RESUMEN
Many microRNAs (miRNAs) are expressed with high spatiotemporal specificity during organismal development, with some being limited to rare cell types, often embedded in complex tissues. Yet, most miRNA profiling efforts remain at the tissue and organ levels. To overcome challenges in accessing the microRNomes from tissue-embedded cells, we had previously developed mime-seq (miRNome by methylation-dependent sequencing), a technique in which cell-specific miRNA methylation in C. elegans and Drosophila enabled chemo-selective sequencing without the need for cell sorting or biochemical purification. Here, we present mime-seq 2.0 for profiling miRNAs from specific mouse cell types. We engineered a chimeric RNA methyltransferase that is tethered to Argonaute protein and efficiently methylates miRNAs at their 3'-terminal 2'-OH in mouse and human cell lines. We also generated a transgenic mouse for conditional expression of this methyltransferase, which can be used to direct methylation of miRNAs in a cell type of choice. We validated the use of this mouse model by profiling miRNAs from B cells and bone marrow plasma cells.
Asunto(s)
MicroARNs , Animales , MicroARNs/genética , MicroARNs/metabolismo , Ratones , Humanos , Ratones Transgénicos , Metilación , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Análisis de Secuencia de ARN/métodos , Metiltransferasas/genética , Metiltransferasas/metabolismo , Línea Celular , Linfocitos B/metabolismoRESUMEN
The B cell regulator Pax5 consists of multiple domains whose function we analyzed in vivo by deletion in Pax5. While B lymphopoiesis was minimally affected in mice with homozygous deletion of the octapeptide or partial homeodomain, both sequences were required for optimal B cell development. Deletion of the C-terminal regulatory domain 1 (CRD1) interfered with B cell development, while elimination of CRD2 modestly affected B-lymphopoiesis. Deletion of CRD1 and CRD2 arrested B cell development at an uncommitted pro-B cell stage. Most Pax5-regulated genes required CRD1 or both CRD1 and CRD2 for their activation or repression as these domains induced or eliminated open chromatin at Pax5-activated or Pax5-repressed genes, respectively. Co-immunoprecipitation experiments demonstrated that the activating function of CRD1 is mediated through interaction with the chromatin-remodeling BAF, H3K4-methylating Set1A-COMPASS, and H4K16-acetylating NSL complexes, while its repressing function depends on recruitment of the Sin3-HDAC and MiDAC complexes. These data provide novel molecular insight into how different Pax5 domains regulate gene expression to promote B cell commitment and development.
Asunto(s)
Linfocitos B , Células Precursoras de Linfocitos B , Animales , Ratones , Homocigoto , Eliminación de Secuencia , Cromatina , Factor de Transcripción PAX5/genéticaRESUMEN
While extended loop extrusion across the entire Igh locus controls VH -DJH recombination, local regulatory sequences, such as the PAIR elements, may also activate VH gene recombination in pro-B-cells. Here, we show that PAIR-associated VH 8 genes contain a conserved putative regulatory element (V8E) in their downstream sequences. To investigate the function of PAIR4 and its V8.7E, we deleted 890 kb containing all 14 PAIRs in the Igh 5' region, which reduced distal VH gene recombination over a 100-kb distance on either side of the deletion. Reconstitution by insertion of PAIR4-V8.7E strongly activated distal VH gene recombination. PAIR4 alone resulted in lower induction of recombination, indicating that PAIR4 and V8.7E function as one regulatory unit. The pro-B-cell-specific activity of PAIR4 depends on CTCF, as mutation of its CTCF-binding site led to sustained PAIR4 activity in pre-B and immature B-cells and to PAIR4 activation in T-cells. Notably, insertion of V8.8E was sufficient to activate VH gene recombination. Hence, enhancers of the PAIR4-V8.7E module and V8.8E element activate distal VH gene recombination and thus contribute to the diversification of the BCR repertoire in the context of loop extrusion.
Asunto(s)
Células Precursoras de Linfocitos B , Secuencias Reguladoras de Ácidos Nucleicos , Secuencias Reguladoras de Ácidos Nucleicos/genética , Sitios de Unión , Recombinación GenéticaRESUMEN
Extended loop extrusion across the immunoglobulin heavy-chain (Igh) locus facilitates VH-DJH recombination following downregulation of the cohesin-release factor Wapl by Pax5, resulting in global changes in the chromosomal architecture of pro-B cells. Here, we demonstrate that chromatin looping and VK-JK recombination at the Igk locus were insensitive to Wapl upregulation in pre-B cells. Notably, the Wapl protein was expressed at a 2.2-fold higher level in pre-B cells compared with pro-B cells, which resulted in a distinct chromosomal architecture with normal loop sizes in pre-B cells. High-resolution chromosomal contact analysis of the Igk locus identified multiple internal loops, which likely juxtapose VK and JK elements to facilitate VK-JK recombination. The higher Wapl expression in Igµ-transgenic pre-B cells prevented extended loop extrusion at the Igh locus, leading to recombination of only the 6 most 3' proximal VH genes and likely to allelic exclusion of all other VH genes in pre-B cells. These results suggest that pro-B and pre-B cells with their distinct chromosomal architectures use different chromatin folding principles for V gene recombination, thereby enabling allelic exclusion at the Igh locus, when the Igk locus is recombined.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina , Células Precursoras de Linfocitos B , Recombinación V(D)J , Cromatina/genética , Cromatina/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Recombinación Genética , Recombinación V(D)J/genéticaRESUMEN
Plasma cells (PCs) and their progenitors plasmablasts (PBs) are essential for the acute and long-term protection of the host against infections by providing vast levels of highly specific antibodies. Several transcription factors, like Blimp1 and Irf4, are already known to be essential for PC and PB differentiation and survival. We set out to identify additional genes, that are essential for PB development by CRISPR-Cas9 screening of 3,000 genes for the loss of PBs by employing the in vitro-inducible germinal center B cell (iGB) culture system and Rosa26Cas9/+ mice. Identified hits in the screen were Mau2 and Nipbl, which are known to contribute to the loop extrusion function of the cohesin complex. Other examples of promising hits were Taf6, Stat3, Ppp6c and Pgs1. We thus provide a new set of genes, which are important for PB development.
Asunto(s)
Sistemas CRISPR-Cas , Células Plasmáticas , Animales , Linfocitos B , Diferenciación Celular/genética , Centro Germinal , RatonesRESUMEN
The genetic causes of primary antibody deficiencies and autism spectrum disorder (ASD) are largely unknown. Here, we report a patient with hypogammaglobulinemia and ASD who carries biallelic mutations in the transcription factor PAX5. A patient-specific Pax5 mutant mouse revealed an early B cell developmental block and impaired immune responses as the cause of hypogammaglobulinemia. Pax5 mutant mice displayed behavioral deficits in all ASD domains. The patient and the mouse model showed aberrant cerebellar foliation and severely impaired sensorimotor learning. PAX5 deficiency also caused profound hypoplasia of the substantia nigra and ventral tegmental area due to loss of GABAergic neurons, thus affecting two midbrain hubs, controlling motor function and reward processing, respectively. Heterozygous Pax5 mutant mice exhibited similar anatomic and behavioral abnormalities. Lineage tracing identified Pax5 as a crucial regulator of cerebellar morphogenesis and midbrain GABAergic neurogenesis. These findings reveal new roles of Pax5 in brain development and unravel the underlying mechanism of a novel immunological and neurodevelopmental syndrome.
Asunto(s)
Agammaglobulinemia , Trastorno del Espectro Autista , Animales , Trastorno del Espectro Autista/genética , Heterocigoto , Ratones , Mutación/genética , Factor de Transcripción PAX5/genéticaRESUMEN
While PAX5 is an important tumor suppressor gene in B-cell acute lymphoblastic leukemia (B-ALL), it is also involved in oncogenic translocations coding for diverse PAX5 fusion proteins. PAX5-JAK2 encodes a protein consisting of the PAX5 DNA-binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of the PAX5-JAK2 fusion protein in a mouse model expressing it from the endogenous Pax5 locus, resulting in inactivation of one of the two Pax5 alleles. Pax5Jak2/+ mice rapidly developed an aggressive B-ALL in the absence of another cooperating exogenous gene mutation. The DNA-binding function and kinase activity of Pax5-Jak2 as well as IL-7 signaling contributed to leukemia development. Interestingly, all Pax5Jak2/+ tumors lost the remaining wild-type Pax5 allele, allowing efficient DNA-binding of Pax5-Jak2. While we could not find evidence for a nuclear role of Pax5-Jak2 as an epigenetic regulator, high levels of active phosphorylated STAT5 and increased expression of STAT5 target genes were seen in Pax5Jak2/+ B-ALL tumors, implying that nuclear Pax5-Jak2 phosphorylates STAT5. Together, these data reveal Pax5-Jak2 as an important nuclear driver of leukemogenesis by maintaining phosphorylated STAT5 levels in the nucleus.
Asunto(s)
Janus Quinasa 2 , Leucemia de Células B , Factor de Transcripción PAX5 , Factor de Transcripción STAT5 , Animales , Janus Quinasa 2/genética , Leucemia de Células B/genética , Ratones , Mutación , Factor de Transcripción PAX5/genética , Factor de Transcripción STAT5/genética , Translocación GenéticaRESUMEN
Antibody secretion by plasma cells provides acute and long-term protection against pathogens. The high secretion potential of plasma cells depends on the unfolded protein response, which is controlled by the transcription factor Xbp1. Here, we analyzed the Xbp1-dependent gene expression program of plasma cells and identified Bhlha15 (Mist1) as the most strongly activated Xbp1 target gene. As Mist1 plays an important role in other secretory cell types, we analyzed in detail the phenotype of Mist1-deficient plasma cells in Cd23-Cre Bhlha15 fl/fl mice under steady-state condition or upon NP-KLH immunization. Under both conditions, Mist1-deficient plasma cells were 1.4-fold reduced in number and exhibited increased IgM production and antibody secretion compared to control plasma cells. At the molecular level, Mist1 regulated a largely different set of target genes compared with Xbp1. Notably, expression of the Blimp1 protein, which is known to activate immunoglobulin gene expression and to contribute to antibody secretion, was 1.3-fold upregulated in Mist1-deficient plasma cells, which led to a moderate downregulation of most Blimp1-repressed target genes in the absence of Mist1. Importantly, a 2-fold reduction of Blimp1 (Prdm1) expression was sufficient to restore the cell number and antibody expression of plasma cells in Prdm1 Gfp/+ Cd23-Cre Bhlha15 fl/fl mice to the same level seen in control mice. Together, these data indicate that Mist1 restricts antibody secretion by restraining Blimp1 expression, which likely contributes to the viability of plasma cells.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Plasmáticas , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Animales , Ratones , Anticuerpos/metabolismo , Regulación de la Expresión Génica , Células Plasmáticas/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismoRESUMEN
The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction, which relies on a complex interplay between specialized effector B and CD4 T lymphocytes, the GC B cells and T follicular helper (TFH) cells. Intriguingly, several positive key regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation, thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by the accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues.
Asunto(s)
Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Susceptibilidad a Enfermedades , Centro Germinal/inmunología , Centro Germinal/metabolismo , Proteínas de Homeodominio/genética , Activación de Linfocitos/inmunología , Células T Auxiliares Foliculares/metabolismo , Animales , Linfocitos B/inmunología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Inmunofenotipificación , Activación de Linfocitos/genética , Linfoma de Células B/etiología , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Ratones , Ratones Noqueados , Células T Auxiliares Foliculares/inmunologíaRESUMEN
Cell fate decisions during early B cell activation determine the outcome of responses to pathogens and vaccines. We examined the early B cell response to T-dependent antigen in mice by single-cell RNA sequencing. Early after immunization, a homogeneous population of activated precursors (APs) gave rise to a transient wave of plasmablasts (PBs), followed a day later by the emergence of germinal center B cells (GCBCs). Most APs rapidly exited the cell cycle, giving rise to non-GC-derived early memory B cells (eMBCs) that retained an AP-like transcriptional profile. Rapid decline of antigen availability controlled these events; provision of excess antigen precluded cell cycle exit and induced a new wave of PBs. Fate mapping revealed a prominent contribution of eMBCs to the MBC pool. Quiescent cells with an MBC phenotype dominated the early response to immunization in primates. A reservoir of APs/eMBCs may enable rapid readjustment of the immune response when failure to contain a threat is manifested by increased antigen availability.
Asunto(s)
Linfocitos B/inmunología , Centro Germinal/inmunología , Inmunidad Humoral/inmunología , Memoria Inmunológica/inmunología , Activación de Linfocitos/inmunología , Animales , Presentación de Antígeno/inmunología , Diferenciación Celular/inmunología , Ratones , Células Plasmáticas/inmunología , Células Precursoras de Linfocitos B/inmunologíaRESUMEN
The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving Pten-targeting microRNAs. Additional PTEN loss in Pten,Pax5 double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.
Asunto(s)
Linfocitos B/inmunología , Factor de Transcripción PAX5/inmunología , Fosfohidrolasa PTEN/inmunología , Fosfatidilinositol 3-Quinasas/inmunología , Animales , Diferenciación Celular , Regulación hacia Abajo , Femenino , Masculino , Ratones Transgénicos , Factor de Transcripción PAX5/genética , Fosfohidrolasa PTEN/genética , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal , Receptores Toll-Like/inmunologíaRESUMEN
Type 2 innate lymphoid cells (ILC2s) play a critical role early in the response to infection by helminths and in the development of allergic reactions. ILC2s are also involved in the physiologic regulation of adipose tissue and its metabolic response to cold shock. We find that the metabolic sensor peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in ILC2s of the lung and adipose tissue and increases responsiveness to IL-33. In turn, activation of ILC2 by IL-33 leads to increased expression of PPARγ, a prerequisite for proliferation and expression of the effector cytokines IL-5 and IL-13. In contrast, pharmacological inhibition of PPARγ leads to decreased expression of CD36 and fatty acid uptake, a necessary source of energy for ILC2s and of potential ligands for PPARγ. As a consequence, treatment of mice with a PPARγ antagonist reduces the severity of an ILC2-dependent acute airway inflammation. Together, our results demonstrate the critical role of the metabolic sensor PPARγ for the functions of ILC2s.
Asunto(s)
Tejido Adiposo/metabolismo , Interleucina-33/metabolismo , Pulmón/metabolismo , Linfocitos/inmunología , PPAR gamma/metabolismo , Neumonía/inmunología , Hipersensibilidad Respiratoria/inmunología , Tejido Adiposo/inmunología , Animales , Antígenos CD36/metabolismo , Células Cultivadas , Citocinas/metabolismo , Regulación hacia Abajo , Humanos , Pulmón/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , PPAR gamma/genética , Células Th2/inmunologíaRESUMEN
Jagunal homolog 1 (JAGN1) has been identified as a critical regulator of neutrophil biology in mutant mice and rare-disease patients carrying JAGN1 mutations. Here, we report that Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) of antibody-producing cells as well as decreased antibody production and secretion. Consequently, mice lacking Jagn1 in B cells exhibit reduced serum immunoglobulin (Ig) levels at steady state and fail to mount an efficient humoral immune response upon immunization with specific antigens or when challenged with viral infections. We also demonstrate that Jagn1 deficiency in B cells results in aberrant IgG N-glycosylation leading to enhanced Fc receptor binding. Jagn1 deficiency in particular affects fucosylation of IgG subtypes in mice as well as rare-disease patients with loss-of-function mutations in JAGN1. Moreover, we show that ER stress affects antibody glycosylation. Our data uncover a novel and key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans.
Asunto(s)
Estrés del Retículo Endoplásmico/inmunología , Inmunidad Humoral , Inmunoglobulina G/inmunología , Proteínas de la Membrana/inmunología , Animales , Estrés del Retículo Endoplásmico/genética , Glicosilación , Humanos , Inmunoglobulina G/genética , Mutación con Pérdida de Función , Proteínas de la Membrana/genética , Ratones Noqueados , Receptores Fc/genética , Receptores Fc/inmunologíaRESUMEN
B cell and plasma cell fates are controlled by different transcriptional networks, as exemplified by the mutually exclusive expression and cross-antagonism of the B cell identity factor Pax5 and the plasma cell regulator Blimp1. It has been postulated that repression of Pax5 by Blimp1 is essential for plasma cell development. Here, we challenged this hypothesis by analyzing the IghPax5/+ mouse, which expressed a Pax5 minigene from the immunoglobulin heavy-chain locus. Despite high Pax5 expression, plasma cells efficiently developed in young IghPax5/+ mice at steady state and upon immunization, while their number moderately declined in older mice. Although Pax5 significantly deregulated the plasma cell expression program, key plasma cell regulators were normally expressed in IghPax5/+ plasma cells. While IgM and IgA secretion by IghPax5/+ plasma cells was normal, IgG secretion was modestly decreased. Hence, Pax5 repression is not essential for robust plasma cell development and antibody secretion, although it is required for optimal IgG production and accumulation of long-lived plasma cells.
Asunto(s)
Linfocitos B/inmunología , Diferenciación Celular/genética , Factor de Transcripción PAX5/metabolismo , Células Plasmáticas/inmunología , Traslado Adoptivo , Animales , Diferenciación Celular/inmunología , Inmunización , Cambio de Clase de Inmunoglobulina/genética , Inmunoglobulina G/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Inmunoglobulina M/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor de Transcripción PAX5/genética , Factor 1 de Unión al Dominio 1 de Regulación Positiva/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Linfocitos T/inmunologíaRESUMEN
Nuclear processes, such as V(D)J recombination, are orchestrated by the three-dimensional organization of chromosomes at multiple levels, including compartments1 and topologically associated domains (TADs)2,3 consisting of chromatin loops4. TADs are formed by chromatin-loop extrusion5-7, which depends on the loop-extrusion function of the ring-shaped cohesin complex8-12. Conversely, the cohesin-release factor Wapl13,14 restricts loop extension10,15. The generation of a diverse antibody repertoire, providing humoral immunity to pathogens, requires the participation of all V genes in V(D)J recombination16, which depends on contraction of the 2.8-Mb-long immunoglobulin heavy chain (Igh) locus by Pax517,18. However, how Pax5 controls Igh contraction in pro-B cells remains unknown. Here we demonstrate that locus contraction is caused by loop extrusion across the entire Igh locus. Notably, the expression of Wapl is repressed by Pax5 specifically in pro-B and pre-B cells, facilitating extended loop extrusion by increasing the residence time of cohesin on chromatin. Pax5 mediates the transcriptional repression of Wapl through a single Pax5-binding site by recruiting the polycomb repressive complex 2 to induce bivalent chromatin at the Wapl promoter. Reduced Wapl expression causes global alterations in the chromosome architecture, indicating that the potential to recombine all V genes entails structural changes of the entire genome in pro-B cells.
Asunto(s)
Genes de las Cadenas Pesadas de las Inmunoglobulinas/genética , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Factor de Transcripción PAX5/metabolismo , Proteínas/genética , Proteínas Represoras/metabolismo , Recombinación V(D)J/genética , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Cadenas Pesadas de Inmunoglobulina/química , Región Variable de Inmunoglobulina/química , Ratones , Complejo Represivo Polycomb 2/metabolismo , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Regiones Promotoras Genéticas/genética , CohesinasRESUMEN
The establishment of a diverse B cell antigen receptor (BCR) repertoire by V(D)J recombination also generates autoreactive B cells. Anergy is one tolerance mechanism; it renders autoreactive B cells insensitive to stimulation by self-antigen, whereas Toll-like receptor (TLR) signaling can reactivate anergic B cells. Here, we describe a critical role of the transcription factor Ikaros in controlling BCR anergy and TLR signaling. Mice with specific deletion of Ikaros in mature B cells developed systemic autoimmunity. Ikaros regulated many anergy-associated genes, including Zfp318, which is implicated in the attenuation of BCR responsiveness by promoting immunoglobulin D expression in anergic B cells. TLR signaling was hyperactive in Ikaros-deficient B cells, which failed to upregulate feedback inhibitors of the MyD88-nuclear factor κB signaling pathway. Systemic inflammation was lost on expression of a non-self-reactive BCR or loss of MyD88 in Ikaros-deficient B cells. Thus, Ikaros acts as a guardian preventing autoimmunity by promoting BCR anergy and restraining TLR signaling.
Asunto(s)
Autoinmunidad/genética , Linfocitos B/inmunología , Anergia Clonal/genética , Factor de Transcripción Ikaros/metabolismo , Receptores Toll-Like/metabolismo , Animales , Linfocitos B/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/inmunología , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/inmunología , Inmunoglobulina D/inmunología , Inmunoglobulina D/metabolismo , Ratones , Modelos Animales , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptores Toll-Like/inmunologíaRESUMEN
Induction of the transcription factor Irf8 in the common dendritic cell progenitor (CDP) is required for classical type 1 dendritic cell (cDC1) fate specification, but the mechanisms controlling this induction are unclear. In the present study Irf8 enhancers were identified via chromatin profiling of dendritic cells and CRISPR/Cas9 genome editing was used to assess their roles in Irf8 regulation. An enhancer 32 kilobases (kb) downstream of the Irf8 transcriptional start site (+32-kb Irf8) that was active in mature cDC1s was required for the development of this lineage, but not for its specification. Instead, a +41-kb Irf8 enhancer, previously thought to be active only in plasmacytoid dendritic cells, was found to also be transiently accessible in cDC1 progenitors, and deleting this enhancer prevented the induction of Irf8 in CDPs and abolished cDC1 specification. Thus, cryptic activation of the +41-kb Irf8 enhancer in dendritic cell progenitors is responsible for cDC1 fate specification.
Asunto(s)
Células Dendríticas/citología , Elementos de Facilitación Genéticos/genética , Factores Reguladores del Interferón/metabolismo , Macrófagos/citología , Monocitos/citología , Animales , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Linaje de la Célula , Células Dendríticas/inmunología , Regulación de la Expresión Génica , Factores Reguladores del Interferón/genética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Células Madre/citología , Células Tumorales CultivadasRESUMEN
Tissues in multicellular organisms are populated by resident macrophages, which perform both generic and tissue-specific functions. The latter are induced by signals from the microenvironment and rely on unique tissue-specific molecular programs requiring the combinatorial action of tissue-specific and broadly expressed transcriptional regulators. Here, we identify the transcription factors Bhlhe40 and Bhlhe41 as novel regulators of alveolar macrophages (AMs)-a population that provides the first line of immune defense and executes homeostatic functions in lung alveoli. In the absence of these factors, AMs exhibited decreased proliferation that resulted in a severe disadvantage of knockout AMs in a competitive setting. Gene expression analyses revealed a broad cell-intrinsic footprint of Bhlhe40/Bhlhe41 deficiency manifested by a downregulation of AM signature genes and induction of signature genes of other macrophage lineages. Genome-wide characterization of Bhlhe40 DNA binding suggested that these transcription factors directly repress the expression of lineage-inappropriate genes in AMs. Taken together, these results identify Bhlhe40 and Bhlhe41 as key regulators of AM self-renewal and guardians of their identity.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Perfilación de la Expresión Génica/métodos , Proteínas de Homeodominio/genética , Macrófagos Alveolares/citología , Acetilación , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Proliferación Celular , Autorrenovación de las Células , Supervivencia Celular , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Macrófagos Alveolares/metabolismo , Ratones , Especificidad de Órganos , Fenotipo , Análisis de Secuencia de ARNRESUMEN
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.