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1.
Genes Dev ; 36(7-8): 433-450, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35450882

RESUMO

Somatic hypermutation (SHM) produces point mutations in immunoglobulin (Ig) genes in B cells when uracils created by the activation-induced deaminase are processed in a mutagenic manner by enzymes of the base excision repair (BER) and mismatch repair (MMR) pathways. Such uracil processing creates DNA strand breaks and is susceptible to the generation of deleterious deletions. Here, we demonstrate that the DNA repair factor HMCES strongly suppresses deletions without significantly affecting other parameters of SHM in mouse and human B cells, thereby facilitating the production of antigen-specific antibodies. The deletion-prone repair pathway suppressed by HMCES operates downstream from the uracil glycosylase UNG and is mediated by the combined action of BER factor APE2 and MMR factors MSH2, MSH6, and EXO1. HMCES's ability to shield against deletions during SHM requires its capacity to form covalent cross-links with abasic sites, in sharp contrast to its DNA end-joining role in class switch recombination but analogous to its genome-stabilizing role during DNA replication. Our findings lead to a novel model for the protection of Ig gene integrity during SHM in which abasic site cross-linking by HMCES intercedes at a critical juncture during processing of vulnerable gapped DNA intermediates by BER and MMR enzymes.


Assuntos
Genes de Imunoglobulinas , Hipermutação Somática de Imunoglobulina , Animais , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/genética , Proteínas de Ligação a DNA , Genes de Imunoglobulinas/genética , Switching de Imunoglobulina/genética , Camundongos , Hipermutação Somática de Imunoglobulina/genética , Uracila
2.
Eur J Immunol ; 50(3): 380-395, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31821534

RESUMO

Secondary diversification of the Ig repertoire occurs through somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR)-three processes that are initiated by activation-induced cytidine deaminase (AID). AID targets Ig genes at orders of magnitude higher than the rest of the genome, but the basis for this specificity is poorly understood. We have previously demonstrated that enhancers and enhancer-like sequences from Ig genes are capable of stimulating SHM of neighboring genes in a capacity distinct from their roles in increasing transcription. Here, we use an in vitro proteomics approach to identify E-box, MEF2, Ets, and Ikaros transcription factor family members as potential binders of these enhancers. ChIP assays in the hypermutating Ramos B cell line confirmed that many of these factors bound the endogenous Igλ enhancer and/or the IgH intronic enhancer (Eµ) in vivo. Further investigation using SHM reporter assays identified binding sites for E2A and MEF2B in Eµ and demonstrated an association between loss of factor binding and decreases in the SHM stimulating activity of Eµ mutants. Our results provide novel insights into trans-acting factors that dictate SHM targeting and link their activity to specific DNA binding sites within Ig enhancers.


Assuntos
Hipermutação Somática de Imunoglobulina/fisiologia , Animais , Galinhas , Genes de Imunoglobulinas , Humanos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
3.
bioRxiv ; 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38659853

RESUMO

Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2-knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

4.
iScience ; 24(5): 102411, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-33997693

RESUMO

Enhanced stemness in colorectal cancer has been reported and it contributes to aggressive progression, but the underlying mechanisms remain unclear. Here we report a Wnt ligand, Dickkopf-2 (DKK2) is essential for developing colorectal cancer stemness. Genetic depletion of DKK2 in intestinal epithelial or stem cells reduced tumorigenesis and expression of the stem cell marker genes including LGR5 in a model of colitis-associated cancer. Sequential mutations in APC, KRAS, TP53, and SMAD4 genes in colonic organoids revealed a significant increase of DKK2 expression by APC knockout and further increased by additional KRAS and TP53 mutations. Moreover, DKK2 activates proto-oncogene tyrosine-protein kinse Src followed by increased LGR5 expressing cells in colorectal cancer through degradation of HNF4α1 protein. These findings suggest that DKK2 is required for colonic epithelial cells to enhance LGR5 expression during the progression of colorectal cancer.

5.
J Clin Invest ; 130(8): 4411-4422, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32484799

RESUMO

Patients with common variable immunodeficiency associated with autoimmune cytopenia (CVID+AIC) generate few isotype-switched B cells with severely decreased frequencies of somatic hypermutations (SHMs), but their underlying molecular defects remain poorly characterized. We identified a CVID+AIC patient who displays a rare homozygous missense M466V mutation in ß-catenin-like protein 1 (CTNNBL1). Because CTNNBL1 binds activation-induced cytidine deaminase (AID) that catalyzes SHM, we tested AID interactions with the CTNNBL1 M466V variant. We found that the M466V mutation interfered with the association of CTNNBL1 with AID, resulting in decreased AID in the nuclei of patient EBV-transformed B cell lines and of CTNNBL1 466V/V Ramos B cells engineered to express only CTNNBL1 M466V using CRISPR/Cas9 technology. As a consequence, the scarce IgG+ memory B cells from the CTNNBL1 466V/V patient showed a low SHM frequency that averaged 6.7 mutations compared with about 18 mutations per clone in healthy-donor counterparts. In addition, CTNNBL1 466V/V Ramos B cells displayed a decreased incidence of SHM that was reduced by half compared with parental WT Ramos B cells, demonstrating that the CTNNBL1 M466V mutation is responsible for defective SHM induction. We conclude that CTNNBL1 plays an important role in regulating AID-dependent antibody diversification in humans.


Assuntos
Proteínas Reguladoras de Apoptose , Linfócitos B , Imunodeficiência de Variável Comum , Homozigoto , Memória Imunológica/genética , Mutação de Sentido Incorreto , Proteínas Nucleares , Hipermutação Somática de Imunoglobulina , Substituição de Aminoácidos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem Celular , Pré-Escolar , Imunodeficiência de Variável Comum/genética , Imunodeficiência de Variável Comum/imunologia , Imunodeficiência de Variável Comum/patologia , Citidina Desaminase/genética , Citidina Desaminase/imunologia , Feminino , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia
6.
Cell Rep ; 29(12): 3902-3915.e8, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31851922

RESUMO

Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes but also causes mutations in other parts of the genome. We have used lentiviral SHM reporter vectors to identify regions of the genome that are susceptible ("hot") and resistant ("cold") to SHM, revealing that SHM susceptibility and resistance are often properties of entire topologically associated domains (TADs). Comparison of hot and cold TADs reveals that while levels of transcription are equivalent, hot TADs are enriched for the cohesin loader NIPBL, super-enhancers, markers of paused/stalled RNA polymerase 2, and multiple important B cell transcription factors. We demonstrate that at least some hot TADs contain enhancers that possess SHM targeting activity and that insertion of a strong Ig SHM-targeting element into a cold TAD renders it hot. Our findings lead to a model for SHM susceptibility involving the cooperative action of cis-acting SHM targeting elements and the dynamic and architectural properties of TADs.


Assuntos
Elementos Facilitadores Genéticos/genética , Hipermutação Somática de Imunoglobulina/genética , Linhagem Celular Tumoral , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Células HEK293 , Humanos , Lentivirus , Masculino , Mutação/genética , Plasmídeos/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo
7.
Autoimmun Rev ; 9(8): 583-7, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20433954

RESUMO

Programmed death 1 (PD-1) and its ligands (PD-L1 and PD-L2) are responsible for inhibitory T cell signaling that helps mediate the mechanisms of tolerance and immune homeostasis. The PD-1:PD-L signaling pathway has been shown to play an important role in a variety of diseases, including autoimmune conditions, chronic infection, and cancer. Recently, investigators have explored the role of sex hormones in modulating the pathway in autoimmune conditions. Exploring the effects of sex hormones on the PD-1:PD-L pathway could shed light on the gender biased nature of many autoimmune conditions as well as aide in the development of therapeutics targeting the immune system.


Assuntos
Antígenos CD/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Autoimunidade/imunologia , Hormônios Esteroides Gonadais/imunologia , Caracteres Sexuais , Animais , Antígeno B7-H1 , Feminino , Humanos , Tolerância Imunológica/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Masculino , Proteína 2 Ligante de Morte Celular Programada 1 , Receptor de Morte Celular Programada 1 , Transdução de Sinais/imunologia , Linfócitos T/imunologia
8.
Autoimmun Rev ; 9(8): 560-8, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20385256

RESUMO

While CD4(+)CD25(high) regulatory T cells (Tregs) have garnered much attention for their role in the maintenance of immune homeostasis, recent findings have shown that subsets of CD8(+) T cells (CD8(+) Tregs) display immunoregulatory functions as well. Both CD4(+) Tregs and CD8(+) Tregs appear impaired in number and/or function in several autoimmune diseases and in experimental animal models of autoimmunity, suggesting the possibility of immunotherapeutic targeting of these cells for improved management of autoimmune conditions. Our group has developed a strategy to induce CD8(+) Tregs in autoimmune mice through the use of a tolerogenic self-peptide, and new information has been gained on the phenotype, function and role of induced CD8(+) Tregs in autoimmunity. Here we present an overview of the role and mechanisms of action of CD8(+) Tregs in autoimmunity, with a special focus on lupus. We also discuss the potential role of CD8(+) Tregs in other diseases, including chronic infection and cancer.


Assuntos
Autoimunidade/imunologia , Linfócitos T CD8-Positivos/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Humanos , Camundongos
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