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TLX1 and NOTCH coregulate transcription in T cell acute lymphoblastic leukemia cells.

Riz I, Hawley TS, Luu TV, Lee NH, Hawley RG - Mol. Cancer (2010)

Bottom Line: Gain- and loss-of-function experiments confirmed that MYC was an essential mediator of TLX1/NOTCH transcriptional output and growth promotion in ALL-SIL cells, with TLX1 contributing to the NOTCH-MYC regulatory axis by posttranscriptional enhancement of MYC protein levels.Functional classification of the TLX1/NOTCH-coregulated targets also showed enrichment for genes associated with other human cancers as well as those involved in developmental processes.In particular, we found that TLX1, NOTCH and MYC coregulate CD1B and RAG1, characteristic markers of early cortical thymocytes, and that concerted downregulation of the TLX1 and NOTCH pathways resulted in their irreversible repression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Regenerative Biology, The George Washington University Medical Center, Washington, DC, USA.

ABSTRACT

Background: The homeobox gene TLX1 (for T-cell leukemia homeobox 1, previously known as HOX11) is inappropriately expressed in a major subgroup of T cell acute lymphoblastic leukemia (T-ALL) where it is strongly associated with activating NOTCH1 mutations. Despite the recognition that these genetic lesions cooperate in leukemogenesis, there have been no mechanistic studies addressing how TLX1 and NOTCH1 functionally interact to promote the leukemic phenotype.

Results: Global gene expression profiling after downregulation of TLX1 and inhibition of the NOTCH pathway in ALL-SIL cells revealed that TLX1 synergistically regulated more than 60% of the NOTCH-responsive genes. Structure-function analysis demonstrated that TLX1 binding to Groucho-related TLE corepressors was necessary for maximal transcriptional regulation of the NOTCH-responsive genes tested, implicating TLX1 modulation of the NOTCH-TLE regulatory network. Comparison of the dataset to publicly available biological databases indicated that the TLX1/NOTCH-coregulated genes are frequently targeted by MYC. Gain- and loss-of-function experiments confirmed that MYC was an essential mediator of TLX1/NOTCH transcriptional output and growth promotion in ALL-SIL cells, with TLX1 contributing to the NOTCH-MYC regulatory axis by posttranscriptional enhancement of MYC protein levels. Functional classification of the TLX1/NOTCH-coregulated targets also showed enrichment for genes associated with other human cancers as well as those involved in developmental processes. In particular, we found that TLX1, NOTCH and MYC coregulate CD1B and RAG1, characteristic markers of early cortical thymocytes, and that concerted downregulation of the TLX1 and NOTCH pathways resulted in their irreversible repression.

Conclusions: We found that TLX1 and NOTCH synergistically regulate transcription in T-ALL, at least in part via the sharing of a TLE corepressor and by augmenting expression of MYC. We conclude that the TLX1/NOTCH/MYC network is a central determinant promoting the growth and survival of TLX1+ T-ALL cells. In addition, the TLX1/NOTCH/MYC transcriptional network coregulates genes involved in T cell development, such as CD1 and RAG family members, and therefore may prescribe the early cortical stage of differentiation arrest characteristic of the TLX1 subgroup of T-ALL.

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Schematic summary of regulatory network controlling TLX1/NOTCH signature genes. NOTCH1 activates transcription of downstream transcriptional regulators MYC and HES. TLX1 augments MYC protein levels. HES represses transcription via interaction with TLE [41-43]. TLX1 interacts with TLE to mediate repression and activation of transcription [49]. MYC binds TLE directly in vitro [45]. However, it is not known whether TLE is required for MYC repressor activity (indicated by "?"). We speculate that the genes coregulated by the TLX1/TLE/NOTCH/MYC network are critical for TLX1/NOTCH transforming function in T-ALL.
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Figure 6: Schematic summary of regulatory network controlling TLX1/NOTCH signature genes. NOTCH1 activates transcription of downstream transcriptional regulators MYC and HES. TLX1 augments MYC protein levels. HES represses transcription via interaction with TLE [41-43]. TLX1 interacts with TLE to mediate repression and activation of transcription [49]. MYC binds TLE directly in vitro [45]. However, it is not known whether TLE is required for MYC repressor activity (indicated by "?"). We speculate that the genes coregulated by the TLX1/TLE/NOTCH/MYC network are critical for TLX1/NOTCH transforming function in T-ALL.

Mentions: Several lines of evidence indicate that TLX1 functions as a transcriptional regulator that can either activate or repress gene expression [49,50,53-55,60,73-76]. The situation is complicated by the fact that TLX1 may switch its mode of regulation of the same gene depending on as yet ill-defined tissue-specific factors that may include the availability of transcriptional cofactors, the presence or state of activation of cis-regulatory DNA elements, and/or the expression levels of the TLX1 protein itself (our unpublished observations and [54]). Recently, for two of the best characterized DNA binding-dependent TLX1 targets, Aldh1a1 and Fhl1, we reported that TLX1 activates transcription via interaction with the transcriptional corepressor TLE [49]. However, the significance of this observation for TLX1-associated leukemogenesis as well as the leukemia-specific downstream targets of TLX1 still remained elusive. In the present work, using a human cell line derived from a TLX1+ T-ALL patient sample we have identified several genes that were upregulated by TLX1 in a DNA binding-dependent manner. Although statistical significance was only reached for a subset of genes examined, an intact Eh1 TLE-binding motif was necessary for maximal effect in all cases with the exception of L1TD1. Additionally, we identified a potential direct TLX1 target gene, GAS1, which was repressed by TLX1 in a classical TLE and DNA binding-dependent manner [48]. Interestingly, we found that our attempts to overexpress TLE1 by retroviral-mediated gene delivery resulted in massive cell death in TLX1 knockdown derivatives but not in parental ALL-SIL cells (our unpublished observations). Taken together, our data suggest that TLX1 interaction with TLE may be an integral component of TLX1 leukemic function (Figure 6). To explain how TLX1-TLE binding may cause transcriptional activation, we can envisage at least two mechanisms: 1) TLX1-TLE-mediated repression of unknown intermediary transcriptional repressors (referred to as the repressor-of-repressor mechanism [77]); and/or 2) A derepression strategy involving competitive sequestration of TLE from other TLE-dependent transcriptional repressors. To illustrate the validity of the second mechanism, we previously used a well-established HES1-TLE-dependent repressor model involving the ASCL1/HASH1 gene and showed that ectopic expression of TLX1 dismissed TLE from the ASCL1 promoter, preventing HES1-mediated ASCL1 repression [49].


TLX1 and NOTCH coregulate transcription in T cell acute lymphoblastic leukemia cells.

Riz I, Hawley TS, Luu TV, Lee NH, Hawley RG - Mol. Cancer (2010)

Schematic summary of regulatory network controlling TLX1/NOTCH signature genes. NOTCH1 activates transcription of downstream transcriptional regulators MYC and HES. TLX1 augments MYC protein levels. HES represses transcription via interaction with TLE [41-43]. TLX1 interacts with TLE to mediate repression and activation of transcription [49]. MYC binds TLE directly in vitro [45]. However, it is not known whether TLE is required for MYC repressor activity (indicated by "?"). We speculate that the genes coregulated by the TLX1/TLE/NOTCH/MYC network are critical for TLX1/NOTCH transforming function in T-ALL.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Schematic summary of regulatory network controlling TLX1/NOTCH signature genes. NOTCH1 activates transcription of downstream transcriptional regulators MYC and HES. TLX1 augments MYC protein levels. HES represses transcription via interaction with TLE [41-43]. TLX1 interacts with TLE to mediate repression and activation of transcription [49]. MYC binds TLE directly in vitro [45]. However, it is not known whether TLE is required for MYC repressor activity (indicated by "?"). We speculate that the genes coregulated by the TLX1/TLE/NOTCH/MYC network are critical for TLX1/NOTCH transforming function in T-ALL.
Mentions: Several lines of evidence indicate that TLX1 functions as a transcriptional regulator that can either activate or repress gene expression [49,50,53-55,60,73-76]. The situation is complicated by the fact that TLX1 may switch its mode of regulation of the same gene depending on as yet ill-defined tissue-specific factors that may include the availability of transcriptional cofactors, the presence or state of activation of cis-regulatory DNA elements, and/or the expression levels of the TLX1 protein itself (our unpublished observations and [54]). Recently, for two of the best characterized DNA binding-dependent TLX1 targets, Aldh1a1 and Fhl1, we reported that TLX1 activates transcription via interaction with the transcriptional corepressor TLE [49]. However, the significance of this observation for TLX1-associated leukemogenesis as well as the leukemia-specific downstream targets of TLX1 still remained elusive. In the present work, using a human cell line derived from a TLX1+ T-ALL patient sample we have identified several genes that were upregulated by TLX1 in a DNA binding-dependent manner. Although statistical significance was only reached for a subset of genes examined, an intact Eh1 TLE-binding motif was necessary for maximal effect in all cases with the exception of L1TD1. Additionally, we identified a potential direct TLX1 target gene, GAS1, which was repressed by TLX1 in a classical TLE and DNA binding-dependent manner [48]. Interestingly, we found that our attempts to overexpress TLE1 by retroviral-mediated gene delivery resulted in massive cell death in TLX1 knockdown derivatives but not in parental ALL-SIL cells (our unpublished observations). Taken together, our data suggest that TLX1 interaction with TLE may be an integral component of TLX1 leukemic function (Figure 6). To explain how TLX1-TLE binding may cause transcriptional activation, we can envisage at least two mechanisms: 1) TLX1-TLE-mediated repression of unknown intermediary transcriptional repressors (referred to as the repressor-of-repressor mechanism [77]); and/or 2) A derepression strategy involving competitive sequestration of TLE from other TLE-dependent transcriptional repressors. To illustrate the validity of the second mechanism, we previously used a well-established HES1-TLE-dependent repressor model involving the ASCL1/HASH1 gene and showed that ectopic expression of TLX1 dismissed TLE from the ASCL1 promoter, preventing HES1-mediated ASCL1 repression [49].

Bottom Line: Gain- and loss-of-function experiments confirmed that MYC was an essential mediator of TLX1/NOTCH transcriptional output and growth promotion in ALL-SIL cells, with TLX1 contributing to the NOTCH-MYC regulatory axis by posttranscriptional enhancement of MYC protein levels.Functional classification of the TLX1/NOTCH-coregulated targets also showed enrichment for genes associated with other human cancers as well as those involved in developmental processes.In particular, we found that TLX1, NOTCH and MYC coregulate CD1B and RAG1, characteristic markers of early cortical thymocytes, and that concerted downregulation of the TLX1 and NOTCH pathways resulted in their irreversible repression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Regenerative Biology, The George Washington University Medical Center, Washington, DC, USA.

ABSTRACT

Background: The homeobox gene TLX1 (for T-cell leukemia homeobox 1, previously known as HOX11) is inappropriately expressed in a major subgroup of T cell acute lymphoblastic leukemia (T-ALL) where it is strongly associated with activating NOTCH1 mutations. Despite the recognition that these genetic lesions cooperate in leukemogenesis, there have been no mechanistic studies addressing how TLX1 and NOTCH1 functionally interact to promote the leukemic phenotype.

Results: Global gene expression profiling after downregulation of TLX1 and inhibition of the NOTCH pathway in ALL-SIL cells revealed that TLX1 synergistically regulated more than 60% of the NOTCH-responsive genes. Structure-function analysis demonstrated that TLX1 binding to Groucho-related TLE corepressors was necessary for maximal transcriptional regulation of the NOTCH-responsive genes tested, implicating TLX1 modulation of the NOTCH-TLE regulatory network. Comparison of the dataset to publicly available biological databases indicated that the TLX1/NOTCH-coregulated genes are frequently targeted by MYC. Gain- and loss-of-function experiments confirmed that MYC was an essential mediator of TLX1/NOTCH transcriptional output and growth promotion in ALL-SIL cells, with TLX1 contributing to the NOTCH-MYC regulatory axis by posttranscriptional enhancement of MYC protein levels. Functional classification of the TLX1/NOTCH-coregulated targets also showed enrichment for genes associated with other human cancers as well as those involved in developmental processes. In particular, we found that TLX1, NOTCH and MYC coregulate CD1B and RAG1, characteristic markers of early cortical thymocytes, and that concerted downregulation of the TLX1 and NOTCH pathways resulted in their irreversible repression.

Conclusions: We found that TLX1 and NOTCH synergistically regulate transcription in T-ALL, at least in part via the sharing of a TLE corepressor and by augmenting expression of MYC. We conclude that the TLX1/NOTCH/MYC network is a central determinant promoting the growth and survival of TLX1+ T-ALL cells. In addition, the TLX1/NOTCH/MYC transcriptional network coregulates genes involved in T cell development, such as CD1 and RAG family members, and therefore may prescribe the early cortical stage of differentiation arrest characteristic of the TLX1 subgroup of T-ALL.

Show MeSH
Related in: MedlinePlus