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1.
Immunity ; 45(6): 1219-1231, 2016 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-27913094

RESUMEN

Hematopoietic stem cells (HSCs) self-renew in bone marrow niches formed by mesenchymal progenitors and endothelial cells expressing the chemokine CXCL12, but whether a separate niche instructs multipotent progenitor (MPP) differentiation remains unclear. We show that MPPs resided in HSC niches, where they encountered lineage-instructive differentiation signals. Conditional deletion of the chemokine receptor CXCR4 in MPPs reduced differentiation into common lymphoid progenitors (CLPs), which decreased lymphopoiesis. CXCR4 was required for CLP positioning near Interleukin-7+ (IL-7) cells and for optimal IL-7 receptor signaling. IL-7+ cells expressed CXCL12 and the cytokine SCF, were mesenchymal progenitors capable of differentiation into osteoblasts and adipocytes, and comprised a minor subset of sinusoidal endothelial cells. Conditional Il7 deletion in mesenchymal progenitors reduced B-lineage committed CLPs, while conditional Cxcl12 or Scf deletion from IL-7+ cells reduced HSC and MPP numbers. Thus, HSC maintenance and multilineage differentiation are distinct cell lineage decisions that are both controlled by HSC niches.


Asunto(s)
Diferenciación Celular/fisiología , Células Madre Hematopoyéticas/citología , Células Madre Multipotentes/citología , Nicho de Células Madre/fisiología , Animales , Linaje de la Célula/fisiología , Separación Celular , Quimiocina CXCL2/metabolismo , Citometría de Flujo , Interleucina-7/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos
2.
Development ; 143(21): 4027-4037, 2016 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-27633995

RESUMEN

The thymus and parathyroids develop from third pharyngeal pouch (3rd pp) endoderm. Our previous studies show that Shh null mice have smaller, aparathyroid primordia in which thymus fate specification extends into the pharynx. SHH signaling is active in both dorsal pouch endoderm and neighboring neural crest (NC) mesenchyme. It is unclear which target tissue of SHH signaling is required for the patterning defects in Shh mutants. Here, we used a genetic approach to ectopically activate or delete the SHH signal transducer Smo in either pp endoderm or NC mesenchyme. Although no manipulation recapitulated the Shh null phenotype, manipulation of SHH signaling in either the endoderm or NC mesenchyme had direct and indirect effects on both cell types during fate specification and organogenesis. SHH pathway activation throughout pouch endoderm activated ectopic Tbx1 expression and partially suppressed the thymus-specific transcription factor Foxn1, identifying Tbx1 as a key target of SHH signaling in the 3rd pp. However, ectopic SHH signaling was insufficient to expand the GCM2-positive parathyroid domain, indicating that multiple inputs, some of which might be independent of SHH signaling, are required for parathyroid fate specification. These data support a model in which SHH signaling plays both positive and negative roles in patterning and organogenesis of the thymus and parathyroids.


Asunto(s)
Tipificación del Cuerpo/genética , Proteínas Hedgehog/fisiología , Organogénesis/genética , Glándulas Paratiroides/embriología , Timo/embriología , Animales , Células Cultivadas , Embrión de Mamíferos , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos/genética , Glándulas Paratiroides/metabolismo , Transducción de Señal/genética , Timo/metabolismo
3.
PLoS Genet ; 12(1): e1005777, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26789196

RESUMEN

Medullary thymic epithelial cells (mTECs) are essential for establishing central tolerance by expressing a diverse array of self-peptides that delete autoreactive thymocytes and/or divert thymocytes into the regulatory T cell lineage. Activation of the NFκB signaling pathway in mTEC precursors is indispensable for mTEC maturation and proliferation resulting in proper medullary region formation. Here we show that the Stat3-mediated signaling pathway also plays a key role in mTEC development and homeostasis. Expression of a constitutively active Stat3 transgene targeted to the mTEC compartment increases mTEC cellularity and bypasses the requirement for signals from positively selected thymocytes to drive medullary region formation. Conversely, conditional deletion of Stat3 disrupts medullary region architecture and reduces the number of mTECs. Stat3 signaling does not affect mTEC proliferation, but rather promotes survival of immature MHCIIloCD80lo mTEC precursors. In contrast to striking alterations in the mTEC compartment, neither enforced expression nor deletion of Stat3 affects cTEC cellularity or organization. These results demonstrate that in addition to the NFkB pathway, Stat3-mediated signals play an essential role in regulating mTEC cellularity and medullary region homeostasis.


Asunto(s)
Diferenciación Celular/genética , Células Epiteliales/metabolismo , FN-kappa B/genética , Factor de Transcripción STAT3/biosíntesis , Animales , Antígenos CD40/genética , Desarrollo Embrionario/genética , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Activación de Linfocitos , Ratones , Factor de Transcripción STAT3/genética , Transducción de Señal , Linfocitos T/metabolismo , Timocitos/metabolismo , Timo/crecimiento & desarrollo , Timo/metabolismo , Transgenes
4.
Development ; 141(15): 2950-8, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25053428

RESUMEN

The thymus and parathyroid glands arise from a shared endodermal primordium in the third pharyngeal pouch (3rd pp). Thymus fate is specified in the ventral 3rd pp between E9.5 and E11, whereas parathyroid fate is specified in the dorsal domain. The molecular mechanisms that specify fate and regulate thymus and parathyroid development are not fully delineated. Previous reports suggested that Tbx1 is required for thymus organogenesis because loss of Tbx1 in individuals with DiGeorge syndrome and in experimental Tbx1 deletion mutants is associated with thymus aplasia or hypoplasia. However, the thymus phenotype is likely to be secondary to defects in pharyngeal pouch formation. Furthermore, the absence of Tbx1 expression in the thymus-fated domain of the wild-type 3rd pp suggested that Tbx1 is instead a negative regulator of thymus organogenesis. To test this hypothesis, we generated a novel mouse strain in which expression of a conditional Tbx1 allele was ectopically activated in the thymus-fated domain of the 3rd pp. Ectopic Tbx1 expression severely repressed expression of Foxn1, a transcription factor that marks the thymus-fated domain and is required for differentiation and proliferation of thymic epithelial cell (TEC) progenitors. By contrast, ectopic Tbx1 did not alter the expression pattern of Gcm2, a transcription factor restricted to the parathyroid-fated domain and required for parathyroid development. Ectopic Tbx1 expression impaired TEC proliferation and arrested TEC differentiation at an early progenitor stage. The results support the hypothesis that Tbx1 negatively regulates TEC growth and differentiation, and that extinction of Tbx1 expression in 3rd pp endoderm is a prerequisite for thymus organogenesis.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Organogénesis , Proteínas de Dominio T Box/fisiología , Timo/embriología , Alelos , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Endodermo , Femenino , Factores de Transcripción Forkhead/fisiología , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Proteínas Nucleares/fisiología , Células Madre/citología , Proteínas de Dominio T Box/genética , Factores de Tiempo , Factores de Transcripción/fisiología
5.
J Immunol ; 192(2): 630-40, 2014 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-24337745

RESUMEN

A critical process during thymic development of the T cell repertoire is the induction of self-tolerance. Tolerance in developing T cells is highly dependent on medullary thymic epithelial cells (mTEC), and mTEC development in turn requires signals from mature single-positive thymocytes, a bidirectional relationship termed thymus crosstalk. We show that CD28-CD80/86 and CD40-CD40L costimulatory interactions, which mediate negative selection and self-tolerance, upregulate expression of LTα, LTß, and receptor activator for NF-κB in the thymus and are necessary for medullary development. Combined absence of CD28-CD80/86 and CD40-CD40L results in profound deficiency in mTEC development comparable to that observed in the absence of single-positive thymocytes. This requirement for costimulatory signaling is maintained even in a TCR transgenic model of high-affinity TCR-ligand interactions. CD4 thymocytes maturing in the altered thymic epithelial environment of CD40/CD80/86 knockout mice are highly autoreactive in vitro and are lethal in congenic adoptive transfer in vivo, demonstrating a critical role for these costimulatory pathways in self-tolerance as well as thymic epithelial development. These findings demonstrate that cooperativity between CD28-CD80/86 and CD40-CD40L pathways is required for normal medullary epithelium and for maintenance of self-tolerance in thymocyte development.


Asunto(s)
Antígeno B7-1/inmunología , Antígeno B7-2/inmunología , Antígenos CD28/inmunología , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Epitelio/inmunología , Autotolerancia/inmunología , Timocitos/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Células Epiteliales/inmunología , Células Asesinas Naturales/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , FN-kappa B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Regulación hacia Arriba/inmunología
6.
J Immunol ; 190(2): 597-604, 2013 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-23248263

RESUMEN

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that methylates histones and transcriptional regulators. We previously reported that the absence of CARM1 partially blocks thymocyte differentiation at embryonic day 18.5 (E18.5). In this study, we find that reduced thymopoiesis in Carm1(-/-) mice is due to a defect in the fetal hematopoietic compartment rather than in the thymic stroma. To determine the cellular basis for impaired thymopoiesis, we examined the number and function of fetal liver (FL) and bone marrow cells. Despite markedly reduced cellularity of hematopoietic progenitors in E18.5 bone marrow, the number of long-term hematopoietic stem cells and downstream subsets was not reduced in Carm1(-/-) E14.5 or E18.5 FL. Nevertheless, competitive reconstitution assays revealed a deficit in the ability of Carm1(-/-) FL cells to contribute to hematopoiesis. Furthermore, impaired differentiation of Carm1(-/-) FL cells in a CARM1-sufficient host showed that CARM1 is required cell autonomously in hematopoietic cells. Coculture of Carm1(-/-) FL cells on OP9-DL1 monolayers showed that CARM1 is required for survival of hematopoietic progenitors under conditions that promote differentiation. Taken together, this report demonstrates that CARM1 is a key epigenetic regulator of hematopoiesis that affects multiple lineages at various stages of differentiation.


Asunto(s)
Feto/metabolismo , Hematopoyesis/genética , Proteína-Arginina N-Metiltransferasas/genética , Timocitos/metabolismo , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Diferenciación Celular/genética , Supervivencia Celular/genética , Feto/embriología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Noqueados , Proteína-Arginina N-Metiltransferasas/deficiencia , Proteína-Arginina N-Metiltransferasas/metabolismo , Receptores Notch/metabolismo , Células del Estroma/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Timocitos/citología , Timo/embriología , Timo/metabolismo
7.
Blood ; 120(24): 4675-83, 2012 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-22955921

RESUMEN

Nonhematopoietic stromal cells of secondary lymphoid organs form important scaffold and fluid transport structures, such as lymph node (LN) trabeculae, lymph vessels, and conduits. Furthermore, through the production of chemokines and cytokines, these cells generate a particular microenvironment that determines lymphocyte positioning and supports lymphocyte homeostasis. IL-7 is an important stromal cell-derived cytokine that has been considered to be derived mainly from T-cell zone fibroblastic reticular cells. We show here that lymphatic endothelial cells (LECs) are a prominent source of IL-7 both in human and murine LNs. Using bacterial artificial chromosome transgenic IL-7-Cre mice, we found that fibroblastic reticular cells and LECs strongly up-regulated IL-7 expression during LN remodeling after viral infection and LN reconstruction after avascular transplantation. Furthermore, IL-7-producing stromal cells contributed to de novo formation of LyveI-positive lymphatic structures connecting reconstructed LNs with the surrounding tissue. Importantly, diphtheria toxin-mediated depletion of IL-7-producing stromal cells completely abolished LN reconstruction. Taken together, this study identifies LN LECs as a major source of IL-7 and shows that IL-7-producing stromal cells are critical for reconstruction and remodeling of the distinct LN microenvironment.


Asunto(s)
Células Endoteliales/metabolismo , Interleucina-7/metabolismo , Ganglios Linfáticos/metabolismo , Células del Estroma/metabolismo , Adulto , Animales , Línea Celular , Proliferación Celular , Células Cultivadas , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica , Humanos , Inmunohistoquímica , Interleucina-7/genética , Riñón/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ganglios Linfáticos/embriología , Ganglios Linfáticos/trasplante , Vasos Linfáticos/citología , Vasos Linfáticos/metabolismo , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Elife ; 122024 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-38635416

RESUMEN

Transposable elements (TEs) are repetitive sequences representing ~45% of the human and mouse genomes and are highly expressed by medullary thymic epithelial cells (mTECs). In this study, we investigated the role of TEs on T-cell development in the thymus. We performed multiomic analyses of TEs in human and mouse thymic cells to elucidate their role in T-cell development. We report that TE expression in the human thymus is high and shows extensive age- and cell lineage-related variations. TE expression correlates with multiple transcription factors in all cell types of the human thymus. Two cell types express particularly broad TE repertoires: mTECs and plasmacytoid dendritic cells (pDCs). In mTECs, transcriptomic data suggest that TEs interact with transcription factors essential for mTEC development and function (e.g., PAX1 and REL), and immunopeptidomic data showed that TEs generate MHC-I-associated peptides implicated in thymocyte education. Notably, AIRE, FEZF2, and CHD4 regulate small yet non-redundant sets of TEs in murine mTECs. Human thymic pDCs homogenously express large numbers of TEs that likely form dsRNA, which can activate innate immune receptors, potentially explaining why thymic pDCs constitutively secrete IFN ɑ/ß. This study highlights the diversity of interactions between TEs and the adaptive immune system. TEs are genetic parasites, and the two thymic cell types most affected by TEs (mTEcs and pDCs) are essential to establishing central T-cell tolerance. Therefore, we propose that orchestrating TE expression in thymic cells is critical to prevent autoimmunity in vertebrates.


Asunto(s)
Proteína AIRE , Elementos Transponibles de ADN , Ratones , Humanos , Animales , Timo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Timocitos/metabolismo , Células Epiteliales/metabolismo , Diferenciación Celular/genética , Ratones Endogámicos C57BL
9.
J Biol Chem ; 287(1): 429-437, 2012 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-22072714

RESUMEN

In epigenetic signaling pathways, histone tails are heavily modified, resulting in the recruitment of effector molecules that can influence transcription. One such molecule, plant homeodomain finger protein 20 (PHF20), uses a Tudor domain to read dimethyl lysine residues and is a known component of the MOF (male absent on the first) histone acetyltransferase protein complex, suggesting it plays a role in the cross-talk between lysine methylation and histone acetylation. We sought to investigate the biological role of PHF20 by generating a knockout mouse. Without PHF20, mice die shortly after birth and display a wide variety of phenotypes within the skeletal and hematopoietic systems. Mechanistically, PHF20 is not required for maintaining the global H4K16 acetylation levels or locus specific histone acetylation but instead works downstream in transcriptional regulation of MOF target genes.


Asunto(s)
Regulación de la Expresión Génica/genética , Histona Acetiltransferasas/metabolismo , Proteínas de Homeodominio/genética , Lisina/metabolismo , Animales , Proteínas de Unión al ADN , Femenino , Técnicas de Inactivación de Genes , Histonas/química , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Factores de Transcripción , Transcripción Genética/genética
10.
Proc Natl Acad Sci U S A ; 107(30): 13414-9, 2010 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-20616004

RESUMEN

Thymus organogenesis requires coordinated interactions of multiple cell types, including neural crest (NC) cells, to orchestrate the formation, separation, and subsequent migration of the developing thymus from the third pharyngeal pouch to the thoracic cavity. The molecular mechanisms driving these processes are unclear; however, NC-derived mesenchyme has been shown to play an important role. Here, we show that, in the absence of ephrin-B2 expression on thymic NC-derived mesenchyme, the thymus remains in the cervical area instead of migrating into the thoracic cavity. Analysis of individual NC-derived thymic mesenchymal cells shows that, in the absence of ephrin-B2, their motility is impaired as a result of defective EphB receptor signaling. This implies a NC-derived cell-specific role of EphB-ephrin-B2 interactions in the collective migration of the thymic rudiment during organogenesis.


Asunto(s)
Efrina-B2/metabolismo , Organogénesis , Receptores de la Familia Eph/metabolismo , Timo/embriología , Animales , Movimiento Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Efrina-B2/genética , Femenino , Citometría de Flujo , Inmunohistoquímica , Masculino , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Ratones Noqueados , Microscopía Confocal , Sistema Nervioso/citología , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Unión Proteica , Timo/citología , Timo/inervación
11.
Aging Cell ; 22(8): e13870, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37221658

RESUMEN

Age-related thymus involution results in decreased T-cell production, contributing to increased susceptibility to pathogens and reduced vaccine responsiveness. Elucidating mechanisms underlying thymus involution will inform strategies to restore thymopoiesis with age. The thymus is colonized by circulating bone marrow (BM)-derived thymus seeding progenitors (TSPs) that differentiate into early T-cell progenitors (ETPs). We find that ETP cellularity declines as early as 3 months (3MO) of age in mice. This initial ETP reduction could reflect changes in thymic stromal niches and/or pre-thymic progenitors. Using a multicongenic progenitor transfer approach, we demonstrate that the number of functional TSP/ETP niches does not diminish with age. Instead, the number of pre-thymic lymphoid progenitors in the BM and blood is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM lymphoid progenitors and in ETPs diminishes by 3MO, suggesting reduced niche quality in the BM and thymus contribute to the early decline in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to an initial reduction in ETPs in young adulthood, setting the stage for progressive age-associated thymus involution.


Asunto(s)
Médula Ósea , Linfocitos T , Ratones , Animales , Timo , Transducción de Señal , Ratones Endogámicos C57BL , Diferenciación Celular
12.
Cancer Discov ; 13(5): 1230-1249, 2023 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-37067911

RESUMEN

Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li-Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies. SIGNIFICANCE: New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li-Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are "basal" p53 activities. See related commentary by Stieg et al., p. 1046. See related article by Choe et al., p. 1250. This article is highlighted in the In This Issue feature, p. 1027.


Asunto(s)
Síndrome de Li-Fraumeni , Animales , Ratones , Síndrome de Li-Fraumeni/genética , Síndrome de Li-Fraumeni/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Activación Transcripcional , Receptores Activados del Proliferador del Peroxisoma/genética , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Muerte Celular
13.
Aging Cell ; 21(6): e13624, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35561351

RESUMEN

One of the earliest hallmarks of immune aging is thymus involution, which not only reduces the number of newly generated and exported T cells, but also alters the composition and organization of the thymus microenvironment. Thymic T-cell export continues into adulthood, yet the impact of thymus involution on the quality of newly generated T-cell clones is not well established. Notably, the number and proportion of medullary thymic epithelial cells (mTECs) and expression of tissue-restricted antigens (TRAs) decline with age, suggesting the involuting thymus may not promote efficient central tolerance. Here, we demonstrate that the middle-aged thymic environment does not support rapid motility of medullary thymocytes, potentially diminishing their ability to scan antigen presenting cells (APCs) that display the diverse self-antigens that induce central tolerance. Consistent with this possibility, thymic slice assays reveal that the middle-aged thymic environment does not support efficient negative selection or regulatory T-cell (Treg) induction of thymocytes responsive to either TRAs or ubiquitous self-antigens. This decline in central tolerance is not universal, but instead impacts lower-avidity self-antigens that are either less abundant or bind to TCRs with moderate affinities. Additionally, the decline in thymic tolerance by middle age is accompanied by both a reduction in mTECs and hematopoietic APC subsets that cooperate to drive central tolerance. Thus, age-associated changes in the thymic environment result in impaired central tolerance against moderate-avidity self-antigens, potentially resulting in export of increasingly autoreactive naive T cells, with a deficit of Treg counterparts by middle age.


Asunto(s)
Células Presentadoras de Antígenos , Tolerancia Central , Células Presentadoras de Antígenos/metabolismo , Autoantígenos/metabolismo , Células Epiteliales/metabolismo , Linfocitos T Reguladores , Timocitos , Timo
14.
J Biol Chem ; 285(2): 1147-52, 2010 Jan 08.
Artículo en Inglés | MEDLINE | ID: mdl-19897492

RESUMEN

CARM1 is one of nine protein arginine methyltransferases that methylate arginine residues in proteins. CARM1 is recruited by many different transcription factors as a positive regulator. Gene targeting of CARM1 in mice has been performed, and knock-out mice, which are smaller than their wild-type littermates, die just after birth. It has been proposed that CARM1 has functions that are independent of its enzymatic activity. Indeed, CARM1 is found to interact with a number of proteins and may have a scaffolding function in this context. However, CARM1 methylates histone H3, PABP1, AIB1, and a number of splicing factors, which strongly suggests that its impact on transcription and splicing is primarily through its ability to modify these substrates. To unequivocally establish the importance of CARM1 enzymatic activity in vivo, we generated an enzyme-dead knock-in of this protein arginine methyltransferase. We determined that knock-in cells and mice have defects similar to those seen in their knock-out counterparts with respect to the time of embryo lethality, T cell development, adipocyte differentiation, and transcriptional coactivator activity. CARM1 requires its enzymatic activity for all of its known cellular functions. Thus, small molecule inhibitors of CARM1 will incapacitate all of the enzyme's cellular functions.


Asunto(s)
Proteína-Arginina N-Metiltransferasas/metabolismo , Empalme del ARN/fisiología , Transcripción Genética/fisiología , Adipocitos/citología , Adipocitos/metabolismo , Animales , Diferenciación Celular/fisiología , Técnicas de Sustitución del Gen , Histonas/genética , Histonas/metabolismo , Metilación , Ratones , Ratones Noqueados , Coactivador 3 de Receptor Nuclear/genética , Coactivador 3 de Receptor Nuclear/metabolismo , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Linfocitos T/citología , Linfocitos T/metabolismo
15.
Front Immunol ; 12: 676236, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33968086

RESUMEN

Thymic epithelial cells (TECs) and hematopoietic antigen presenting cells (HAPCs) in the thymus microenvironment provide essential signals to self-reactive thymocytes that induce either negative selection or generation of regulatory T cells (Treg), both of which are required to establish and maintain central tolerance throughout life. HAPCs and TECs are comprised of multiple subsets that play distinct and overlapping roles in central tolerance. Changes that occur in the composition and function of TEC and HAPC subsets across the lifespan have potential consequences for central tolerance. In keeping with this possibility, there are age-associated changes in the cellular composition and function of T cells and Treg. This review summarizes changes in T cell and Treg function during the perinatal to adult transition and in the course of normal aging, and relates these changes to age-associated alterations in thymic HAPC and TEC subsets.


Asunto(s)
Envejecimiento/inmunología , Tolerancia Central , Timo/inmunología , Factores de Edad , Células Presentadoras de Antígenos/inmunología , Células Epiteliales/inmunología , Humanos , Linfocitos T Reguladores/inmunología
16.
Dev Biol ; 327(1): 216-27, 2009 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-19135046

RESUMEN

Embryos that are homozygous for Splotch, a null allele of Pax3, have a severe neural crest cell (NCC) deficiency that generates a complex phenotype including spina bifida, exencephaly and cardiac outflow tract abnormalities. Contrary to the widely held perception that thymus aplasia or hypoplasia is a characteristic feature of Pax3(Sp/Sp) embryos, we find that thymic rudiments are larger and parathyroid rudiments are smaller in E11.5-12.5 Pax3(Sp/Sp) compared to Pax3(+/+) embryos. The thymus originates from bilateral third pharyngeal pouch primordia containing endodermal progenitors of both thymus and parathyroid glands. Analyses of Foxn1 and Gcm2 expression revealed a dorsal shift in the border between parathyroid- and thymus-fated domains at E11.5, with no change in the overall cellularity or volume of each shared primordium. The border shift increases the allocation of third pouch progenitors to the thymus domain and correspondingly decreases allocation to the parathyroid domain. Initial patterning in the E10.5 pouch was normal suggesting that the observed change in the location of the organ domain interface arises during border refinement between E10.5 and E11.5. Given the well-characterized NCC defects in Splotch mutants, these findings implicate NCCs in regulating patterning of third pouch endoderm into thymus- versus parathyroid-specified domains, and suggest that organ size is determined in part by the number of progenitor cells specified to a given fate.


Asunto(s)
Factores de Transcripción Paired Box/genética , Glándulas Paratiroides/embriología , Timo/embriología , Animales , Tipificación del Cuerpo , Embrión de Mamíferos , Desarrollo Embrionario , Inducción Embrionaria , Factores de Transcripción Forkhead/genética , Ratones , Ratones Mutantes , Proteínas Nucleares/genética , Factor de Transcripción PAX3 , Glándulas Paratiroides/citología , Timo/citología , Factores de Transcripción/genética
17.
Genesis ; 47(4): 281-7, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19263498

RESUMEN

IL-7 is a cytokine that is required for T-cell development and homeostasis as well as for lymph node organogenesis. Despite the importance of IL-7 in the immune system and its potential therapeutic relevance, questions remain regarding the sites of IL-7 synthesis, specific cell types involved and molecular mechanisms regulating IL-7 expression. To address these issues, we generated two bacterial artificial chromosome (BAC) transgenic mouse lines in which IL-7 regulatory elements drive expression of either Cre recombinase or a human CD25 (hCD25) cell surface reporter molecule. Expression of the IL-7.hCD25 BAC transgene, detected by reactivity with anti-hCD25 antibody, mimicked endogenous IL-7 expression. Fetal and adult tissues from crosses between IL-7.Cre transgenic mice and Rosa26R or R26-EYFP reporters demonstrated X-gal or YFP staining in tissues known to express endogenous IL-7 at some stage during development. These transgenic lines provide novel genetic tools to identify IL-7 producing cells in various tissues and to manipulate gene expression selectively in IL-7 expressing cells.


Asunto(s)
Integrasas/metabolismo , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Interleucina-7/metabolismo , Animales , Animales Recién Nacidos , Cromosomas Artificiales Bacterianos/genética , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Integrasas/genética , Subunidad alfa del Receptor de Interleucina-2/genética , Interleucina-7/genética , Masculino , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timo/citología , Timo/embriología , Timo/metabolismo
18.
Mol Carcinog ; 48(6): 508-16, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18942117

RESUMEN

Cyclin D1b is an alternative transcript of the cyclin D1 gene (CCND1) expressed in human tumors. Its abundance is regulated by a single base pair polymorphism at the exon 4/intron 4 boundary (nucleotide 870). Epidemiological studies have shown a correlation between the presence of the G870A allele (that favors the splicing for cyclin D1b) with increased risk and less favorable outcome in several forms of cancer. More recently, it has been shown that, unlike cyclin D1a, the alternative transcript D1b by itself has the capacity to transform fibroblasts in vitro. In order to study the oncogenic potential of cyclin D1b, we developed transgenic mice expressing human cyclin D1b under the control of the bovine K5 promoter (K5D1b mice). Seven founders were obtained and none of them presented any significant phenotype or developed spontaneous tumors. Interestingly, K5D1b mice do not develop the fatal thymic hyperplasia, which is characteristic of the cyclin D1a transgenic mice (K5D1a). Susceptibility to skin carcinogenesis was tested in K5D1b mice using two-stage carcinogenesis protocols. In two independent experiments, K5D1b mice developed higher papilloma multiplicity as compared with wild-type littermates. However, when K5D1b mice were crossed with cyclin D1KO mice, the expression of cyclin D1b was unable to rescue the carcinogenesis-resistant phenotype of the cyclin D1 KO mice. To further explore the role of cyclin D1b in mouse models of carcinogenesis we carried out in silico analysis and in vitro experiments to evaluate the existence of a mouse homologous of the human cyclin D1b transcript. We were unable to find any evidence of an alternatively spliced transcript in mouse Ccnd1. These results show that human cyclin D1b has different biological functions than cyclin D1a and confirm its oncogenic properties.


Asunto(s)
Transformación Celular Neoplásica/genética , Ciclina D1/genética , Neoplasias Cutáneas/genética , Timo/patología , Animales , Secuencia de Bases , Cartilla de ADN , Exones , Hiperplasia , Intrones , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple
19.
Sci Rep ; 8(1): 14335, 2018 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-30254371

RESUMEN

Thymic epithelial cells (TEC) are essential for thymocyte differentiation and repertoire selection. Despite their indispensable role in generating functional T cells, the molecular mechanisms that orchestrate TEC development from endodermal progenitors in the third pharyngeal pouch (3rd PP) are not fully understood. We recently reported that the T-box transcription factor TBX1 negatively regulates TEC development. Although initially expressed throughout the 3rd PP, Tbx1 becomes downregulated in thymus-fated progenitors and when ectopically expressed impairs TEC progenitor proliferation and differentiation. Here we show that ectopic Tbx1 expression in thymus fated endoderm increases expression of Polycomb repressive complex 2 (PRC2) target genes in TEC. PRC2 is an epigenetic modifier that represses gene expression by catalyzing trimethylation of lysine 27 on histone H3. The increased expression of PRC2 target genes suggests that ectopic Tbx1 interferes with PRC2 activity and implicates PRC2 as an important regulator of TEC development. To test this hypothesis, we used Foxn1Cre to delete Eed, a PRC2 component required for complex stability and function in thymus fated 3rd PP endoderm. Proliferation and differentiation of fetal and newborn TEC were disrupted in the conditional knockout (EedCKO) mutants leading to severely dysplastic adult thymi. Consistent with PRC2-mediated transcriptional silencing, the majority of differentially expressed genes (DEG) were upregulated in EedCKO TEC. Moreover, a high frequency of EedCKO DEG overlapped with DEG in TEC that ectopically expressed Tbx1. These findings demonstrate that PRC2 plays a critical role in TEC development and suggest that Tbx1 expression must be downregulated in thymus fated 3rd PP endoderm to ensure optimal PRC2 function.


Asunto(s)
Células Epiteliales/citología , Complejo Represivo Polycomb 2/metabolismo , Timo/citología , Animales , Diferenciación Celular , Linaje de la Célula , Eliminación de Gen , Regulación de la Expresión Génica , Histonas/metabolismo , Metilación , Ratones , Complejo Represivo Polycomb 2/deficiencia , Complejo Represivo Polycomb 2/genética , Proteínas de Dominio T Box/genética
20.
Cell Host Microbe ; 21(4): 478-493.e7, 2017 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-28407484

RESUMEN

Humans with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a T cell-driven autoimmune disease caused by impaired central tolerance, are susceptible to chronic fungal infection and esophageal squamous cell carcinoma (ESCC). However, the relationship between autoreactive T cells and chronic fungal infection in ESCC development remains unclear. We find that kinase-dead Ikkα knockin mice develop APECED-like phenotypes, including impaired central tolerance, autoreactive T cells, chronic fungal infection, and ESCCs expressing specific human ESCC markers. Using this model, we investigated the link between ESCC and fungal infection. Autoreactive CD4 T cells permit fungal infection and incite tissue injury and inflammation. Antifungal treatment or autoreactive CD4 T cell depletion rescues, whereas oral fungal administration promotes, ESCC development. Inhibition of inflammation or epidermal growth factor receptor (EGFR) activity decreases fungal burden. Fungal infection is highly associated with ESCCs in non-autoimmune human patients. Therefore, autoreactive T cells and chronic fungal infection, fostered by inflammation and epithelial injury, promote ESCC development.


Asunto(s)
Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/patología , Neoplasias Esofágicas/etiología , Neoplasias Esofágicas/patología , Poliendocrinopatías Autoinmunes/complicaciones , Animales , Autoinmunidad , Linfocitos T CD4-Positivos/inmunología , Candidiasis/complicaciones , Carcinogénesis , Modelos Animales de Enfermedad , Receptores ErbB/metabolismo , Ratones
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