RESUMEN
Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/αA, NFATc1/ßC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.
Asunto(s)
Factores de Transcripción NFATC/metabolismo , Proteínas Nucleares/metabolismo , Linfocitos T/metabolismo , Humanos , Células Jurkat , Espectrometría de Masas , Factores de Transcripción NFATC/genética , Proteínas Nucleares/genéticaRESUMEN
Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl- /H+ -exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).
Asunto(s)
Células Madre Pluripotentes Inducidas , Osteopetrosis , Canales de Cloruro/genética , Humanos , Leucocitos Mononucleares , Mutación , Osteoclastos , Osteopetrosis/genéticaRESUMEN
Osteosclerotic metaphyseal dysplasia (OSMD) is a rare autosomal recessive sclerosing skeletal dysplasia. We report on a 34-year-old patient with sandwich vertebrae, platyspondyly, osteosclerosis of the tubular bones, pathologic fractures, and anemia. In the third decade, he developed osteonecrosis of the jaws, which was progressive in spite of repeated surgical treatment over a period of 11 years. An iliac crest bone biopsy revealed the presence of hypermineralized cartilage remnants, large multinucleated osteoclasts with abnormal morphology, and inadequate bone resorption typical for osteoclast-rich osteopetrosis. After exclusion of mutations in TCIRG1 and CLCN7 we performed trio-based exome sequencing. The novel homozygous splice-site mutation c.261G>A in the gene LRRK1 was found and co-segregated with the phenotype in the family. cDNA sequencing showed nearly complete skipping of exon 3 leading to a frameshift (p.Ala34Profs*33). Osteoclasts differentiated from the patient's peripheral blood monocytes were extremely large. Instead of resorption pits these cells were only capable of superficial erosion. Phosphorylation of L-plastin at position Ser5 was strongly reduced in patient-derived osteoclasts showing a loss of function of the mutated LRRK1 kinase protein. Our analysis indicates a strong overlap of LRRK1-related OSMD with other forms of intermediate osteopetrosis, but an exceptional abnormality of osteoclast resorption. Like in other osteoclast pathologies an increased risk for progressive osteonecrosis of the jaws should be considered in OSMD, an intermediate form of osteopetrosis. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.
Asunto(s)
Resorción Ósea , Osteonecrosis , Osteopetrosis , Proteínas Serina-Treonina Quinasas , ATPasas de Translocación de Protón Vacuolares , Adulto , Humanos , Maxilares , Masculino , Mutación , Osteocondrodisplasias , Osteoclastos/metabolismo , Osteopetrosis/diagnóstico por imagen , Osteopetrosis/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
Differentiation toward CD4+ regulatory T (Treg) cells is essentially dependent on an epigenetic program at Treg signature genes, which involves remodeling of the Treg-specific demethylated regions (TSDRs). In particular, the epigenetic status of the conserved non-coding sequence 2 of Foxp3 (Foxp3 TSDR) determines expression stability of the master transcription factor and thus Treg lineage identity. However, the molecular mechanisms controlling the epigenetic remodeling at TSDRs in Treg and conventional T cells are largely unknown. Using a combined approach of DNA pull-down and mass spectrometric analysis, we report a novel regulatory mechanism in which transcription factor Wiz recruits the histone methyltransferase Ehmt1 to Foxp3 TSDR. We show that both Wiz and Ehmt1 are crucial for shaping the region with the repressive histone modification H3K9me2 in conventional T cells. Consistently, knocking out either Ehmt1 or Wiz by CRISPR/Cas resulted in the loss of H3K9me2 and enhanced Foxp3 expression during iTreg differentiation. Moreover, the essential role of the Wiz-Ehmt1 interaction as observed at several TSDRs indicates a global function of Ehmt1 in the Treg differentiation program.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Linfocitos T Reguladores/metabolismo , Animales , Ácido Ascórbico/farmacología , Secuencia de Bases , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Desmetilación , Epigénesis Genética/efectos de los fármacos , Histonas/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Genéticos , Proteínas del Tejido Nervioso/metabolismo , Linfocitos T Reguladores/efectos de los fármacosRESUMEN
Autosomal recessive osteopetrosis (ARO) is a genetic bone disease that can be caused by mutations in the CLCN7 gene preventing osteoclast-mediated bone resorption. We generated a human induced pluripotent stem cell (hiPSC) line, BIHi002-A, from peripheral blood mononuclear cells of an ARO patient carrying the CLCN7 mutations c.875G>A and c.1208G>A using Sendai viral vectors. The pluripotent identity of the BIHi002-A line was confirmed by their expression of typical markers for undifferentiated hiPSCs, their capacity to differentiate into cells of the three germ layers and by PluriTest analysis. The BIHi002-A line provides a tool for disease modelling and therapy development.