RNA-seq of CLB-GA neuroblastoma cell line upon TBX2 knockdown
ABSTRACT: RNA-seq upon TBX2 knockdown in the neuroblastoma cell line CLB-GA. Cells were transduced with two different shRNAs (sh#2 and sh#4) targeting TBX2 and a non-targeting control (NTC), and selected with puromycin. Analysis was performed seven days upon TBX2 knockdown, including three biological replicates per condition.
Project description:RNA-seq upon TBX2, MYCN or combination of TBX2 and MYCN knockdown in the neuroblastoma cell line IMR-5/75. Cells were transduced with two different shRNAs (shTBX2_2 and shTBX2_4) targeting TBX2 and a non-targeting control (NTC), and selected with puromycin. Cells were treated with doxycycline for shMYCN induction (with DOX or not). Analysis was performed three days upon TBX2 knockdown and two days upon MYCN knockdown, including six biological replicates per condition.
Project description:RNA-seq upon TBX2 knockdown in the neuroblastoma cell line CLB-GA. Cells were transduced with two different shRNAs (sh#2 and sh#4) targeting TBX2 and a non-targeting control (NTC), and selected with puromycin. Analysis was performed seven days upon TBX2 knockdown, including three biological replicates per condition.
Project description:<i>Background and Objectives</i>: Therapeutic interventions targeting molecular factors involved in the transition from uterine quiescence to overt labour are not substantially reducing the rate of spontaneous preterm labour. The identification of novel rational therapeutic targets are essential to prevent the most common cause of neonatal mortality. Based on our previous work showing that Tbx2 (T-Box transcription factor 2) is a putative upstream regulator preceding progesterone withdrawal in mouse myometrium, we now investigate the role of TBX2 in human myometrium. <i>Materials and Methods</i>: RNA microarray analysis of (A) preterm human myometrium samples and (B) myometrial cells overexpressing TBX2 in vitro, combined with subsequent analysis of the two publicly available datasets of (C) Chan et al. and (D) Sharp et al. The effect of TBX2 overexpression on cytokines/chemokines secreted to the myometrium cell culture medium were determined by Luminex assay. <i>Results</i>: Analysis shows that overexpression of TBX2 in myometrial cells results in downregulation of TNFα- and interferon signalling. This downregulation is consistent with the decreased expression of cytokines and chemokines of which a subset has been previously associated with the inflammatory pathways relevant for human labour. In contrast, CXCL5 (C-X-C motif chemokine ligand 5), CCL21 and IL-6 (Interleukin 6), previously reported in relation to parturition, do not seem to be under TBX2 control. The combined bioinformatical analysis of the four mRNA datasets identifies a subset of upstream regulators common to both preterm and term labour under control of TBX2. Surprisingly, TBX2 mRNA levels are increased in preterm contractile myometrium. <i>Conclusions</i>: We identified a subset of upstream regulators common to both preterm and term labour that are activated in labour and repressed by TBX2. The increased TBX2 mRNA expression in myometrium collected during a preterm caesarean section while in spontaneous preterm labour compared to tissue harvested during iatrogenic preterm delivery does not fit the bioinformatical model. We can only explain this by speculating that the in vivo activity of TBX2 in human myometrium depends not only on the TBX2 expression levels but also on levels of the accessory proteins necessary for TBX2 activity.
Project description:Tbx2 is a member of a large family of transcription factors defined by homology to the T-box DNA-binding domain. Tbx2 plays a key role in embryonic development, and in cancer through its capacity to suppress senescence and promote invasiveness. Despite its importance, little is known of how Tbx2 is regulated or how it achieves target gene specificity. Here we show that Tbx2 specifically associates with active hypophosphorylated retinoblastoma protein (Rb1), a known regulator of many transcription factors involved in cell cycle progression and cellular differentiation, but not with the Rb1-related proteins p107 or p130. The interaction with Rb1 maps to a domain immediately carboxy-terminal to the T-box and enhances Tbx2 DNA binding and transcriptional repression. Microarray analysis of melanoma cells expressing inducible dominant-negative Tbx2, comprising the T-box and either an intact or mutated Rb1 interaction domain, shows that Tbx2 regulates the expression of many genes involved in cell cycle control and that a mutation which disrupts the Rb1-Tbx2 interaction also affects Tbx2 target gene selectivity. Taken together, the data show that Rb1 is an important determinant of Tbx2 functional specificity.
Project description:The TBX2 transcription factor plays critical roles during embryonic development and it is overexpressed in several cancers, where it contributes to key oncogenic processes including the promotion of proliferation and bypass of senescence. Importantly, based on compelling biological evidences, TBX2 has been considered as a potential target for new anticancer therapies. There has therefore been a substantial interest to identify molecules with TBX2-modulatory activity, but no such substance has been found to date. Here, we adopt a targeted approach based on a reverse-affinity procedure to identify the ability of chromomycins A5 (CA5) and A6 (CA6) to interact with TBX2. Briefly, a TBX2-DNA-binding domain recombinant protein was N-terminally linked to a resin, which in turn, was incubated with either CA5 or CA6. After elution, bound material was analyzed by UPLC-MS and CA5 was recovered from TBX2-loaded resins. To confirm and quantify the affinity (KD) between the compounds and TBX2, microscale thermophoresis analysis was performed. CA5 and CA6 modified the thermophoretic behavior of TBX2, with a KD in micromolar range. To begin to understand whether these compounds exerted their anti-cancer activity through binding TBX2, we next analyzed their cytotoxicity in TBX2 expressing breast carcinoma, melanoma and rhabdomyosarcoma cells. The results show that CA5 was consistently more potent than CA6 in all tested cell lines with IC50 values in the nM range. Of the cancer cell types tested, the melanoma cells were most sensitive. The knockdown of TBX2 in 501mel melanoma cells increased their sensitivity to CA5 by up to 5 times. Furthermore, inducible expression of TBX2 in 501mel cells genetically engineered to express TBX2 in the presence of doxycycline, were less sensitive to CA5 than the control cells. Together, the data presented in this study suggest that, in addition to its already recognized DNA-binding properties, CA5 may be binding the transcription factor TBX2, and it can contribute to its cytotoxic activity.
Project description:Senescence shapes embryonic development, plays a key role in aging, and is a critical barrier to cancer initiation, yet how senescence is regulated remains incompletely understood. TBX2 is an antisenescence T-box family transcription repressor implicated in embryonic development and cancer. However, the repertoire of TBX2 target genes, its cooperating partners, and how TBX2 promotes proliferation and senescence bypass are poorly understood. Here, using melanoma as a model, we show that TBX2 lies downstream from PI3K signaling and that TBX2 binds and is required for expression of E2F1, a key antisenescence cell cycle regulator. Remarkably, TBX2 binding in vivo is associated with CACGTG E-boxes, present in genes down-regulated by TBX2 depletion, more frequently than the consensus T-element DNA binding motif that is restricted to Tbx2 repressed genes. TBX2 is revealed to interact with a wide range of transcription factors and cofactors, including key components of the BCOR/PRC1.1 complex that are recruited by TBX2 to the <i>E2F1</i> locus. Our results provide key insights into how PI3K signaling modulates TBX2 function in cancer to drive proliferation.
Project description:Rhabdomyosarcomas (RMSs) are the most frequent soft tissue sarcomas in children that share many features of developing skeletal muscle. We have discovered that a T-box family member, TBX2, is highly upregulated in tumor cells of both major RMS subtypes. TBX2 is a repressor that is often overexpressed in cancer cells and is thought to function in bypassing cell growth control, including repression of p14 and p21. The cell cycle regulator p21 is required for the terminal differentiation of skeletal muscle cells and is silenced in RMS cells. We have found that TBX2 interacts with the myogenic regulatory factors MyoD and myogenin and inhibits the activity of these factors. TBX2 is expressed in primary myoblasts and C2C12 cells, but is strongly downregulated upon differentiation. TBX2 recruits the histone deacetylase HDAC1 and is a potent inhibitor of the expression of muscle-specific genes and the cell cycle regulators, p21 and p14. TBX2 promotes the proliferation of RMS cells and either depletions of TBX2 or dominant negative TBX2 upregulate p21- and muscle-specific genes. Significantly, depletion or interference with TBX2 completely inhibits tumor growth in a xenograft assay, highlighting the oncogenic role of TBX2 in RMS cells. Thus, the data demonstrate that elevated expression of TBX2 contributes to the pathology of RMS cells by promoting proliferation and repressing differentiation-specific gene expression. These results show that deregulated TBX2 serves as an oncogene in RMS, suggesting that TBX2 may serve as a new diagnostic marker or therapeutic target for RMS tumors.
Project description:TBX2 is a member of the T box transcription factor family. Its expression and potential biological functions in nasopharyngeal cancer (NPC) cells are studied here. We showed that TBX2 mRNA and protein expression was significantly elevated in multiple human NPC tissues, as compared with that in adjacent normal tissues. Knockdown of TBX2 by targeted-siRNA significantly inhibited proliferation and invasion of NPC cells (CNE-1 and HONE-1 lines). Meanwhile, TBX2 knockdown also induced G1-phase cell cycle arrest. At the molecular level, we discovered that expressions of several tumor suppressor genes, including p21, p27, phosphatase with tensin homology (PTEN) and E-Cadherin, were increased dramatically after TBX2 knockdown in above NPC cells. Collectively, our results imply that TBX2 over-expression promotes NPC cell proliferation and invasion, possibly via silencing several key tumor suppressor genes.