Bulk RNA-seq expression profiles of DN T cell subpopulations
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE336271 | GEO | 2026/09/14
REPOSITORIES: GEO
Similar Datasets
Project description:The goal of this study is to resolve the heterogeneity of transcriptomic and chromatin accessibility (ChrAcc) landscape in DN1 thymocytes harboring ETPs. Comparison of molecular features between ETPs with non-ETP DN1 cells predicted contribution by Tcf/Lef family transcription factors, and pre-thymic ablation of Tcf1 and Lef1 impaired ETP formation. Single cell analysis of the factor-targeted ETPs revealed that they converged on Notch1 or Notch pathway effector molecules including Hes1 and Hhex, to support their transcriptional activation.
2025-09-17 | GSE283601 | GEO
Project description:The goal of this study is to resolve the heterogeneity of transcriptomic and chromatin accessibility (ChrAcc) landscape in DN1 thymocytes harboring ETPs. Comparison of molecular features between ETPs with non-ETP DN1 cells predicted contribution by Runx and Tcf/Lef family transcription factors, and pre-thymic ablation of Runx1/3, Tcf1/Lef1, or their common Tle/Groucho cofactors invariably impaired ETP formation. Single cell analysis of the factor-targeted ETPs revealed that these three sets of factors converged on Notch1 or Notch pathway effector molecules including Hes1 and Hhex, to support their transcriptional activation by acting on shared and distinct regulatory elements. Furthermore, Runx and Tcf/Lef deficiency caused thymic expansion of myeloid and B cells, respectively, while loss of Tle/Groucho factors resulted in broader diversion to multiple non-T lineages
2025-09-17 | GSE264410 | GEO
Project description:Upregulation of CD90 Expression Pinpoints T-Lineage Commitment Within DN2a Thymocytes
| PRJNA1482192 | ENA
Project description:The Ets family transcription factor PU.1 is essential for the development and maintenance of several hematopoietic lineages. In the thymus, PU.1 is expressed only in the early ETP/DN1, DN2a and DN2b stages of development. While PU.1 deletion in multipotent precursors leads to a complete block in T-cell development its function in the intrathymic stages in which it is expressed remains undetermined. The goal of this expression profiling study was to determine if PU.1 regulates the expression of T-lineage genes during the early stages of development. To do this, we generated the PU.1-Eng construct which expresses a fusion protein containing the DNA binding ETS domain of PU.1 (aas 159-260) fused to the obligate repressor domain (aas 1-298) of the Drosophila engrailed protein. The PU.1-ETS construct only expresses the ETS domain of PU.1 (aas 159-260) and serves as a control. Fetal liver precursors were isolated from e14.5 embryos and co-cultured with OP9-DL1 cells in the presence of IL-7 and Flt3L (5 ng/ml each) for 4 days to obtain FLDN1, DN2a and DN2b cells. These were infected with vector only, PU.1-ETS and the PU.1-Eng constructs and DN2 cells were sorted after 20 hours of infection. Total RNA was isolated from these cells and polyA+ fraction was used to prepare libraries for high throughput sequencing. Libraries prepared from 2 independent sets of samples were subjected to non-strand specific single-end sequencing. Two sets of samples generated from fetal liver precursor derived DN2 cells expressing PU.1-ETS and PU.1-Eng constructs were used for expression profiling. The LZRS retroviral vector, without any insert, was used to generate the vector control dataset.
2015-04-08 | E-GEOD-65344 | biostudies-arrayexpress
Project description:Understanding the specific cell populations responsible for propagation of leukemia is an important step for development of effective targeted therapies. Recently, the lymphoid-primed multipotent progenitor (LMPP) has been proposed to be a key propagating population in acute myeloid leukemia (AML; PMID 21251617). We have also shown that LMPPs share many functional and gene expression properties with early thymic progenitors (ETPs; PMID 22344248). This finding is of particular interest as ETP leukemias have recently been described: a distinct and poor prognostic disease entity with a transcriptional profile reminiscent of murine ETPs, showing co-expression of hematopoietic stem cell (HSC) and myeloid markers (PMID 19147408). Together, this raises the question whether ETPs can act as a leukemia-initiating/propagating cell population; however, relevant disease models to test this hypothesis are currently lacking. Analysis of the genetic landscape of ETP leukemias has revealed frequent coexistence of inactivating mutations of EZH2 and RUNX1 (PMID 22237106). We therefore generated mice with deletions of Ezh2 and Runx1 specifically targeted to early lymphoid progenitors using Rag1Cre (Ezh2fl/flRunx1fl/flRag1Cre+; DKO mice). As anticipated, HSCs lacked significant recombination in DKO mice whereas close to 100% of purified ETPs (Lin-CD4-CD8-CD44+CD25-Kit+Flt3+) showed deletion of Ezh2 and Runx1. Strikingly, despite a 16-fold reduction in thymus cellularity caused by a block in thymocyte maturation at the DN2-DN3 transition, absolute numbers of ETPs within the thymus of DKO mice were markedly expanded (12-fold; p<0.0001). In contrast, Ezh2 or Runx1 deletion alone had no impact on numbers of ETPs. RNA-sequencing of the expanded ETPs in DKO mice revealed upregulation of HSC- and myeloid-associated transcriptional programs, reminiscent of ETP leukaemia e.g. Pbx1 (log2FC=3.0; p<0.0001) and Csf3r (log2FC=1.9; p=0.0038). Single-cell gene expression analysis confirmed co-expression of HSC and myeloid programs with lymphoid genes within individual DKO ETPs. Further, some key regulators of T-cell maturation which are aberrantly expressed in ETP leukemia were also disrupted in DKO ETPs e.g. Tcf7 (log2FC=-9.5; p<0.0001). Gene expression associated with aberrant Ras signalling was also present. However, despite a continued expansion of the ETP population with age, we did not observe leukemia in DKO mice with over 1 year of follow-up. Since ETP leukemias frequently feature activating mutations in genes regulating RAS signaling, we hypothesised that the expanded “pre-leukemic” ETPs in DKO mice would be primed for leukemic transformation by signalling pathway mutation. We therefore crossed DKO mice with a Flt3ITD/+ knock-in mouse line, as internal tandem duplications (ITD) of FLT3 are frequent in ETP leukemias. Ezh2fl/flRunx1fl/flRag1Cre+Flt3ITD/+ (DKOITD) mice showed dramatically reduced survival (median 9.3 weeks) resulting from an aggressive, fully penetrant acute leukemia showing a predominantly myeloid phenotype (e.g. Mac1) but with co-expression of some lymphoid antigens (e.g. intracellular CD3). Crucially, this leukaemia could be propagated in wild-type recipients upon transplantation of the expanded ETPs. DKOITD ETPs were transcriptionally very similar to DKO ETPs, retaining expression of lymphoid alongside HSC- and myeloid-associated genes. Finally, in a lympho-myeloid cell line model (EML cells) we demonstrated that Ezh2 inactivation-induced loss of H3K27me3 is associated with a corresponding increase in H3K27Ac, a transcriptional activating signal that recruits bromodomain proteins. As such, we reasoned that our ETP leukemia model might be sensitive to bromodomain inhibitors such as JQ1. Indeed, we observed high sensitivity of expanded DKOITD ETPs to JQ1, raising the possibility of a new therapeutic approach for ETP leukemias. This novel mouse model of ETP-propagated leukemia, driven by clinically relevant mutations, provides intriguing evidence that leukemias with a predominant myeloid phenotype, but co-expressing lymphoid genes, may initiate within a bona fide early lymphoid progenitor population. Since the functional characteristics of the cell of origin of a leukaemia may direct its progression and response to therapy, these findings could have important implications for future stratification and treatment of both AML and ETP leukemias.
2018-02-12 | GSE75187 | GEO
Project description:Understanding the specific cell populations responsible for propagation of leukemia is an important step for development of effective targeted therapies. Recently, the lymphoid-primed multipotent progenitor (LMPP) has been proposed to be a key propagating population in acute myeloid leukemia (AML; PMID 21251617). We have also shown that LMPPs share many functional and gene expression properties with early thymic progenitors (ETPs; PMID 22344248). This finding is of particular interest as ETP leukemias have recently been described: a distinct and poor prognostic disease entity with a transcriptional profile reminiscent of murine ETPs, showing co-expression of hematopoietic stem cell (HSC) and myeloid markers (PMID 19147408). Together, this raises the question whether ETPs can act as a leukemia-initiating/propagating cell population; however, relevant disease models to test this hypothesis are currently lacking. Analysis of the genetic landscape of ETP leukemias has revealed frequent coexistence of inactivating mutations of EZH2 and RUNX1 (PMID 22237106). We therefore generated mice with deletions of Ezh2 and Runx1 specifically targeted to early lymphoid progenitors using Rag1Cre (Ezh2fl/flRunx1fl/flRag1Cre+; DKO mice). As anticipated, HSCs lacked significant recombination in DKO mice whereas close to 100% of purified ETPs (Lin-CD4-CD8-CD44+CD25-Kit+Flt3+) showed deletion of Ezh2 and Runx1. Strikingly, despite a 16-fold reduction in thymus cellularity caused by a block in thymocyte maturation at the DN2-DN3 transition, absolute numbers of ETPs within the thymus of DKO mice were markedly expanded (12-fold; p<0.0001). In contrast, Ezh2 or Runx1 deletion alone had no impact on numbers of ETPs. RNA-sequencing of the expanded ETPs in DKO mice revealed upregulation of HSC- and myeloid-associated transcriptional programs, reminiscent of ETP leukaemia e.g. Pbx1 (log2FC=3.0; p<0.0001) and Csf3r (log2FC=1.9; p=0.0038). Single-cell gene expression analysis confirmed co-expression of HSC and myeloid programs with lymphoid genes within individual DKO ETPs. Further, some key regulators of T-cell maturation which are aberrantly expressed in ETP leukemia were also disrupted in DKO ETPs e.g. Tcf7 (log2FC=-9.5; p<0.0001). Gene expression associated with aberrant Ras signalling was also present. However, despite a continued expansion of the ETP population with age, we did not observe leukemia in DKO mice with over 1 year of follow-up. Since ETP leukemias frequently feature activating mutations in genes regulating RAS signaling, we hypothesised that the expanded “pre-leukemic” ETPs in DKO mice would be primed for leukemic transformation by signalling pathway mutation. We therefore crossed DKO mice with a Flt3ITD/+ knock-in mouse line, as internal tandem duplications (ITD) of FLT3 are frequent in ETP leukemias. Ezh2fl/flRunx1fl/flRag1Cre+Flt3ITD/+ (DKOITD) mice showed dramatically reduced survival (median 9.3 weeks) resulting from an aggressive, fully penetrant acute leukemia showing a predominantly myeloid phenotype (e.g. Mac1) but with co-expression of some lymphoid antigens (e.g. intracellular CD3). Crucially, this leukaemia could be propagated in wild-type recipients upon transplantation of the expanded ETPs. DKOITD ETPs were transcriptionally very similar to DKO ETPs, retaining expression of lymphoid alongside HSC- and myeloid-associated genes. Finally, in a lympho-myeloid cell line model (EML cells) we demonstrated that Ezh2 inactivation-induced loss of H3K27me3 is associated with a corresponding increase in H3K27Ac, a transcriptional activating signal that recruits bromodomain proteins. As such, we reasoned that our ETP leukemia model might be sensitive to bromodomain inhibitors such as JQ1. Indeed, we observed high sensitivity of expanded DKOITD ETPs to JQ1, raising the possibility of a new therapeutic approach for ETP leukemias. This novel mouse model of ETP-propagated leukemia, driven by clinically relevant mutations, provides intriguing evidence that leukemias with a predominant myeloid phenotype, but co-expressing lymphoid genes, may initiate within a bona fide early lymphoid progenitor population. Since the functional characteristics of the cell of origin of a leukaemia may direct its progression and response to therapy, these findings could have important implications for future stratification and treatment of both AML and ETP leukemias.
2018-02-12 | GSE75186 | GEO
Project description:Understanding the specific cell populations responsible for propagation of leukemia is an important step for development of effective targeted therapies. Recently, the lymphoid-primed multipotent progenitor (LMPP) has been proposed to be a key propagating population in acute myeloid leukemia (AML; PMID 21251617). We have also shown that LMPPs share many functional and gene expression properties with early thymic progenitors (ETPs; PMID 22344248). This finding is of particular interest as ETP leukemias have recently been described: a distinct and poor prognostic disease entity with a transcriptional profile reminiscent of murine ETPs, showing co-expression of hematopoietic stem cell (HSC) and myeloid markers (PMID 19147408). Together, this raises the question whether ETPs can act as a leukemia-initiating/propagating cell population; however, relevant disease models to test this hypothesis are currently lacking. Analysis of the genetic landscape of ETP leukemias has revealed frequent coexistence of inactivating mutations of EZH2 and RUNX1 (PMID 22237106). We therefore generated mice with deletions of Ezh2 and Runx1 specifically targeted to early lymphoid progenitors using Rag1Cre (Ezh2fl/flRunx1fl/flRag1Cre+; DKO mice). As anticipated, HSCs lacked significant recombination in DKO mice whereas close to 100% of purified ETPs (Lin-CD4-CD8-CD44+CD25-Kit+Flt3+) showed deletion of Ezh2 and Runx1. Strikingly, despite a 16-fold reduction in thymus cellularity caused by a block in thymocyte maturation at the DN2-DN3 transition, absolute numbers of ETPs within the thymus of DKO mice were markedly expanded (12-fold; p<0.0001). In contrast, Ezh2 or Runx1 deletion alone had no impact on numbers of ETPs. RNA-sequencing of the expanded ETPs in DKO mice revealed upregulation of HSC- and myeloid-associated transcriptional programs, reminiscent of ETP leukaemia e.g. Pbx1 (log2FC=3.0; p<0.0001) and Csf3r (log2FC=1.9; p=0.0038). Single-cell gene expression analysis confirmed co-expression of HSC and myeloid programs with lymphoid genes within individual DKO ETPs. Further, some key regulators of T-cell maturation which are aberrantly expressed in ETP leukemia were also disrupted in DKO ETPs e.g. Tcf7 (log2FC=-9.5; p<0.0001). Gene expression associated with aberrant Ras signalling was also present. However, despite a continued expansion of the ETP population with age, we did not observe leukemia in DKO mice with over 1 year of follow-up. Since ETP leukemias frequently feature activating mutations in genes regulating RAS signaling, we hypothesised that the expanded “pre-leukemic” ETPs in DKO mice would be primed for leukemic transformation by signalling pathway mutation. We therefore crossed DKO mice with a Flt3ITD/+ knock-in mouse line, as internal tandem duplications (ITD) of FLT3 are frequent in ETP leukemias. Ezh2fl/flRunx1fl/flRag1Cre+Flt3ITD/+ (DKOITD) mice showed dramatically reduced survival (median 9.3 weeks) resulting from an aggressive, fully penetrant acute leukemia showing a predominantly myeloid phenotype (e.g. Mac1) but with co-expression of some lymphoid antigens (e.g. intracellular CD3). Crucially, this leukaemia could be propagated in wild-type recipients upon transplantation of the expanded ETPs. DKOITD ETPs were transcriptionally very similar to DKO ETPs, retaining expression of lymphoid alongside HSC- and myeloid-associated genes. Finally, in a lympho-myeloid cell line model (EML cells) we demonstrated that Ezh2 inactivation-induced loss of H3K27me3 is associated with a corresponding increase in H3K27Ac, a transcriptional activating signal that recruits bromodomain proteins. As such, we reasoned that our ETP leukemia model might be sensitive to bromodomain inhibitors such as JQ1. Indeed, we observed high sensitivity of expanded DKOITD ETPs to JQ1, raising the possibility of a new therapeutic approach for ETP leukemias. This novel mouse model of ETP-propagated leukemia, driven by clinically relevant mutations, provides intriguing evidence that leukemias with a predominant myeloid phenotype, but co-expressing lymphoid genes, may initiate within a bona fide early lymphoid progenitor population. Since the functional characteristics of the cell of origin of a leukaemia may direct its progression and response to therapy, these findings could have important implications for future stratification and treatment of both AML and ETP leukemias.
2018-02-12 | GSE75185 | GEO
Project description:Understanding the specific cell populations responsible for propagation of leukemia is an important step for development of effective targeted therapies. Recently, the lymphoid-primed multipotent progenitor (LMPP) has been proposed to be a key propagating population in acute myeloid leukemia (AML; PMID 21251617). We have also shown that LMPPs share many functional and gene expression properties with early thymic progenitors (ETPs; PMID 22344248). This finding is of particular interest as ETP leukemias have recently been described: a distinct and poor prognostic disease entity with a transcriptional profile reminiscent of murine ETPs, showing co-expression of hematopoietic stem cell (HSC) and myeloid markers (PMID 19147408). Together, this raises the question whether ETPs can act as a leukemia-initiating/propagating cell population; however, relevant disease models to test this hypothesis are currently lacking. Analysis of the genetic landscape of ETP leukemias has revealed frequent coexistence of inactivating mutations of EZH2 and RUNX1 (PMID 22237106). We therefore generated mice with deletions of Ezh2 and Runx1 specifically targeted to early lymphoid progenitors using Rag1Cre (Ezh2fl/flRunx1fl/flRag1Cre+; DKO mice). As anticipated, HSCs lacked significant recombination in DKO mice whereas close to 100% of purified ETPs (Lin-CD4-CD8-CD44+CD25-Kit+Flt3+) showed deletion of Ezh2 and Runx1. Strikingly, despite a 16-fold reduction in thymus cellularity caused by a block in thymocyte maturation at the DN2-DN3 transition, absolute numbers of ETPs within the thymus of DKO mice were markedly expanded (12-fold; p<0.0001). In contrast, Ezh2 or Runx1 deletion alone had no impact on numbers of ETPs. RNA-sequencing of the expanded ETPs in DKO mice revealed upregulation of HSC- and myeloid-associated transcriptional programs, reminiscent of ETP leukaemia e.g. Pbx1 (log2FC=3.0; p<0.0001) and Csf3r (log2FC=1.9; p=0.0038). Single-cell gene expression analysis confirmed co-expression of HSC and myeloid programs with lymphoid genes within individual DKO ETPs. Further, some key regulators of T-cell maturation which are aberrantly expressed in ETP leukemia were also disrupted in DKO ETPs e.g. Tcf7 (log2FC=-9.5; p<0.0001). Gene expression associated with aberrant Ras signalling was also present. However, despite a continued expansion of the ETP population with age, we did not observe leukemia in DKO mice with over 1 year of follow-up. Since ETP leukemias frequently feature activating mutations in genes regulating RAS signaling, we hypothesised that the expanded “pre-leukemic” ETPs in DKO mice would be primed for leukemic transformation by signalling pathway mutation. We therefore crossed DKO mice with a Flt3ITD/+ knock-in mouse line, as internal tandem duplications (ITD) of FLT3 are frequent in ETP leukemias. Ezh2fl/flRunx1fl/flRag1Cre+Flt3ITD/+ (DKOITD) mice showed dramatically reduced survival (median 9.3 weeks) resulting from an aggressive, fully penetrant acute leukemia showing a predominantly myeloid phenotype (e.g. Mac1) but with co-expression of some lymphoid antigens (e.g. intracellular CD3). Crucially, this leukaemia could be propagated in wild-type recipients upon transplantation of the expanded ETPs. DKOITD ETPs were transcriptionally very similar to DKO ETPs, retaining expression of lymphoid alongside HSC- and myeloid-associated genes. Finally, in a lympho-myeloid cell line model (EML cells) we demonstrated that Ezh2 inactivation-induced loss of H3K27me3 is associated with a corresponding increase in H3K27Ac, a transcriptional activating signal that recruits bromodomain proteins. As such, we reasoned that our ETP leukemia model might be sensitive to bromodomain inhibitors such as JQ1. Indeed, we observed high sensitivity of expanded DKOITD ETPs to JQ1, raising the possibility of a new therapeutic approach for ETP leukemias. This novel mouse model of ETP-propagated leukemia, driven by clinically relevant mutations, provides intriguing evidence that leukemias with a predominant myeloid phenotype, but co-expressing lymphoid genes, may initiate within a bona fide early lymphoid progenitor population. Since the functional characteristics of the cell of origin of a leukaemia may direct its progression and response to therapy, these findings could have important implications for future stratification and treatment of both AML and ETP leukemias.
2018-02-19 | GSE100693 | GEO
Project description:Understanding the specific cell populations responsible for propagation of leukemia is an important step for development of effective targeted therapies. Recently, the lymphoid-primed multipotent progenitor (LMPP) has been proposed to be a key propagating population in acute myeloid leukemia (AML; PMID 21251617). We have also shown that LMPPs share many functional and gene expression properties with early thymic progenitors (ETPs; PMID 22344248). This finding is of particular interest as ETP leukemias have recently been described: a distinct and poor prognostic disease entity with a transcriptional profile reminiscent of murine ETPs, showing co-expression of hematopoietic stem cell (HSC) and myeloid markers (PMID 19147408). Together, this raises the question whether ETPs can act as a leukemia-initiating/propagating cell population; however, relevant disease models to test this hypothesis are currently lacking. Analysis of the genetic landscape of ETP leukemias has revealed frequent coexistence of inactivating mutations of EZH2 and RUNX1 (PMID 22237106). We therefore generated mice with deletions of Ezh2 and Runx1 specifically targeted to early lymphoid progenitors using Rag1Cre (Ezh2fl/flRunx1fl/flRag1Cre+; DKO mice). As anticipated, HSCs lacked significant recombination in DKO mice whereas close to 100% of purified ETPs (Lin-CD4-CD8-CD44+CD25-Kit+Flt3+) showed deletion of Ezh2 and Runx1. Strikingly, despite a 16-fold reduction in thymus cellularity caused by a block in thymocyte maturation at the DN2-DN3 transition, absolute numbers of ETPs within the thymus of DKO mice were markedly expanded (12-fold; p<0.0001). In contrast, Ezh2 or Runx1 deletion alone had no impact on numbers of ETPs. RNA-sequencing of the expanded ETPs in DKO mice revealed upregulation of HSC- and myeloid-associated transcriptional programs, reminiscent of ETP leukaemia e.g. Pbx1 (log2FC=3.0; p<0.0001) and Csf3r (log2FC=1.9; p=0.0038). Single-cell gene expression analysis confirmed co-expression of HSC and myeloid programs with lymphoid genes within individual DKO ETPs. Further, some key regulators of T-cell maturation which are aberrantly expressed in ETP leukemia were also disrupted in DKO ETPs e.g. Tcf7 (log2FC=-9.5; p<0.0001). Gene expression associated with aberrant Ras signalling was also present. However, despite a continued expansion of the ETP population with age, we did not observe leukemia in DKO mice with over 1 year of follow-up. Since ETP leukemias frequently feature activating mutations in genes regulating RAS signaling, we hypothesised that the expanded “pre-leukemic” ETPs in DKO mice would be primed for leukemic transformation by signalling pathway mutation. We therefore crossed DKO mice with a Flt3ITD/+ knock-in mouse line, as internal tandem duplications (ITD) of FLT3 are frequent in ETP leukemias. Ezh2fl/flRunx1fl/flRag1Cre+Flt3ITD/+ (DKOITD) mice showed dramatically reduced survival (median 9.3 weeks) resulting from an aggressive, fully penetrant acute leukemia showing a predominantly myeloid phenotype (e.g. Mac1) but with co-expression of some lymphoid antigens (e.g. intracellular CD3). Crucially, this leukaemia could be propagated in wild-type recipients upon transplantation of the expanded ETPs. DKOITD ETPs were transcriptionally very similar to DKO ETPs, retaining expression of lymphoid alongside HSC- and myeloid-associated genes. Finally, in a lympho-myeloid cell line model (EML cells) we demonstrated that Ezh2 inactivation-induced loss of H3K27me3 is associated with a corresponding increase in H3K27Ac, a transcriptional activating signal that recruits bromodomain proteins. As such, we reasoned that our ETP leukemia model might be sensitive to bromodomain inhibitors such as JQ1. Indeed, we observed high sensitivity of expanded DKOITD ETPs to JQ1, raising the possibility of a new therapeutic approach for ETP leukemias. This novel mouse model of ETP-propagated leukemia, driven by clinically relevant mutations, provides intriguing evidence that leukemias with a predominant myeloid phenotype, but co-expressing lymphoid genes, may initiate within a bona fide early lymphoid progenitor population. Since the functional characteristics of the cell of origin of a leukaemia may direct its progression and response to therapy, these findings could have important implications for future stratification and treatment of both AML and ETP leukemias.
2018-02-19 | GSE100694 | GEO
Project description:The Ets family transcription factor PU.1 is essential for the development and maintenance of several hematopoietic lineages. In the thymus, PU.1 is expressed only in the early ETP/DN1, DN2a and DN2b stages of development. While PU.1 deletion in multipotent precursors leads to a complete block in T-cell development its function in the intrathymic stages in which it is expressed remains undetermined. The goal of this expression profiling study was to determine if PU.1 regulates the expression of T-lineage genes during the early stages of development. To do this, we generated the PU.1-Eng construct which expresses a fusion protein containing the DNA binding ETS domain of PU.1 (aas 159-260) fused to the obligate repressor domain (aas 1-298) of the Drosophila engrailed protein. The PU.1-ETS construct only expresses the ETS domain of PU.1 (aas 159-260) and serves as a control. Fetal liver precursors were isolated from e14.5 embryos and co-cultured with OP9-DL1 cells in the presence of IL-7 and Flt3L (5 ng/ml each) for 4 days to obtain FLDN1, DN2a and DN2b cells. These were infected with vector only, PU.1-ETS and the PU.1-Eng constructs and DN2 cells were sorted after 20 hours of infection. Total RNA was isolated from these cells and polyA+ fraction was used to prepare libraries for high throughput sequencing. Libraries prepared from 2 independent sets of samples were subjected to non-strand specific single-end sequencing.
2015-04-08 | GSE65344 | GEO