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Lineage-specific transcription factors (TFs) regulate differentiation of hematopoietic stem cells (HSCs). They are decisive for the establishment and maintenance of lineage-specific gene expression programs during hematopoiesis. For this they create a regulatory network between TFs, epigenetic cofactors, and microRNAs. They activate cell-type specific genes and repress competing gene expression programs. Disturbance of this process leads to impaired lineage fidelity and diseases of the blood system. The TF T-cell acute leukemia 1 (TAL1) is central for erythroid differentiation and contributes to the formation of distinct gene regulatory complexes in progenitor cells and erythroid cells. A TAL1/E47 heterodimer binds to DNA with the TFs GATA-binding factor 1 and 2 (GATA1/2), the cofactors LIM domain only 1 and 2 (LMO1/2), and LIM domain-binding protein 1 (LDB1) to form a core TAL1 complex. Furthermore, cell-type-dependent interactions of TAL1 with other TFs such as with runt-related transcription factor 1 (RUNX1) and Kruppel-like factor 1 (KLF1) are established. Moreover, TAL1 activity is regulated by the formation of TAL1 isoforms, posttranslational modifications (PTMs), and microRNAs. Here, we describe the function of TAL1 in normal hematopoiesis with a focus on erythropoiesis.
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Eritropoese , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Eritropoese/genética , Humanos , Animais , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genéticaRESUMO
INTRODUCTION: Lung adenocarcinoma (LUAD) is the major type of non-small cell lung cancer with low a survival rate caused by metastasis. SCN4B encoding voltage-gated sodium channel ß subunit is regarded as a metastasis-suppressor gene. We aim to explore how SCN4B influences the progression and prognosis of LUAD. METHODS: The gene expression profiles of 585 LUAD samples in TCGA and GSE31210, GSE116959, and GSE72094 datasets from the GEO database were downloaded for analysis. Differentially expressed genes were obtained through the "limma" package. The "clusterProfiler" package was used to conduct GSEA. Survival analysis was conducted via "survival" and "survminer" packages. Transcription factors regulating SCN4B expression were screened by correlation analysis and further predicted by FIMO. Infiltration of immune cells was analyzed by CIBERSORT. ESTIMATE algorithm was used to evaluate the immune-related scores. RESULTS: SCN4B expressed higher in normal samples than in LUAD samples and higher in female samples than male samples. One hundred and twenty-six pathways were significantly enriched between high and low SCN4B expression groups. Six transcription factors' expressions were positively related to SCN4B expression, and ChIP-seq data from "Cistrome" verified that TAL1 and ERG might bind to the upstream sequence of SCN4B. SCN4B expression was significantly correlated with activated memory CD4 T cells, resting mast cells, and monocytes. TMB status, three scores based on ESTIMATE algorithm, and expression of three immune checkpoints showed significant differences between SCN4B high- and low-expression groups. SCN4B could be considered as an independent prognostic signature of LUAD patients that higher expression represents a better prognosis. CONCLUSION: SCN4B expresses higher in normal samples, and SCN4B is able to be an independent prognostic signature for LUAD patients. TAL1 and ERG may regulate the expression of SCN4B by binding its upstream sequences. Our research is valuable in improving the effectiveness of treatment in LUAD.
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Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Feminino , Masculino , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Prognóstico , Adenocarcinoma de Pulmão/genética , Fatores de Transcrição , Subunidade beta-4 do Canal de Sódio Disparado por VoltagemRESUMO
Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome that targets a highly selective subset of organs causing specific types of tumors. The biological basis for this principle of organ selectivity and tumor specificity is not well understood. VHL-associated hemangioblastomas share similar molecular and morphological features with embryonic blood and vascular precursor cells. Therefore, we suggest that VHL hemangioblastomas are derived from developmentally arrested hemangioblastic lineage keeping their potential of further differentiation. Due to these common features, it is of major interest to investigate whether VHL-associated tumors other than hemangioblastoma also share these pathways and molecular features. The expression of hemangioblast proteins has not yet been assessed in other VHL-related tumors. To gain a better understanding of VHL tumorigenesis, the expression of hemangioblastic proteins in different VHL-associated tumors was investigated. The expression of embryonic hemangioblast proteins Brachyury and TAL1 (T-cell acute lymphocytic leukemia protein 1) was assessed by immunohistochemistry staining on 75 VHL-related tumors of 51 patients: 47 hemangioblastomas, 13 clear cell renal cell carcinomas, 8 pheochromocytomas, 5 pancreatic neuroendocrine tumors, and 2 extra-adrenal paragangliomas. Brachyury and TAL1 expression was, respectively, observed in 26% and 93% of cerebellar hemangioblastomas, 55% and 95% of spinal hemangioblastomas, 23% and 92% of clear cell renal cell carcinomas, 38% and 88% of pheochromocytomas, 60% and 100% of pancreatic neuroendocrine tumors, and 50% and 100% of paragangliomas. We concluded that the expression of hemangioblast proteins in different VHL-associated tumors indicates a common embryological origin of these lesions. This may also explain the specific topographic distribution of VHL-associated tumors.
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BACKGROUND: In this study, we observed that in human colon carcinoma HCT116 cells mRNA level of the human ß-galactoside α2,6-sialyltransferase (hST6Gal I) was decreased by curcumin. FACS analysis using the α2,6-sialyl-specific lectin (SNA) also showed a noticeable decrease in binding to SNA by curcumin. OBJECTIVE: To investigate the mechanism for curcumin-triggered downregulation of hST6Gal I transcription. METHODS: The mRNA levels of nine kinds of hST genes were assessed by RT-PCR after curcumin was treated in HCT116 cells. The level of hST6Gal I product on cell surface was examined by flow cytometry analysis. Luciferase reporter plasmids with 5'-deleted constructs and mutants of the hST6Gal I promoter were transiently transfected into HCT116 cells, and the luciferase activity was measured after treatment with curcumin. RESULTS: Curcumin led to significant transcriptional repression of the hST6Gal I promoter. Promoter analysis using deletion mutants proved that the - 303 to - 189 region of the hST6Gal I promoter is required for transcriptional repression in response to curcumin. Among putative binding sites for transcription factors IK2, GATA1, TCF12, TAL1/E2A, SPT, and SL1 in this region, by site-directed mutagenesis analysis the TAL/E2A binding site (nucleotides - 266/- 246) was proved to be crucial for curcumin-triggered downregulation of hST6Gal I transcription in HCT116 cells. The transcription activity of hST6Gal I gene in HCT116 cells was markedly suppressed by compound C, an AMP-activated protein kinase (AMPK) inhibitor. CONCLUSION: These indicate that gene expression of hST6Gal I in HCT116 cells is controlled through AMPK/TAL/E2A signal pathway.
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Carcinoma , Neoplasias do Colo , Curcumina , Humanos , Curcumina/farmacologia , Proteínas Quinases Ativadas por AMP , beta-D-Galactosídeo alfa 2-6-Sialiltransferase , Células HCT116 , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , RNA Mensageiro/genética , LuciferasesRESUMO
Objective: To assess the clinical characteristics and prognosis of patients with SIL-TAL1-positive T-cell acute lymphoblastic leukemia (T-ALL) . Methods: The clinical data of 19 SIL-TAL1-positive T-ALL patients admitted to the First Affiliated Hospital of Soochow University between January 2014 and February 2022 were retrospectively computed and contrasted with SIL-TAL1-negative T-ALL patients. Results: The median age of the 19 SIL-TAL1-positive T-ALL patients was 15 (7 to 41 years) , including 16 males (84.2%) . SIL-TAL1-positive T-ALL patients had younger age, higher WBC, and hemoglobin compared with SIL-TAL1-negative T-ALL patients. There was no discrepancy in gender distribution, PLT, chromosome abnormality distribution, immunophenotyping, and complete remission (CR) rate. The 3-year overall survival (OS) was 60.9% and 74.4%, respectively (HR=2.070, P=0.071) . The 3-year relapse-free survival (RFS) was 49.2% and 70.6%, respectively (HR=2.275, P=0.040) . The 3-year RFS rate of SIL-TAL1-positive T-ALL patients was considerably lower than SIL-TAL1-negative T-ALL patients. Conclusion: SIL-TAL1-positive T-ALL patients were connected to younger age, higher WBC, higher HGB, and poor outcome.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Adolescente , Adulto , Humanos , Masculino , Adulto Jovem , Aberrações Cromossômicas , Proteínas de Fusão Oncogênica/genética , Prognóstico , Recidiva , Estudos Retrospectivos , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Linfócitos T , Feminino , CriançaRESUMO
Enhancers activate gene transcription remotely, which requires tissue specific transcription factors binding to them. GATA1 and TAL1 are hematopoietic/erythroid-specific factors and often bind together to enhancers, activating target genes. Interestingly, we found that some hematopoietic/erythroid genes are transcribed in a GATA1-dependent but TAL1-independnet manner. They appear to have enhancers within a relatively short distance. In this study, we paired highly transcribed hematopoietic/erythroid genes with the nearest GATA1/TAL1-binding enhancers and analyzed these putative enhancer-gene pairs depending on distance between them. Enhancers located at various distances from genes in the pairs, which was not related to transcription level of the genes. However, genes with enhancers at short distances away tended to be transcriptionally unaffected by TAL1 depletion. Histone H3K27ac extended from the enhancers to target genes. The H3K27ac extension was maintained without TAL1, even though it disappeared owing to the loss of GATA1. Intergenic RNA was highly transcribed from the enhancers to nearby target genes, independent of TAL1. Taken together, TAL1-independent transcription of hematopoietic/erythroid genes appears to be promoted by enhancers present in a short distance. These enhancers are likely to activate nearby target genes by tracking the intervening regions.
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DNA Intergênico , Elementos Facilitadores Genéticos , Hematopoese , Histonas , DNA Intergênico/genética , DNA Intergênico/metabolismo , Hematopoese/genética , Histonas/genética , Histonas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismoRESUMO
Efficient sensory processing of spatial information is facilitated through the organization of neuronal connections into topographic maps of space. In integrative sensory centers, converging topographic maps must be aligned to merge spatially congruent information. The superior colliculus (SC) receives topographically ordered visual inputs from retinal ganglion cells (RGCs) in the eye and layer 5 neurons in the primary visual cortex (L5-V1). Previous studies suggest that RGCs instruct the alignment of later-arriving L5-V1 inputs in an activity-dependent manner. However, the molecular mechanisms underlying this remain unclear. Here, we explored the role of NMDA receptors in visual map alignment in the SC using a conditional genetic knockout approach. We leveraged a novel knock-in mouse line that expresses tamoxifen-inducible Cre recombinase under the control of the Tal1 gene (Tal1CreERT2 ), which we show allows for specific recombination in the superficial layers of the SC. We used Tal1CreERT2 mice of either sex to conditionally delete the obligate GluN1 subunit of the NMDA receptor (SC-cKO) during the period of visual map alignment. We observed a significant disruption of L5-V1 axon terminal organization in the SC of SC-cKO mice. Importantly, retinocollicular topography was unaffected in this context, suggesting that alignment is also disrupted. Time-course experiments suggest that NMDA receptors may play a critical role in the refinement of L5-V1 inputs in the SC. Together, these data implicate NMDA receptors as critical mediators of activity-dependent visual map alignment in the SC.SIGNIFICANCE STATEMENT Alignment of topographic inputs is critical for integration of spatially congruent sensory information; however, little is known about the mechanisms underlying this complex process. Here, we took a conditional genetic approach to explore the role of NMDA receptors in the alignment of retinal and cortical visual inputs in the superior colliculus. We characterize a novel mouse line providing spatial and temporal control of recombination in the superior colliculus and reveal a critical role for NMDA expression in visual map alignment. These data support a role for neuronal activity in visual map alignment and provide mechanistic insight into this complex developmental process.
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Receptores de N-Metil-D-Aspartato , Colículos Superiores , Camundongos , Animais , Colículos Superiores/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Vias Visuais/fisiologia , Sensação , Células Ganglionares da RetinaRESUMO
Spinal cord injury often leads to severe motor and sensory deficits, and prognosis using the currently available therapies remains poor. Therefore, we aimed to explore a novel therapeutic approach for improving the prognosis of spinal cord injury. In this study, we implanted oscillating field stimulation devices and transplanted neural stem cells into the thoracic region (T9-T10) of rats with a spinal cord contusion. Basso-Beattie-Bresnahan scoring revealed that oscillating field stimulation combined with neural stem cells transplantation promoted motor function recovery following spinal cord injury. In addition, we investigated the regulation of oscillating field stimulation on the miR-124/Tal1 axis in neural stem cells. Transfection of lentivirus was performed to investigate the role of Tal1 in neurogenesis of neural stem cells induced by oscillating field stimulation. Quantitative reverse transcription-polymerase chain reaction, immunofluorescence and western blotting showed that oscillating field stimulation promoted neurogenesis of neural stem cells in vitro and in vivo. Hematoxylin and eosin staining showed that oscillating field stimulation combined with neural stem cells transplantation alleviated cavities formation after spinal cord injury. Taking the results together, we concluded that oscillating field stimulation decreased miR-124 expression and increased Tal1 content, thereby promoting the neurogenesis of neural stem cells. The combination of oscillating field stimulation and neural stem cells transplantation improved neurogenesis, and thereby promoted structural and functional recovery after spinal cord injury.
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Background: Transcription factors (TFs) play a crucial role in the occurrence and progression of lung adenocarcinoma (LUAD), and targeting TFs is an important direction for treating LUAD. However, targeting a single TF often fails to achieve satisfactory therapeutic outcomes. Furthermore, the regulatory TF-target gene networks involved in the development of LUAD is complex and not yet fully understood. Methods: In this study, we performed RNA sequencing (RNA-seq) to analyze the transcriptome profile of human LUAD tissues and matched adjacent nontumor tissues. We selected the differentially expressed TFs, performed enrichment analysis and survival curve analysis, and predicted the regulatory networks of the top differential TFs with their target genes. Finally, alternative splicing analyses were also performed. Results: We found that TFs GRHL3, SIX1, SIX2, SPDEF, and ETV4 were upregulated, while TAL1, EPAS1, SOX17, NR4A1, and EGR3 were significantly downregulated in LUAD tissues compared to normal tissues. We propose a potential GRHL3-CDH15-Wnt-ß-catenin pro-oncogenic signaling axis and a potential TAL1-ADAMTS1-vascular antioncogenic signaling axis. In addition, we found that alternative splicing of intron retention (IR), approximate IR (XIR), multi-IR (MIR), approximate MIR (XMIR), and approximate alternative exon ends (XAE) showed abnormally increased frequencies in LUAD tissues. Conclusions: These findings revealed a novel TF-target gene regulatory axis related to tumorigenesis and provided potential therapeutic targets and mechanisms for LUAD.
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Endothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.
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Redes Reguladoras de Genes , Fatores de Transcrição , Endotélio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
T Lymphocyte Acute Lymphoblastic Leukemia (ALL) is an aggressive disease arising from transformation of T lymphocytes during their development. The mutation spectrum of T-ALL has revealed critical regulators of the growth and differentiation of normal and leukemic T lymphocytes. Approximately, 60% of T-ALLs show aberrant expression of the hematopoietic stem cell-associated helix-loop-helix transcription factors TAL1 and LYL1. TAL1 and LYL1 function in multiprotein complexes that regulate gene expression in T-ALL but they also antagonize the function of the E protein homodimers that are critical regulators of T cell development. Mice lacking E2A, or ectopically expressing TAL1, LYL1, or other inhibitors of E protein function in T cell progenitors, also succumb to an aggressive T-ALL-like disease highlighting that E proteins promote T cell development and suppress leukemogenesis. In this review, we discuss the role of E2A in T cell development and how alterations in E protein function underlie leukemogenesis. We focus on the role of TAL1 and LYL1 and the genes that are dysregulated in E2a-/- T cell progenitors that contribute to human T-ALL. These studies reveal novel mechanisms of transformation and provide insights into potential therapeutic targets for intervention in this disease.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Fatores de Transcrição , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Camundongos , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The B-cell CLL/lymphoma 11B gene (BCL11B) plays a crucial role in T-cell development, but its role in T-cell malignancies is still unclear. To study its role in the development of T-cell neoplasms, we generated an inducible BCL11B knockout in a murine T cell leukemia/lymphoma model. Mice, bearing human oncogenes TAL BHLH Transcription Factor 1 (TAL1; SCL) or LIM Domain Only 1 (LMO1), responsible for T-cell acute lymphoblastic leukemia (T-ALL) development, were crossed with BCL11B floxed and with CRE-ER/lox mice. The mice with a single oncogene BCL11Bflox/floxCREtg/tgTAL1tg or BCL11Bflox/floxCREtg/tgLMO1tg were healthy, bred normally, and were used to maintain the mice in culture. When crossed with each other, >90% of the double transgenic mice BCL11Bflox/floxCREtg/tgTAL1tgLMO1tg, within 3 to 6 months after birth, spontaneously developed T-cell leukemia/lymphoma. Upon administration of synthetic estrogen (tamoxifen), which binds to the estrogen receptor and activates the Cre recombinase, the BCL11B gene was knocked out by excision of its fourth exon from the genome. The mouse model of inducible BCL11B knockout we generated can be used to study the role of this gene in cancer development and the potential therapeutic effect of BCL11B inhibition in T-cell leukemia and lymphoma.
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Leucemia de Células T , Fatores de Transcrição , Animais , Modelos Animais de Doenças , Proteínas com Domínio LIM/genética , Leucemia de Células T/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Early T-cell development is precisely controlled by E proteins, that indistinguishably include HEB/TCF12 and E2A/TCF3 transcription factors, together with NOTCH1 and pre-T cell receptor (TCR) signalling. Importantly, perturbations of early T-cell regulatory networks are implicated in leukemogenesis. NOTCH1 gain of function mutations invariably lead to T-cell acute lymphoblastic leukemia (T-ALL), whereas inhibition of E proteins accelerates leukemogenesis. Thus, NOTCH1, pre-TCR, E2A and HEB functions are intertwined, but how these pathways contribute individually or synergistically to leukemogenesis remain to be documented. To directly address these questions, we leveraged Cd3e-deficient mice in which pre-TCR signaling and progression through ß-selection is abrogated to dissect and decouple the roles of pre-TCR, NOTCH1, E2A and HEB in SCL/TAL1-induced T-ALL, via the use of Notch1 gain of function transgenic (Notch1ICtg) and Tcf12+/- or Tcf3+/- heterozygote mice. As a result, we now provide evidence that both HEB and E2A restrain cell proliferation at the ß-selection checkpoint while the clonal expansion of SCL-LMO1-induced pre-leukemic stem cells in T-ALL is uniquely dependent on Tcf12 gene dosage. At the molecular level, HEB protein levels are decreased via proteasomal degradation at the leukemic stage, pointing to a reversible loss of function mechanism. Moreover, in SCL-LMO1-induced T-ALL, loss of one Tcf12 allele is sufficient to bypass pre-TCR signaling which is required for Notch1 gain of function mutations and for progression to T-ALL. In contrast, Tcf12 monoallelic deletion does not accelerate Notch1IC-induced T-ALL, indicating that Tcf12 and Notch1 operate in the same pathway. Finally, we identify a tumor suppressor gene set downstream of HEB, exhibiting significantly lower expression levels in pediatric T-ALL compared to B-ALL and brain cancer samples, the three most frequent pediatric cancers. In summary, our results indicate a tumor suppressor function of HEB/TCF12 in T-ALL to mitigate cell proliferation controlled by NOTCH1 in pre-leukemic stem cells and prevent NOTCH1-driven progression to T-ALL.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfócitos T , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismoRESUMO
T cell acute lymphoblastic leukaemia (T-ALL) is a genetically heterogeneous and aggressive form of malignancy. Although a number of recurrent fusion genes are reported in T-ALL, their involvement in disease stratification and therapeutic intervention is still controversial. Considering the prognostic value of STIL-TAL1 fusion and tyrosine kinase inhibitor (TKI) based therapeutic potential of NUP214-ABL1, the present study aimed to investigate their frequency and clinical correlation in pediatric T-ALL cases. Our cohort consisted of 48 T-ALL pediatric cases (age ≤ 12 years) with a median age of 6 years and male to female ratio of 20.5:1. The median TLC of the study group was noted to be 220,000/ cu mm with a range from 26,810/cu mm to 785,430/cu mm. By MLPA and RT-PCR analysis we observed that 11/48 cases (23%) showed STIL-TAL1 fusion and 4/48 cases (8.3%) had NUP214-ABL1 fusion gene. Both of the fusion genes did not show any significant correlation with any of the clinico-hematological or treatment outcome parameters. However, upon analysis of copy number variations (CNVs) with other clinically relevant genes, we found significant correlation between LEF1 (p = 0.024) and PTEN (p = 0.049) gene deletions with STIL/TAL1 fusion in T-ALL patients. NUP214-ABL1 fusion gene did not reveal any association with either CNVs or with survival. Although limited with the small cohort size and follow up, our study supports the similar frequency of these fusions as compared to other Asian and Western studies and also highlights utility of MLPA technique as a good diagnostic modality to screen for both STIL-TAL1 and NUP214-ABL1 fusions in a single assay with additional data on secondary copy number changes.
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Glioblastomas (GBM) are high-grade brain tumors, containing cells with distinct phenotypes and tumorigenic potentials, notably aggressive and treatment-resistant multipotent glioblastoma stem cells (GSC). The molecular mechanisms controlling GSC plasticity and growth have only partly been elucidated. Contact with endothelial cells and the Notch1 pathway control GSC proliferation and fate. We used three GSC cultures and glioma resections to examine the expression, regulation, and role of two transcription factors, SLUG (SNAI2) and TAL1 (SCL), involved in epithelial to mesenchymal transition (EMT), hematopoiesis, vascular identity, and treatment resistance in various cancers. In vitro, SLUG and a truncated isoform of TAL1 (TAL1-PP22) were strongly upregulated upon Notch1 activation in GSC, together with LMO2, a known cofactor of TAL1, which formed a complex with truncated TAL1. SLUG was also upregulated by TGF-ß1 treatment and by co-culture with endothelial cells. In patient samples, the full-length isoform TAL1-PP42 was expressed in all glioma grades. In contrast, SLUG and truncated TAL1 were preferentially overexpressed in GBMs. SLUG and TAL1 are expressed in the tumor microenvironment by perivascular and endothelial cells, respectively, and to a minor extent, by a fraction of epidermal growth factor receptor (EGFR) -amplified GBM cells. Mechanistically, both SLUG and truncated TAL1 reduced GSC growth after their respective overexpression. Collectively, this study provides new evidence for the role of SLUG and TAL1 in regulating GSC plasticity and growth.
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Tumor-infiltrating immune cells shape the tumor microenvironment and are closely related to clinical outcomes. Several transcription factors (TFs) have also been reported to regulate the antitumor activity and immune cell infiltration. This study aimed to quantify the populations of different immune cells infiltrated in tumor samples based on the bulk RNA sequencing data obtained from 50 cancer patients using the CIBERSORT and the EPIC algorithm. Weighted gene coexpression network analysis (WGCNA) identified eigengene modules strongly associated with tumorigenesis and the activation of CD4+ memory T cells, dendritic cells, and macrophages. TF genes FOXM1, MYBL2, TAL1, and ERG are central in the subnetworks of the eigengene modules associated with immune-related genes. The analysis of The Cancer Genome Atlas (TCGA) cancer data confirmed these findings and further showed that the expression of these potential TF genes regulating immune infiltration, and the immune-related genes that they regulated, was associated with the survival of patients within multiple cancers. Exome-seq was performed on 24 paired samples that also had RNA-seq data. The expression quantitative trait loci (eQTL) analysis showed that mutations were significantly more frequent in the regions flanking the TF genes compared with those of non-TF genes, suggesting a driver role of these TF genes regulating immune infiltration. Taken together, this study presented a practical method for identifying genes that regulate immune infiltration. These genes could be potential biomarkers for cancer prognosis and possible therapeutic targets.
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Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica/genética , Sistema Imunitário/metabolismo , Neoplasias/genética , Análise de Sequência de RNA/métodos , Fatores de Transcrição/genética , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/imunologia , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/imunologia , Humanos , Sistema Imunitário/imunologia , Sistema Imunitário/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Mutação , Neoplasias/imunologia , Neoplasias/metabolismo , Prognóstico , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
The erythroid Krüppel-like factor EKLF/KLF1 is a hematopoietic transcription factor binding to the CACCC DNA motif and participating in the regulation of erythroid differentiation. With combined use of microarray-based gene expression profiling and the promoter-based ChIP-chip assay of E14.5 fetal liver cells from wild type (WT) and EKLF-knockout (Eklf-/-) mouse embryos, we identified the pathways and direct target genes activated or repressed by EKLF. This genome-wide study together with the molecular/cellular analysis of the mouse erythroleukemic cells (MEL) indicate that among the downstream direct target genes of EKLF is Tal1/Scl. Tal1/Scl encodes another DNA-binding hematopoietic transcription factor TAL1/SCL, known to be an Eklf activator and essential for definitive erythroid differentiation. Further identification of the authentic Tal gene promoter in combination with the in vivo genomic footprinting approach and DNA reporter assay demonstrate that EKLF activates the Tal gene through binding to a specific CACCC motif located in its promoter. These data establish the existence of a previously unknow positive regulatory feedback loop between two DNA-binding hematopoietic transcription factors, which sustains mammalian erythropoiesis.
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Eritropoese , Feto/embriologia , Hematopoese Extramedular , Fatores de Transcrição Kruppel-Like/metabolismo , Fígado/embriologia , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Animais , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Knockout , Elementos de Resposta , Proteína 1 de Leucemia Linfocítica Aguda de Células T/genéticaRESUMO
The use of intracellular antibodies as templates to derive surrogate compounds is an important objective because intracellular antibodies can be employed initially for target validation in pre-clinical assays and subsequently employed in compound library screens. LMO2 is a T cell oncogenic protein activated in the majority of T cell acute leukaemias. We have used an inhibitory intracellular antibody fragment as a competitor in a small molecule library screen using competitive surface plasmon resonance (cSPR) to identify compounds that bind to LMO2. We selected four compounds that bind to LMO2 but not when the anti-LMO2 intracellular antibody fragment is bound to it. These findings further illustrate the value of intracellular antibodies in the initial stages of drug discovery campaigns and more generally antibodies, or antibody fragments, can be the starting point for chemical compound development as surrogates of the antibody combining site.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos de Neoplasias/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Proteínas com Domínio LIM/metabolismo , Leucemia de Células T/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/metabolismo , Anticorpos/metabolismo , Ligação Competitiva , Células Cultivadas , Descoberta de Drogas , Humanos , Fragmentos de Imunoglobulinas/genética , Espaço Intracelular , Conformação Proteica , Bibliotecas de Moléculas Pequenas , Ressonância de Plasmônio de Superfície , Proteína 1 de Leucemia Linfocítica Aguda de Células T/metabolismo , Linfócitos T/imunologiaRESUMO
Chromatin immunoprecipitation followed by massively parallel, high throughput sequencing (ChIP-seq) is the method of choice for genome-wide identification of DNA segments bound by specific transcription factors or in chromatin with particular histone modifications. However, the quality of ChIP-seq datasets varies widely, with a substantial fraction being of intermediate to poor quality. Thus, it is important to discern and control the factors that contribute to variation in ChIP-seq. In this study, we focused on sonication, a user-controlled variable, to produce sheared chromatin. We systematically varied the amount of shearing of fixed chromatin from a mouse erythroid cell line, carefully measuring the distribution of resultant fragment lengths prior to ChIP-seq. This systematic study was complemented with a retrospective analysis of additional experiments. We found that the level of sonication had a pronounced impact on the quality of ChIP-seq signals. Over-sonication consistently reduced quality, while the impact of under-sonication differed among transcription factors, with no impact on sites bound by CTCF but frequently leading to the loss of sites occupied by TAL1 or bound by POL2. The bound sites not observed in low-quality datasets were inferred to be a mix of both direct and indirect binding. We leveraged these findings to produce a set of CTCF ChIP-seq datasets in rare, primary hematopoietic progenitor cells. Our observation that the amount of chromatin sonication is a key variable in success of ChIP-seq experiments indicates that monitoring the level of sonication can improve ChIP-seq quality and reproducibility and facilitate ChIP-seq in rare cell types.