Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes.
Project description:A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes. Mouse hematopoietic stem cells were purified from bone marrow cells using negative and positive selection with a Magnetic-Activated Cell Sorter (MACS). total RNA and mRNA were purified from the purified cells using Trizol reagent and magnetic oligo dT beads. Double strand cDNAs were synthesized using a cDNA synthesis kit and anchored oligo dT primers. After NlaIII digestion, 3’ cDNAs were isolated and amplified through 16-cycle PCR. SAGE tags were released from the 3’ cDNA after linker ligation. Ditags were formed, concatemerized and cloned into a pZERO vector. Sequencing reactions were performed with the ET sequencing terminator kit. Sequences were collected using a Megabase 1000 sequencer. SAGE tag sequences were extracted using SAGE 2000 software.
Project description:Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell (HSC) self-renewal and expansion ex vivo and in vivo. In order to investigate the largely unknown downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line, EML. Gene expression differences were compared between KLS (c-Kit+, Lin-, Sca-1+)-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) to control KLS-EML cells that were transduced with vector alone. ChIP-chip was used to identify promoter regions bound by HOXB4.
Project description:To unravel the molecular mechanism by which HOXB4 promotes the expansion of early hematopoietic progenitors within differentiating ES cells, we analzed the gene expression profiles of embryoid bodies (EBs) in which transcription of HOXB4 had been induced or not induced. A substantial number of the identified HOXB4 target genes are involved in signaling pathways important for controlling self-renewal, maintenance and differentiation of stem cells. Furthermore, we demonstrate that HOXB4 activity and FGF-signaling are intertwined. HOXB4-mediated expansion of ES cell-derived early progenitors was enhanced by specific and complete inhibition of FGF-receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2) indicating a dominant negative effect of FGF-signaling on the earliest hematopoietic cells. Taken together, we show that modulation of FGF signaling is an essential feature of HOXB4 activity in the context of embryonic hematopoiesis. Keywords: plus/minus induction of HOXB4 gene expression by treatment with doxycycline (Dox)
Project description:Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell (HSC) self-renewal and expansion ex vivo and in vivo. In order to investigate the largely unknown downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line, EML. Gene expression differences were compared between KLS (c-Kit+, Lin-, Sca-1+)-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) to control KLS-EML cells that were transduced with vector alone. ChIP-chip was used to identify promoter regions bound by HOXB4. We overexpressed HOXB4 in EML cells. We isolated 3 separate single cell clones as assessed by Southern Blot Analysis (3 clones for EML-HOXB4 and 3 clones for control EML-GFP cells). RNA was isolated from the KLS (c-Kit+, Lin-, Sca-1+) fraction of each single cell clone population and processed for hybridization to array chips using established lab protocols. Chip-Chip analysis of the three HOXB4 overexpressing clones was performed to identify HOXB4 bound promoters.
Project description:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.
Project description:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network. Cells from three time points of the developmental processes were collected, including days 6, 16, and 26. Deep sequencing were done for both IP and input DNA from cells from each time point.
Project description:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network.