Project description:BCL11A is a critical mediator of hemoglobin switching and gamma-globin silencing. In this study, we showed the BCL11A is required in vivo for developmental silencing of gamma-globin genes in adult animals. We used microarray to determine the changes in gene expression profile after loss of BCL11A in adult erythroid cells CD71+Ter119+ erythroid progenitor cells were FACS-sorted from bone marrows of 6-week old control (Bcl11a +/+) and BCL11A knockout (Bcl11a fl/fl EpoR-Cre+) mice.
Project description:Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map transfactor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1. (Blood.2011;118(17):e139-e148) We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts and RNA-Seq analysis of the transcriptome.
Project description:With the aim of finding small molecules that stimulate erythropoiesis earlier than erythropoietin and that enhance CFU-E production, we studied the mechanism by which glucocorticoids increase CFU-E formation. Using BFU-E and CFU-E progenitors purified by a new technique, we demonstrate that glucocorticoids stimulate the earliest (BFU-E) progenitors to undergo limited self-renewal, which increases formation of CFU-E cells > 20-fold. Interestingly, glucocorticoids induce expression of genes in BFU-E cells that contain promoter regions highly enriched for hypoxia-induced factor 1 alpha (HIF1a) binding sites. This suggests activation of HIF1a may enhance or replace the effect of glucocorticoids on BFU-E self-renewal. Indeed, HIF1a activation by a prolyl hydroxylase inhibitor (PHI) synergizes with glucocorticoids and enhances production of CFU-Es 170-fold. Since PHIs are able to increase erythroblast production at very low concentrations of glucocorticoids, PHI-induced stimulation of BFU-E progenitors thus represents a conceptually new therapeutic window for treating Epo-resistant anemia. RNA-Seq was performed on enriched populations of BFU-E, CFU-E and Ter119+ as well as BFU-E enriched cells treated with Dex and DMOG
Project description:Using RNA-seq technology, we quantitatively determined the expression profile of microRNAs during mouse terminal erythroid differentiation. CFU-E erythroid progenitors were isolated from E14.5 fetal liver as the Ter119, B220, Mac-1, CD3 and Gr-1 negative, C-Kit positive and 20% high CD71 population. Mature Ter119+ erythroblasts were isolated from E14.5 fetal liver as C-Kit negative and Ter119 positive population. Consistent with nuclear condensation and global gene expression shut down during terminal erythroid differentiation, we found that the majority of microRNAs are downregulated in more mature Ter119+ erythroblasts compared with CFU-E erythroid progenitors. Examination of microRNA expression profiles in 2 cell types
Project description:EKLF is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf -/-) mice die at day 14.5 of gestation from severe anemia. In this study, we demonstrate that early progenitor cells fail to undergo terminal erythroid differention in Eklf -/- embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis, transcriptional profiling was performed with RNA from wild type and Eklf -/- early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation, with the critical regulator of the cell cycle, E2f2, at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf -/- early erythroid progenitor cells, which showed a delay in the G1-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier, EKLF binding-sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation. RNA was isolated from flow-sorted early erythroid progenitors in 13.5 day old fetal livers from EKLF knock out mice (n=3 fetal livers) and wild-type control mice (n=3 fetal livers) for gene expression analysis
Project description:Cyclin-Dependent Kinase 9 (CDK9) as part of the PTEFb complex promotes transcriptional elongation by promoting RNAPII pause release. We now report that, paradoxically, CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell screen, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression and cell differentiation, along with activation of endogenous retrovirus (ERV) genes. CDK9 inhibition dephosphorylates the SWI/SNF protein SMARCA4 and represses HP1α expression, both of which contribute to gene reactivation. Based on gene activation, we developed the highly selective and potent CDK9 inhibitor MC180295 (IC50 =5.1nM) that has broad anti-cancer activity in-vitro and is effective in in-vivo cancer models. Additionally, CDK9 inhibition sensitizes with the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.
Project description:We report genome-wide maps of transcription in mouse erythroid cells. We used an approach to survey poly(A)+ (mRNA) (GSE26877) and non-polyadenylated RNA poly(A)- (GSE27920) separately. This provides an alternative framework for comprehensive transcriptome profiling in mammalian cells. Two Samples. mRNA-seq from wild type murine Ter119+ cells and mRNA-seq from ?MCS-P6MCS-R3-/- Ter119+ cells