A conditionally-active form of MEK1 results in autocrine transformation of human and mouse hematopoietic cells

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.

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Comparison of Expression and Epigenetic Profiles in Human and Mouse Erythropoiesis and Megakaryopoiesis Using a Systems Biology Model

Blood ◽

10.1182/blood.v126.23.2383.2383 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2383-2383

Author(s):

Jens Lichtenberg ◽

Elisabeth F. Heuston ◽

Cheryl A. Keller ◽

Ross C. Hardison ◽

David M. Bodine

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Transcription Factor ◽

Systems Biology ◽

Hematopoietic Cells ◽

Gene Body ◽

Cell Type ◽

Human And Mouse ◽

Different Levels ◽

Transcription Factor Occupancy

Abstract To date numerous datasets of gene expression and epigenetic profiles for mouse and human hematopoietic cells have been generated. While individual data sets for a particular cell type have been correlated, no approach exists to harness all expression and epigenetic profiles for the different types of hematopoietic cells. Our goal is to develop a systems biology platform to compare epigenetic profiles of hematopoietic cells towards a better understanding of epigenetic mechanisms governing hematopoiesis. To provide the necessary foundation to support systematic studies of hematopoiesis, we have developed the Systems Biology Repository (SBR, http://sbrblood.nhgri.nih.gov), a data "ranch" for organizing and analyzing transcriptome and epigenome data cells throughout differentiation. To populate SBR, we extracted, curated, annotated, and integrated all human and mouse hematopoietic datasets available through the Encyclopedia of DNA Elements (ENCODE), the Gene Expression Omnibus (GEO) and the Short Read Repository (SRR). These include genome-wide profiles of DNA methylation, histone methylation and acetylation, transcription factor occupancy (ChIPSeq), chromatin accessibility (DNaseISeq, ATACSeq, FAIRESeq), and coding as well as non-coding transcriptional profiles (RNASeq). To demonstrate the utility of SBR, we conducted three different analyses. The first was a vertical study of HistoneSeq (H3K4me1, H3K4me2, H3K4me3, and H3K27ac), DNA methylation and RNASeq profiles during mouse erythroid differentiation. We found a global decrease in DNA methylation from hematopoietic stem and progenitor cells (HSC) through common myeloid progenitors (CMP), erythroid progenitor cells (MEP) and erythroblasts (ERY; 92936 peaks in HSC to 14422 in ERY). The number of expressed genes (using a tags per million cutoff of 10) increased in erythroid progenitors (8901 in HSC to 10778 in CMP and 10670 in MEP) before decreasing in ERY (8654). 62% of histone marks delineating active enhancers (H3K27ac, H3K4me1) are present in both HSC and ERY, while 48% arise de novo during differentiation. In contrast, only 16% of active promoter specific histone marks (H3K4me2, H3K4me3) are present in both HSC and ERY. For a horizontal analysis we compared the DNA methylation, RNASeq, histone modification (H3K4me1, H3K4me2, H3K4me3, and H3K27ac) and transcription factor binding (GATA1 and NFE2) profiles of erythroblasts (ERY) and megakaryocytes (MEG). We found a similar relationship between gene expression and the histone and DNA methylation profiles in each cell type but differences between expression and in transcription factor occupancy. DNA methylation and H3K4me3 was enriched in the gene body of expressed genes (>36%) for both ERY (p ≤ 0.001) and MEG (p ≤ 0.01). In contrast DNA methylation was enriched in the upstream and downstream regions of non-coding RNA genes (p ≤ 0.001). Transcription factor occupancy was cell type specific: 79% of GATA1 sites are in ERY and 72% of NFE2 sites are in MEG. In erythroblasts, DNA methylation and GATA1 binding in the gene body are associated with gene silencing (4 fold difference, p ≤ 0.001), while in megakaryocytes, DNA methylation and NFE2 binding in the gene body are associated with gene activation (8 fold difference, p ≤ 0.001). We used the Mouse Genome Informatics homology map data to perform a cross-species comparison of the expression profiles of mouse and human multipotent progenitors (MPP), proerythroblasts and orthochromatic erythroblasts. We found a total of 5247 genes expressed at significantly different levels (p ≤ 0.001) between human and mouse MPP, while only 2010 genes were expressed at significantly similar levels (p ≤ 0.001). At the proerythroblast and orthochromatic erythroblast stages 7696 genes and 6571 genes were expressed at significantly different levels (p ≤ 0.001) between human and mouse respectively, while 2024 and 2560 genes were expressed at significantly similar levels (p ≤ 0.001). These data are consistent with previous studies showing differences in the transcriptional profiles of mouse and human hematopoietic cells. In summary, SBR provides a foundation to model the genetic and epigenetic landscape in both the mouse and human hematopoietic system, and enables functional correlations to be made between the species. As SBR is expanded to include data from patient cells, it will be possible to model epigenetic changes associated with disease. Disclosures No relevant conflicts of interest to declare.

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Synergy between AUUUA motif disruption and enhancer insertion results in autocrine transformation of interleukin-3-dependent hematopoietic cells

Blood ◽

10.1182/blood.v86.8.3139.bloodjournal8683139 ◽

1995 ◽

Vol 86 (8) ◽

pp. 3139-3150 ◽

Cited By ~ 1

Author(s):

MW Mayo ◽

XY Wang ◽

PA Algate ◽

GF Arana ◽

PE Hoyle ◽

...

Keyword(s):

Mrna Stability ◽

Untranslated Region ◽

Stability Region ◽

Rank Order ◽

Hematopoietic Cells ◽

Transformation Process ◽

Wild Type ◽

Transcriptional Enhancer ◽

Interleukin 3 ◽

Autocrine Transformation

Previously, we characterized the transposition of an intracisternal type A particle (IAP) to the 32 untranslated region (UTR) of the interleukin-3 (IL-3) gene, which displaced two of the six AUUUA motifs associated with mRNA stability in an IL-3-secreting clone. To determine whether this rearrangement was involved in the autocrine transformation of the parental IL-3-dependent FL5.12 cell line, the germline (gIL-3) and rearranged IL-3 (rIL-3) genes were isolated and subcloned into a gene transfer vector. Moreover, the IAP-long terminal repeat (LTR) and the IL-3 32 UTR AUUUA motifs were deleted (rIL-3 + delta LTR and gIL-3 + delta AUUUA) in some IL-3 constructs to ascertain their role in the transformation process. The IAP-LTR was also added to these constructs (rIL-3 + delta LTR + IAP-LTR, gIL-3 + delta AUUUA + IAP-LTR, and gIL-3 + IAP-LTR), to determine whether it was necessary for autocrine transformation. The ability of the modified IL-3 genes to abrogate the IL-3 dependency of FL5.12 cells had the following rank order: rIL-3 was greater than rIL-3 + delta LTR + IAP-LTR, which was greater than gIL-3 + delta AUUUA + IAP-LTR, which was greater than gIL-3 + delta AUUUA, which was equal to rIL-3 + delta LTR, which was greater than gIL-3. The half-life of IL-3 mRNA was 20-fold longer in cells containing a mutated as opposed to a wild-type AUUUA region. All of the factor-independent cells that expressed the IL-3 transgenes secreted IL-3 and were tumorigenic after injection into BALB/c nude mice. These results indicated that two events could synergize in the autocrine transformation of hematopoietic cells: (1) addition of a transcriptional enhancer present in a retroviral LTR, and (2) disruption of an mRNA stability region.

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Expression of the mammalian c-fes protein in hematopoietic cells and identification of a distinct fes-related protein

Molecular and Cellular Biology ◽

10.1128/mcb.5.10.2543-2551.1985 ◽

1985 ◽

Vol 5 (10) ◽

pp. 2543-2551

Author(s):

I MacDonald ◽

J Levy ◽

T Pawson

Keyword(s):

Protein Kinase ◽

Kinase Activity ◽

Human Peripheral Blood ◽

Hematopoietic Cells ◽

Specific Protein ◽

Protein Kinase Activity ◽

Sarcoma Virus ◽

Human And Mouse ◽

Specific Protein Kinase

The avian c-fps and mammalian c-fes proto-oncogenes are cognate cellular sequences. Antiserum raised against the P140gag-fps transforming protein of Fujinami avian sarcoma virus specifically recognized a 92,000-Mr protein in human and mouse hematopoietic cells which was closely related in structure to Snyder-Theilen feline sarcoma virus P87gag-fes. This polypeptide was apparently the product of the human c-fes gene and was therefore designated p92c-fes. Human p92c-fes was associated with a tyrosine-specific protein kinase activity in vitro and was capable of both autophosphorylation and phosphorylation of enolase as an exogenous protein substrate. The synthesis of human and mouse p92c-fes was largely, though not entirely, confined to myeloid cells. p92c-fes was expressed to relatively high levels in a multipotential murine myeloid cell line, in more mature human and mouse granulocyte-macrophage progenitors, and in differentiated macrophage like cells as well as in the mononuclear fraction of normal and leukemic human peripheral blood. p92c-fes was not found in erythroid cells, with the exception of a human erythroleukemia line which retains the capacity to differentiate into macrophage like cells. These results suggest a normal role for the p92c-fes tyrosine kinase in hematopoiesis, particularly in granulocyte-macrophage differentiation. In addition, a distinct 94,000-Mr polypeptide, antigenically related to p92c-fes, was identified in a number of hematopoietic and nonhematopoietic human and mouse cells and was also found to be associated with a tyrosine-specific protein kinase activity.

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Stem Cell Mobilization and Egress of AML Blasts from the Bone Marrow Are Both Regulated by MT-MMP-Mediated Activation of MMP-2.

Blood ◽

10.1182/blood.v106.11.1368.1368 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1368-1368

Author(s):

Leah A. Marquez-Curtis ◽

Neeta Shirvaikar ◽

Loree M. Larratt ◽

A. Robert Turner ◽

Mariusz Z. Ratajczak ◽

...

Keyword(s):

Bone Marrow ◽

Steady State ◽

Stromal Cells ◽

Proteolytic Enzymes ◽

Active Form ◽

Hematopoietic Cells ◽

Common Mechanism ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Cell Mobilization

Abstract The cell-surface proteolytic enzymes membrane-type (MT)-matrix metalloproteinases (MMPs) activate secreted latent forms of MMPs and play a key role in cell migration and tumor cell invasion and metastasis. Previously we reported that mobilized peripheral blood (mPB) CD34+ cells as well as AML blasts secrete inactive (pro)MMP-2, in contrast to normal steady-state bone marrow (BM) CD34+ cells which do not. In this study we hypothesized that the egress from BM of normal hematopoietic stem/progenitor cells during G-CSF-induced mobilization or of AML blasts shares a common mechanism that involves activation of secreted proMMP-2 by MT-MMPs. We evaluated the expression of MT-MMPs (MT1-MMP, MT2-, MT3- MT4-, MT5-, MT6-) in normal and leukemic hematopoietic cells, as well as in BM stromal cells, and determined their role in proMMP-2 activation and migration. We found MT1-MMP expression (mRNA and protein) in mPB CD34+ cells and in the majority of AML samples (from 21 out of 26 patients) and upregulation of MT1-MMP by G-CSF in steady-state BM CD34+ cells. Moreover, we confirmed the secretion of proMMP-2 in media conditioned by hematopoietic (mPB CD34+, AML) cells and stromal cells. However, we detected the active form of MMP-2 in co-cultures of the hematopoietic (mPB CD34+, AML) cells with stromal cells, as well as in co-cultures of steady-state BM CD34+ cells stimulated with G-CSF with stroma. In cultures of stromal cells alone G-CSF had no effect on the expression of either MT1-MMP or MMP-2. To evaluate the role of MT-MMPs in proMMP activation, we examined the leukemic KG-1 cell line, which we found not to express any MT-MMPs, and did not detect active MMP-2 in co-cultures with stroma. On the other hand, primary AML samples that did not express MT1-MMP but expressed MT2-, MT4- or MT5-MMPs activated proMMP-2 in co-cultures with stroma. Moreover, the MT1-MMP inhibitor epigallocatechin-3-gallate significantly reduced trans-Matrigel migration of mPB CD34+ (by 50%) and AML cells (by 70–90%). Hence we suggest that in the BM microenvironment (i) MT1-MMP localized on the surface of hematopoietic cells activates MMP-2, inducing a highly proteolytic microenvironment, and (ii) MT1-MMP upregulation by G-CSF in BM CD34+ cells can result in CD34+ cell mobilization from BM. Similarly, the constitutive high expression of MT1-MMP and other MT-MMPs in AML blasts also contributes to MMP-2 activation in the BM microenvironment and may be conducive to the egress of AML blasts from the BM.

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Synergy between AUUUA motif disruption and enhancer insertion results in autocrine transformation of interleukin-3-dependent hematopoietic cells

Blood ◽

10.1182/blood.v86.8.3139.3139 ◽

1995 ◽

Vol 86 (8) ◽

pp. 3139-3150 ◽

Cited By ~ 16

Author(s):

MW Mayo ◽

XY Wang ◽

PA Algate ◽

GF Arana ◽

PE Hoyle ◽

...

Keyword(s):

Mrna Stability ◽

Untranslated Region ◽

Stability Region ◽

Rank Order ◽

Hematopoietic Cells ◽

Transformation Process ◽

Wild Type ◽

Transcriptional Enhancer ◽

Interleukin 3 ◽

Autocrine Transformation

Abstract Previously, we characterized the transposition of an intracisternal type A particle (IAP) to the 32 untranslated region (UTR) of the interleukin-3 (IL-3) gene, which displaced two of the six AUUUA motifs associated with mRNA stability in an IL-3-secreting clone. To determine whether this rearrangement was involved in the autocrine transformation of the parental IL-3-dependent FL5.12 cell line, the germline (gIL-3) and rearranged IL-3 (rIL-3) genes were isolated and subcloned into a gene transfer vector. Moreover, the IAP-long terminal repeat (LTR) and the IL-3 32 UTR AUUUA motifs were deleted (rIL-3 + delta LTR and gIL-3 + delta AUUUA) in some IL-3 constructs to ascertain their role in the transformation process. The IAP-LTR was also added to these constructs (rIL-3 + delta LTR + IAP-LTR, gIL-3 + delta AUUUA + IAP-LTR, and gIL-3 + IAP-LTR), to determine whether it was necessary for autocrine transformation. The ability of the modified IL-3 genes to abrogate the IL-3 dependency of FL5.12 cells had the following rank order: rIL-3 was greater than rIL-3 + delta LTR + IAP-LTR, which was greater than gIL-3 + delta AUUUA + IAP-LTR, which was greater than gIL-3 + delta AUUUA, which was equal to rIL-3 + delta LTR, which was greater than gIL-3. The half-life of IL-3 mRNA was 20-fold longer in cells containing a mutated as opposed to a wild-type AUUUA region. All of the factor-independent cells that expressed the IL-3 transgenes secreted IL-3 and were tumorigenic after injection into BALB/c nude mice. These results indicated that two events could synergize in the autocrine transformation of hematopoietic cells: (1) addition of a transcriptional enhancer present in a retroviral LTR, and (2) disruption of an mRNA stability region.

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Small peptide analogs to stromal derived factor–1 enhance chemotactic migration of human and mouse hematopoietic cells

Experimental Hematology ◽

10.1016/j.exphem.2004.01.011 ◽

2004 ◽

Vol 32 (5) ◽

pp. 470-475 ◽

Cited By ~ 29

Author(s):

Ruikun Zhong ◽

Ping Law ◽

Donald Wong ◽

Ahmed Merzouk ◽

Hassan Salari ◽

...

Keyword(s):

Hematopoietic Cells ◽

Small Peptide ◽

Chemotactic Migration ◽

Peptide Analogs ◽

Human And Mouse

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Rap1 Gtpases Regulate the Retention and Engraftment of Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2018-99-117197 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 326-326

Author(s):

Baskar Ramdas ◽

Joydeep Ghosh ◽

Raghuveer Singh Mali ◽

Zollman Amy ◽

Nadia Carlesso ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Active Form ◽

Hematopoietic Cells ◽

Hematopoietic Stem ◽

Cell Counts ◽

Vascular Niche ◽

Stem And Progenitor Cells

Abstract Signaling molecules that control the homing and mobilization of hematopoietic stem and progenitor cells (HSC/Ps) are poorly understood. Rap1, a small-molecular-weight GTP-binding protein belongs to the Ras-like superfamily of GTPases and regulates several signal transduction cascades. Rap1 cycles between a GDP-bound inactive and a GTP-bound active form and exists in two isoforms - Rap1a and Rap1b, which have been implicated in the regulation of actin based functions in non-hematopoietic cells. Although Rap1 has been involved in regulating several hematologic disorders including chronic lymphocytic leukemia, myeloproliferative stem cell disorders, polycythemia vera and sickle cell anemia, its role in the development and function of HSC/Ps has not been investigated. We have generated a mouse model in which both Rap1a and Rap1b isoforms were conditionally deleted in HSC/Ps individually or in combination (double knockout; DKO). Our results demonstrate that deletion of both isoforms of Rap1 results in profound mobilization of primitive hematopoietic stem cells in peripheral blood. In the bone marrow, Rap1ab deficiency shows increased frequency of LSK cells, HPC-1 (LSK CD150-CD48+), HPC-2 (LSK CD150+CD48+) along with an increase in granulocyte-macrophage progenitor cell (GMP) population. Furthermore, spleen size and cellularity were significantly enhanced in DKO mice relative to controls. We hypothesized that Rap1 plays an essential role in regulating the retention of HSC/Ps in the bone marrow (BM) and that loss of Rap1 might inhibit the interaction of HSC/Ps with the BM niche cells, leading to egress of HSC/Ps and thus creating empty space(s) in the marrow for enhanced engraftment of donor derived cells when transplanted under non-myeloablative conditions. To test this, we performed BM transplantation using Rap1ab DKO mice as recipients and WT GFP expressing HSC/Ps as donors in the absence of any myeloablative conditioning. Our long-term engrfatment results showed significantly greater donor derived reconstitution of GFP positive cells in peripheral blood of DKO recipients compared to WT controls (WT: 19.2% vs DKO: 82.18% n=3, *p<0.05), suggesting that loss of Rap1ab creates functional open niche(s) in the BM due to mobilization of endogenous HSC/Ps. To better understand the mechanism behind this observation and to determine whether the GFP donor cells localize closer to the endosteal or vascular niche, we transplanted GFP positive cells into unconditioned (non-myeloablative) WT and Rap1ab DKO mice as described above. We measured the median distance of engrafted GFP cells from the bone surface and vasculature as a measure of proximity utilizing intravital microscopy. DKO recipients, transplanted with WT HSC/Ps preferentially localized to the vascular niche compared to control WT recipients (WT: 8µm vs DKO: 3 µm) and compared to osteoblastic niche, which was comparable in the two recipients, suggesting that GFP+ donor HSC/Ps preferentially localize and engraft near vascular niches providing indirect evidence to suggest that loss of Rap1ab leads to egress of hematopoietic cells from the vascular niche as opposed to osteoblastic niche. We next assessed the potential of Rap1ab deficient cells to engraft in a lethally irradiated host in a competitive repopulation assay. Rap1ab DKO HSC/Ps showed a defect in engraftment as well as multi-lineage reconstitution when transplanted into lethally irradiated hosts compared to WT controls. The defect in engraftment was largely due to impaired homing of DKO HSC/Ps. To assess which specific isoform of Rap1 is essential for mobilization and engraftment/homing of HSC/Ps, we induced deletion in Rap1a and Rap1b separately (single knock out mice) and assessed these mice for peripheral blood cell counts. We found no significant changes in the peripheral WBC counts in single Rap1a KO mice relative to controls; and only a modest increase in single Rap1b KO mice; suggesting that mobilization of HSC/Ps was relatively unperturbed in these mice and requires the loss of both isoforms of Rap1. In contrast, engraftment of HSC/Ps derived from the single KOs of Rap1a and Rap1b was impaired to the same extent as DKO HSC/Ps. These data suggest that loss of single Rap1 isoform contributes similarly to the engraftment of HSC/Ps, whereas the combined loss of both isoforms is required for efficient mobilization of HSC/Ps. Disclosures No relevant conflicts of interest to declare.

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Some Structural Features of Metaphase Chromosomes of Mice and Men

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060969 ◽

1967 ◽

Vol 25 ◽

pp. 190-191

Author(s):

Godfrey C. Hoskins ◽

Betty B. Hoskins

Keyword(s):

Electron Microscopy ◽

Electron Micrographs ◽

Structural Features ◽

Metaphase Chromosomes ◽

The Future ◽

Of Mice And Men ◽

Mouse Cells ◽

Human And Mouse

Metaphase chromosomes from human and mouse cells in vitro are isolated by micrurgy, fixed, and placed on grids for electron microscopy. Interpretations of electron micrographs by current methods indicate the following structural features.Chromosomal spindle fibrils about 200Å thick form fascicles about 600Å thick, wrapped by dense spiraling fibrils (DSF) less than 100Å thick as they near the kinomere. Such a fascicle joins the future daughter kinomere of each metaphase chromatid with those of adjacent non-homologous chromatids to either side. Thus, four fascicles (SF, 1-4) attach to each metaphase kinomere (K). It is thought that fascicles extend from the kinomere poleward, fray out to let chromosomal fibrils act as traction fibrils against polar fibrils, then regroup to join the adjacent kinomere.

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Binucleate Spermiogenesis in the Mouse

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010009381x ◽

1972 ◽

Vol 30 ◽

pp. 112-113

Author(s):

John J. Wolosewick

Keyword(s):

Electron Microscopy ◽

Phosphate Buffer ◽

Seminiferous Epithelium ◽

Mouse Testis ◽

Germinal Epithelium ◽

Intercellular Bridges ◽

Adult Mice ◽

Cervical Dislocation ◽

Human And Mouse

Classically, the male germinal epithelium is depicted as synchronously developing uninucleate spermatids conjoined by intercellular bridges. Recently, binucleate and multinucleate spermatids from human and mouse testis have been reported. The present paper describes certain developmental events in one type of binucleate spermatid in the seminiferous epithelium of the mouse.Testes of adult mice (ABP Jax) were removed from the animals after cervical dislocation and placed into 2.5% glutaraldehyde/Millonig's phosphate buffer (pH 7.2). Testicular capsules were gently split and separated, exposing the tubules. After 15 minutes the tissue was carefully cut into cubes (approx. 1mm), fixed for an additional 45 minutes and processed for electron microscopy.

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