RESUMO
IKZF1 (IKAROS) is essential for normal lymphopoiesis in both humans and mice. Previous Ikzf1 mouse models have demonstrated the dual role for IKZF1 in both B and T cell development and have indicated differential requirements of each zinc finger. Furthermore, mutations in IKZF1 are known to cause common variable immunodeficiency in patients characterized by a loss of B cells and reduced Ab production. Through N-ethyl-N-nitrosourea mutagenesis, we have discovered a novel Ikzf1 mutant mouse with a missense mutation (L132P) in zinc finger 1 (ZF1) located in the DNA binding domain. Unlike other previously reported murine Ikzf1 mutations, this L132P point mutation (Ikzf1L132P ) conserves overall protein expression and has a B cell-specific phenotype with no effect on T cell development, indicating that ZF1 is not required for T cells. Mice have reduced Ab responses to immunization and show a progressive loss of serum Igs compared with wild-type littermates. IKZF1L132P overexpressed in NIH3T3 or HEK293T cells failed to localize to pericentromeric heterochromatin and bind target DNA sequences. Coexpression of wild-type and mutant IKZF1, however, allows for localization to pericentromeric heterochromatin and binding to DNA indicating a haploinsufficient mechanism of action for IKZF1L132P Furthermore, Ikzf1+/L132P mice have late onset defective Ig production, similar to what is observed in common variable immunodeficiency patients. RNA sequencing revealed a total loss of Hsf1 expression in follicular B cells, suggesting a possible functional link for the humoral immune response defects observed in Ikzf1L132P/L132P mice.
Assuntos
Linfócitos B/imunologia , Imunodeficiência de Variável Comum/genética , Fator de Transcrição Ikaros/genética , Mutação Puntual/genética , Animais , Formação de Anticorpos , Células HEK293 , Haploinsuficiência , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Humanos , Fator de Transcrição Ikaros/metabolismo , Imunoglobulinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Células NIH 3T3RESUMO
The molecular mechanisms tuning cholinergic interneuron (CIN) activity, although crucial for striatal function and behavior, remain largely unexplored. Previous studies report that the Etv1/Er81 transcription factor is vital for regulating neuronal maturation and activity. While Er81 is known to be expressed in the striatum during development, its specific role in defining CIN properties and the resulting consequences on striatal function is unknown. We report here that Er81 is expressed in CINs and its specific ablation leads to prominent changes in their molecular, morphologic, and electrophysiological features. In particular, the lack of Er81 amplifies intrinsic delayed-rectifier and hyperpolarization-activated currents, which subsequently alters the tonic and phasic activity of CINs. We further reveal that Er81 expression is required for normal CIN pause and time-locked responses to sensorimotor inputs in awake mice. Overall, this study uncovers a new cell type-specific control of CIN function in the striatum which drives habit formation in adult male mice.SIGNIFICANCE STATEMENT Although previous studies have shown that cholinergic interneurons drive striatal activity and habit formation, the underlying molecular mechanisms controlling their function are unknown. Here we reveal that key cholinergic interneuron physiological properties are controlled by Er81, a transcription factor regulating neuronal activity and development in a cell-specific manner. Moreover, our findings uncover a link between the Er81-dependent molecular control of cholinergic interneuron function and habit formation in mice. These insights will contribute to the future enhancement of our understanding of disorders that involve behavioral inflexibility, such as autism and addiction.
Assuntos
Neurônios Colinérgicos/metabolismo , Corpo Estriado/metabolismo , Proteínas de Ligação a DNA/metabolismo , Hábitos , Interneurônios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Masculino , Camundongos , Camundongos KnockoutRESUMO
Special AT-binding protein 1 (SATB1) is a chromatin-binding protein that has been shown to be a key regulator of T-cell development and CD4+ T-cell fate decisions and function. The underlying function for SATB1 in peripheral CD8+ T-cell differentiation processes is largely unknown. To address this, we examined SATB1-binding patterns in naïve and effector CD8+ T cells demonstrating that SATB1 binds to noncoding regulatory elements linked to T-cell lineage-specific gene programs, particularly in naïve CD8+ T cells. We then assessed SATB1 function using N-ethyl-N-nitrosourea-mutant mice that exhibit a point mutation in the SATB1 DNA-binding domain (termed Satb1m1Anu/m1Anu ). Satb1m1Anu/m1Anu mice exhibit diminished SATB1-binding, naïve, Satb1m1Anu/m1Anu CD8+ T cells exhibiting transcriptional and phenotypic characteristics reminiscent of effector T cells. Upon activation, the transcriptional signatures of Satb1m1Anu/m1Anu and wild-type effector CD8+ T cells converged. While there were no overt differences, primary respiratory infection of Satb1m1Anu/m1Anu mice with influenza A virus (IAV) resulted in a decreased proportion and number of IAV-specific CD8+ effector T cells recruited to the infected lung when compared with wild-type mice. Together, these data suggest that SATB1 has a major role in an appropriate transcriptional state within naïve CD8+ T cells and ensures appropriate CD8+ T-cell effector gene expression upon activation.
Assuntos
Vírus da Influenza A , Proteínas de Ligação à Região de Interação com a Matriz , Animais , Linfócitos T CD8-Positivos , Diferenciação Celular , Ativação Linfocitária , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , CamundongosRESUMO
T-cell immunity requires extremely rapid clonal proliferation of rare, antigen-specific T lymphocytes to form effector cells. Here we identify a critical role for ETAA1 in this process by surveying random germ line mutations in mice using exome sequencing and bioinformatic annotation to prioritize mutations in genes of unknown function with potential effects on the immune system, followed by breeding to homozygosity and testing for immune system phenotypes. Effector CD8+ and CD4+ T-cell formation following immunization, lymphocytic choriomeningitis virus (LCMV) infection, or herpes simplex virus 1 (HSV1) infection was profoundly decreased despite normal immune cell development in adult mice homozygous for two different Etaa1 mutations: an exon 2 skipping allele that deletes Gly78-Leu119, and a Cys166Stop truncating allele that eliminates most of the 877-aa protein. ETAA1 deficiency decreased clonal expansion cell autonomously within the responding T cells, causing no decrease in their division rate but increasing TP53-induced mRNAs and phosphorylation of H2AX, a marker of DNA replication stress induced by the ATM and ATR kinases. Homozygous ETAA1-deficient adult mice were otherwise normal, healthy, and fertile, although slightly smaller, and homozygotes were born at lower frequency than expected, consistent with partial lethality after embryonic day 12. Taken together with recently reported evidence in human cancer cell lines that ETAA1 activates ATR kinase through an exon 2-encoded domain, these findings reveal a surprisingly specific requirement for this ATR activator in adult mice restricted to rapidly dividing effector T cells. This specific requirement may provide new ways to suppress pathological T-cell responses in transplantation or autoimmunity.
Assuntos
Antígenos de Superfície/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Divisão Celular/imunologia , Imunidade Celular , Mutação , Animais , Antígenos de Superfície/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Divisão Celular/genética , Herpes Simples/genética , Herpes Simples/imunologia , Herpes Simples/patologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/patologia , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Mutantes , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
Each person's genome sequence has thousands of missense variants. Practical interpretation of their functional significance must rely on computational inferences in the absence of exhaustive experimental measurements. Here we analyzed the efficacy of these inferences in 33 de novo missense mutations revealed by sequencing in first-generation progeny of N-ethyl-N-nitrosourea-treated mice, involving 23 essential immune system genes. PolyPhen2, SIFT, MutationAssessor, Panther, CADD, and Condel were used to predict each mutation's functional importance, whereas the actual effect was measured by breeding and testing homozygotes for the expected in vivo loss-of-function phenotype. Only 20% of mutations predicted to be deleterious by PolyPhen2 (and 15% by CADD) showed a discernible phenotype in individual homozygotes. Half of all possible missense mutations in the same 23 immune genes were predicted to be deleterious, and most of these appear to become subject to purifying selection because few persist between separate mouse substrains, rodents, or primates. Because defects in immune genes could be phenotypically masked in vivo by compensation and environment, we compared inferences by the same tools with the in vitro phenotype of all 2,314 possible missense variants in TP53; 42% of mutations predicted by PolyPhen2 to be deleterious (and 45% by CADD) had little measurable consequence for TP53-promoted transcription. We conclude that for de novo or low-frequency missense mutations found by genome sequencing, half those inferred as deleterious correspond to nearly neutral mutations that have little impact on the clinical phenotype of individual cases but will nevertheless become subject to purifying selection.
Assuntos
Mutação de Sentido Incorreto , Animais , Códon , Biologia Computacional , Simulação por Computador , Exoma , Variação Genética , Genoma , Genoma Humano , Genótipo , Humanos , Sistema Imunitário , Síndromes de Imunodeficiência/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Neoplasias/genética , Fenótipo , Software , Proteína Supressora de Tumor p53/genéticaRESUMO
Chronic intestinal pseudo-obstruction (CIPO) is a rare but life-threatening disease characterized by severe intestinal dysmotility. Histopathologic studies in CIPO patients have identified several different mechanisms that appear to be involved in the dysmotility, including defects in neurons, smooth muscle, or interstitial cells of Cajal. Currently there are few mouse models of the various forms of CIPO. We generated a mouse with a point mutation in the RNA recognition motif of the Nup35 gene, which encodes a component of the nuclear pore complex. Nup35 mutants developed a severe megacolon and exhibited a reduced lifespan. Histopathologic examination revealed a degenerative myopathy that developed after birth and specifically affected smooth muscle in the colon; smooth muscle in the small bowel and the bladder were not affected. Furthermore, no defects were found in enteric neurons or interstitial cells of Cajal. Nup35 mice are likely to be a valuable model for the subtype of CIPO characterized by degenerative myopathy. Our study also raises the possibility that Nup35 polymorphisms could contribute to some cases of CIPO.
Assuntos
Pseudo-Obstrução do Colo/genética , Modelos Animais de Doenças , Doenças Musculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Mutação Puntual , Animais , Doença Crônica , Pseudo-Obstrução do Colo/patologia , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Músculo Liso/patologia , Doenças Musculares/patologiaRESUMO
IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.
Assuntos
Processamento Alternativo , Linfócitos B/metabolismo , Imunoglobulina D/genética , Cadeias Pesadas de Imunoglobulinas/genética , Dedos de Zinco , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Homologia de Sequência de AminoácidosRESUMO
DOCK8 deficiency in humans and mice leads to multiple defects in immune cell numbers and function. Patients with this immunodeficiency have a high morbidity and mortality, and are distinguished by chronic cutaneous viral infections, including those caused by herpes simplex virus (HSV). The underlying mechanism of the specific susceptibility to these chronic cutaneous viral infections is currently unknown, largely because the effect of DOCK8 deficiency has not been studied in suitable models. A better understanding of these mechanisms is required to underpin the development of more specific therapies. Here we show that DOCK8-deficient mice have poor control of primary cutaneous herpes simplex lesions and this is associated with increased virus loads. Furthermore, DOCK8-deficient mice showed a lack of CD4(+) T-cell infiltration into HSV-infected skin.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Quimiotaxia de Leucócito , Fatores de Troca do Nucleotídeo Guanina/deficiência , Herpes Simples/genética , Herpes Simples/imunologia , Simplexvirus/imunologia , Pele/imunologia , Pele/patologia , Animais , Linhagem Celular , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Herpes Simples/patologia , Herpes Simples/virologia , Imunidade , Camundongos , Camundongos Knockout , Pele/virologia , Carga ViralRESUMO
A rising concern in autism spectrum disorder (ASD) is the heightened sensitivity to trauma, the potential consequences of which have been overlooked, particularly upon the severity of the ASD traits. We first demonstrate a reciprocal relationship between ASD and post-traumatic stress disorder (PTSD) and reveal that exposure to a mildly stressful event induces PTSD-like memory in four mouse models of ASD. We also establish an unanticipated consequence of stress, as the formation of PTSD-like memory leads to the aggravation of core autistic traits. Such a susceptibility to developing PTSD-like memory in ASD stems from hyperactivation of the prefrontal cortex and altered fine-tuning of parvalbumin interneuron firing. Traumatic memory can be treated by recontextualization, reducing the deleterious effects on the core symptoms of ASD in the Cntnap2 KO mouse model. This study provides a neurobiological and psychological framework for future examination of the impact of PTSD-like memory in autism.
RESUMO
Interneurons are fundamental cells for maintaining the excitation-inhibition balance in the brain in health and disease. While interneurons have been shown to play a key role in the pathophysiology of autism spectrum disorder (ASD) in adult mice, little is known about how their maturation is altered in the developing striatum in ASD. Here, we aimed to track striatal developing interneurons and elucidate the molecular and physiological alterations in the Cntnap2 knockout mouse model. Using Stereo-seq and single-cell RNA sequencing data, we first characterized the pattern of expression of Cntnap2 in the adult brain and at embryonic stages in the medial ganglionic eminence (MGE), a transitory structure producing most cortical and striatal interneurons. We found that Cntnap2 is enriched in the striatum, compared to the cortex, particularly in the developing striatal cholinergic interneurons. We then revealed enhanced MGE-derived cell proliferation, followed by increased cell loss during the canonical window of developmental cell death in the Cntnap2 knockout mice. We uncovered specific cellular and molecular alterations in the developing Lhx6-expressing cholinergic interneurons of the striatum, which impacts interneuron firing properties during the first postnatal week. Overall, our work unveils some of the mechanisms underlying the shift in the developmental trajectory of striatal interneurons which greatly contribute to the ASD pathogenesis.
RESUMO
Notch signalling is critical to help direct T-cell lineage commitment in early T-cell progenitors and in the development of αß T-cells. Epithelial and stromal cell populations in the thymus express the Notch DSL (Delta, Serrate and Lag2)ligands Delta-like 1 (Dll1), Delta-like 4 (Dll4), Jagged 1 and Jagged 2, and induce Notch signalling in thymocytes that express the Notch receptor. At present there is nothing known about the role of the Delta-like 3 (Dll3) ligand in the immune system. Here we describe a novel cell autonomous role for Dll3 in αß T-cell development. We show that Dll3 cannot activate Notch when expressed in trans but like other Notch ligands it can inhibit Notch signalling when expressed in cis with the receptor. The loss of Dll3 leads to an increase in Hes5 expression in double positive thymocytes and their increased production of mature CD4(+) and CD8(+) T cells. Studies using competitive irradiation chimeras proved that Dll3 acts in a cell autonomous manner to regulate positive selection but not negative selection of autoreactive T cells. Our results indicate that Dll3 has a unique function during T-cell development that is distinct from the role played by the other DSL ligands of Notch and is in keeping with other recent studies indicating that Dll1 and Dll3 ligands have non-overlapping roles during embryonic development.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Membrana/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores Notch/metabolismo , Linfócitos T/imunologia , Animais , Diferenciação Celular , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ligantes , Células Progenitoras Linfoides/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/citologia , Timócitos/imunologia , Timócitos/metabolismoRESUMO
Hematopoietic stem cells (HSCs) are rare, ancestral cells that underlie the development, homeostasis, aging, and regeneration of the blood. Here we show that the chromatin-associated protein Ikaros is a crucial self-renewal regulator of the long-term (LT) reconstituting subset of HSCs. Ikaros, and associated family member proteins, are highly expressed in self-renewing populations of stem cells. Ikaros point mutant mice initially develop LT-HSCs with the surface phenotype cKit+Thy1.1(lo)Lin(-/lo)Sca1+Flk2-CD150+ during fetal ontogeny but are unable to maintain this pool, rapidly losing it within two days of embryonic development. A synchronous loss of megakaryocyte/erythrocyte progenitors results, along with a fatal, fetal anemia. At this time, mutation of Ikaros exerts a differentiation defect upon common lymphoid progenitors that cannot be rescued with an ectopic Notch signal in vitro, with hematopoietic cells preferentially committing to the NK lineage. Althoughdispensable for the initial embryonic development of blood, Ikaros is clearly needed for maintenance of this tissue. Achieving successful clinical tissue regeneration necessitates understanding degeneration, and these data provide a striking example by a discrete genetic lesion in the cells underpinning tissue integrity during a pivotal timeframe of organogenesis.
Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Fator de Transcrição Ikaros/metabolismo , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Linhagem da Célula , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Fator de Transcrição Ikaros/genética , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Tecido Linfoide/embriologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Gravidez , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Fatores de TempoRESUMO
The chromosomal region 10q24 is involved in reciprocal translocations with one of the T-cell receptor loci in a significant proportion of human T-cell acute lymphoblastic leukemias. The breakpoints of these rearrangements cluster immediately upstream of the TLX1 homeobox gene and lead to its transcriptional activation. Genomic analysis using sequences located on the opposite side of the breakpoint cluster region identified a novel gene composed of three exons that is oriented in a head-to-head manner with TLX1. The novel gene, named TDI (TLX1 divergent) codes for a 1.9 kb transcript with an atypically long 5' leader sequence. Although predicted to be a transcriptional regulator of 13.4 kDa, the TDI protein has no significant sequence similarity to any known protein. The TLX1 and TDI genes are separated by a short spacer of only 161 bp that contains numerous GC boxes and a centrally located CCAAT box embedded within a CpG island. Using luciferase as the reporter in transient transfection assays, the intergenic region was found to be a functional promoter with robust bidirectional activity. TLX1 and TDI thus appear to represent another example of a divergently transcribed gene pair whose expression is regulated by a common promoter. Our finding that TDI is transcriptionally co-activated in leukemic cells that aberrantly express TLX1, additionally suggests that it may have the potential to act as a co-operating oncogene in leukemogenesis.
Assuntos
Proteínas de Homeodomínio/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Transcrição Gênica/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Células K562 , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transfecção , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
To understand the interactions between Notch1 and Ikaros in the evolution of T cell acute lymphoblastic leukemia (T-ALL), we traced the evolution of T-ALL in mice with an inherited Ikaros mutation, Ikzf1(Plstc) which inactivates DNA binding. DNA-binding Ikaros repressed Notch1 response in transfected cell lines and in CD4(+)8(+) (DP) thymocytes from young pre-leukemic Ikzf1(Plstc) heterozygous mice. In DP thymocytes, a 50-1000 fold escalation in mRNA for Notch1 target genes Hes1 and Dtx1 preceded thymic lymphoma or leukemia and was closely correlated with the first detectable differentiation abnormalities, loss of heterozygosity (LOH) eliminating wild-type Ikzf1, and multiple missense and truncating Notch1 mutations. These findings illuminate the early stages of leukemogenesis by demonstrating progressive exaggeration of Notch1 responsiveness at the DP thymocyte stage brought about by multiple mutations acting in concert upon the Notch1 pathway.