an unexpected redundancy of MafB function in macrophage differentiation and a previously unknown role in actin-dependent macrophage morphology.


Asunto(s)
Actinas/metabolismo , Macrófagos/citología , Factor de Transcripción MafB/deficiencia , Animales , Animales Recién Nacidos , Diferenciación Celular , Embrión de Mamíferos/citología , Feto/citología , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Sistema Hematopoyético/citología , Hígado/citología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-maf/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/citología , Irradiación Corporal Total
10.
Nat Genet ; 49(7): 1073-1081, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28581502

RESUMEN

Gene expression in mammals is precisely regulated by the combination of promoters and gene-distal regulatory regions, known as enhancers. Several studies have suggested that some promoters might have enhancer functions. However, the extent of this type of promoters and whether they actually function to regulate the expression of distal genes have remained elusive. Here, by exploiting a high-throughput enhancer reporter assay, we unravel a set of mammalian promoters displaying enhancer activity. These promoters have distinct genomic and epigenomic features and frequently interact with other gene promoters. Extensive CRISPR-Cas9 genomic manipulation demonstrated the involvement of these promoters in the cis regulation of expression of distal genes in their natural loci. Our results have important implications for the understanding of complex gene regulation in normal development and disease.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Células 3T3 , Animales , Sistemas CRISPR-Cas , Epigenómica , Ontología de Genes , Células HeLa , Humanos , Interferón-alfa/farmacología , Células K562 , Mamíferos/genética , Ratones
11.
Science ; 351(6274): aad5510, 2016 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-26797145

RESUMEN

Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells.


Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Células Madre Embrionarias/citología , Elementos de Facilitación Genéticos/fisiología , Regulación de la Expresión Génica , Macrófagos/citología , Animales , Proliferación Celular , Células Cultivadas , Regulación hacia Abajo , Redes Reguladoras de Genes , Factor de Transcripción MafB/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-maf/metabolismo , Análisis de la Célula Individual , Activación Transcripcional
12.
Gene ; 363: 85-96, 2005 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-16236469

RESUMEN

Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity on histone and various other cellular substrates. Functional analyses of such proteins have been carried out in a number of prokaryotes and eukaryotes organisms but until now, none have described an essential function for any SIR2 genes. Here using genetic approach, we report that a cytosolic SIR2 homolog in Leishmania is determinant to parasite survival. L. infantum promastigote tolerates deletion of one wild-type LiSIR2 allele (LiSIR2+/-) but achievement of null chromosomal mutants (LiSIR2-/-) requires episomal rescue. Accordingly, plasmid cure shows that these parasites maintain episome even in absence of drug pressure. Though single LiSIR2 gene disruption (LiSIR2+/-) does not affect the growth of parasite in the promastigote form, axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Taken together these data support a stage specific requirement and/or activity of the Leishmania cytosolic SIR2 protein and reveal an unrelated essential function for the life cycle of this unicellular pathogenic organism. The lack of an effective vaccine against leishmaniasis, and the need for alternative drug treatments, makes LiSIR2 protein a new attractive therapeutic target.


Asunto(s)
Proliferación Celular , Supervivencia Celular , Citosol/enzimología , Leishmania infantum/citología , Sirtuinas/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Leishmania infantum/enzimología , Ratones , Ratones Endogámicos BALB C
13.
Kinetoplastid Biol Dis ; 4(1): 1, 2005 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-15667659

RESUMEN

BACKGROUND: In yeast and Caenorhabditis elegans, Silent Information Regulator (SIR2) proteins have been shown to be involved in ageing regulation. In Leishmania, the LmSIR2rp was originally isolated from the excreted/secreted material of the Leishmania parasites. Among the function(s) of this protein in Leishmania biology, we have documented its implication in parasite survival, and in particular in Leishmania amastigotes. In this paper we question the role of the excreted/secreted form of the protein. In particular we wonder if the Leishmania Sir2 homologue is involved in some aspect of its biological function(s), in various components and pathways, which could promote the host cell survival. To test this hypothesis we have mimicked an intracellular release of the protein through constitutive expression in mouse L929 fibrosarcoma cells. RESULTS: Our results demonstrate that the LmSIR2 protein was properly expressed by fibroblasts and that LmSIR2 is localized both in the cytoplasm and the nucleus of all the transformed cell clones. Unexpectedly, we found that cells expressing LmSIR2 presents reduced saturation cell density ranging from 40% to 60% and expressed an acidic (pH6.0) beta-galactosidase activity, which is known to be a senescence biomarker. As a consequence, we observed that LmSIR2 positive fibroblasts were more permissive towards Leihmania infection. CONCLUSIONS: LmSIR2 is able to substantially interfere with the host cell physiology. Thus, it is tempting to speculate that these modifications could help Leishmania to survive for a long period in a cell with reduced capacity to multiply or respond to immunologic stimuli. The potential implications of our finding during the in vivo infection process are discussed.

14.
Acta Trop ; 94(2): 107-15, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15860278

RESUMEN

Silent information regulator 2 (SIR2) proteins are NAD-dependant deacetylases found in organisms ranging from bacteria to human. In eukaryotes, these proteins are involved in many biological processes including transcriptional repression, metabolism, ageing, or apoptosis. Here, we have shown that Sirtinol, a commercially available inhibitor of SIR2 deacetylases, significantly inhibits the in vitro proliferation of Leishmania infantum axenic amastigotes in a dose-dependent manner. This activity is stage specific since sirtinol did not affect the in vitro growth of parasite promastigotes. Growth arrest in amastigotes is associated with genomic DNA fragmentation, a process reminiscent of apoptosis. Interestingly parasites carrying extra copies of the LmSIR2 gene were less susceptible to the sirtinol mediated cell death. Altogether, these results constitute novel evidences that Leishmania SIR2 proteins play a role in the control of the parasite apoptotic phenomenon.


Asunto(s)
Benzamidas/farmacología , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Leishmania infantum/efectos de los fármacos , Naftoles/farmacología , Sirtuinas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , ADN Protozoario/efectos de los fármacos , ADN Protozoario/metabolismo , Citometría de Flujo , Histona Desacetilasas/biosíntesis , Histona Desacetilasas/metabolismo , Humanos , Leishmania infantum/enzimología , Leishmania infantum/genética , Leishmania infantum/crecimiento & desarrollo , Leishmaniasis Visceral/parasitología , Datos de Secuencia Molecular , Alineación de Secuencia , Sirtuinas/biosíntesis , Sirtuinas/metabolismo
15.
Nat Commun ; 6: 6905, 2015 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-25872643

RESUMEN

Cell-type specific regulation of gene expression requires the activation of promoters by distal genomic elements defined as enhancers. The identification and the characterization of enhancers are challenging in mammals due to their genome complexity. Here we develop CapStarr-Seq, a novel high-throughput strategy to quantitatively assess enhancer activity in mammals. This approach couples capture of regions of interest to previously developed Starr-seq technique. Extensive assessment of CapStarr-seq demonstrates accurate quantification of enhancer activity. Furthermore, we find that enhancer strength is associated with binding complexity of tissue-specific transcription factors and super-enhancers, while additive enhancer activity isolates key genes involved in cell identity and function. The CapStarr-Seq thus provides a fast and cost-effective approach to assess the activity of potential enhancers for a given cell type and will be helpful in decrypting transcription regulation mechanisms.


Asunto(s)
Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica/genética , Expresión Génica/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Factores de Transcripción/genética , Animales , Inmunoprecipitación de Cromatina , Masculino , Ratones , Células 3T3 NIH , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN/métodos
16.
Structure ; 22(3): 466-77, 2014 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-24530283

RESUMEN

The ability of basic leucine zipper transcription factors for homo- or heterodimerization provides a paradigm for combinatorial control of eukaryotic gene expression. It has been unclear, however, how facultative dimerization results in alternative DNA-binding repertoires on distinct regulatory elements. To unravel the molecular basis of such coupled preferences, we determined two high-resolution structures of the transcription factor MafB as a homodimer and as a heterodimer with c-Fos bound to variants of the Maf-recognition element. The structures revealed several unexpected and dimer-specific coiled-coil-heptad interactions. Based on these findings, we have engineered two MafB mutants with opposite dimerization preferences. One of them showed a strong preference for MafB/c-Fos heterodimerization and enabled selection of heterodimer-favoring over homodimer-specific Maf-recognition element variants. Our data provide a concept for transcription factor design to selectively activate dimer-specific pathways and binding repertoires.


Asunto(s)
Factor de Transcripción MafB/química , Factor de Transcripción MafB/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Sitios de Unión , ADN/metabolismo , Factor de Transcripción MafB/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Ingeniería de Proteínas/métodos , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-fos/química , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo
17.
J Exp Med ; 211(9): 1821-32, 2014 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-25135298

RESUMEN

V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus, Dδ2-Dδ3 rearrangements occur at a very immature thymic, CD34(+)/CD1a(-)/CD7(+dim) stage, before Dδ2(Dδ3)-Jδ1 rearrangements. These strictly ordered rearrangements are regulated by mechanisms acting beyond chromatin accessibility. Importantly, direct Dδ2-Jδ1 rearrangements are prohibited by a B12/23 restriction and ordered human TCR-δ gene assembly requires RUNX1 protein, which binds to the Dδ2-23RSS, interacts with RAG1, and enhances RAG1 deposition at this site. This RUNX1-mediated V(D)J recombinase targeting imposes the use of two Dδ gene segments in human TCR-δ chains. Absence of this RUNX1 binding site in the homologous mouse Dδ1-23RSS provides a molecular explanation for the lack of ordered TCR-δ gene assembly in mice and may underlie differences in early lymphoid differentiation between these species.


Asunto(s)
Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Reordenamiento Génico de la Cadena delta de los Receptores de Antígenos de los Linfocitos T , Proteínas de Homeodominio/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Diferenciación Celular , Línea Celular , ADN/genética , ADN/metabolismo , Células HEK293 , Humanos , Cinética , Linfopoyesis , Ratones , Datos de Secuencia Molecular , Especificidad de la Especie , Subgrupos de Linfocitos T/citología , VDJ Recombinasas/metabolismo
18.
Nucleus ; 3(2): 126-31, 2012 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-22555596

RESUMEN

Gene-distal cis-regulatory sequences, such as enhancers, are key contributors of tissue-specific gene expression. In particular, enhancers can be located up to hundreds of kilobases from the promoters that they control, making their identification challenging. Thanks to the recent technological advances to map histone modifications and chromatin-associated factors genome-wide, several studies have begun to characterize chromatin signatures of active enhancers. Here, we discuss some of these results and how they provide new insights into the tissue-specific organization of enhancer repertoires.


Asunto(s)
Cromatina/genética , Elementos de Facilitación Genéticos/genética , Animales , Cromatina/metabolismo , Genómica , Histonas/química , Histonas/metabolismo , Humanos , ARN Polimerasa II/metabolismo , Transcripción Genética
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