160 research outputs found
The Role of Translation Initiation Regulation in Haematopoiesis
Organisation of RNAs into functional subgroups that are translated in response to extrinsic and intrinsic factors underlines a relatively unexplored gene expression modulation that drives cell fate in the same manner as regulation of the transcriptome by transcription factors. Recent studies on the molecular mechanisms of inflammatory responses and haematological disorders indicate clearly that the regulation of mRNA translation at the level of translation initiation, mRNA stability, and protein isoform synthesis is implicated in the tight regulation of gene expression. This paper outlines how these posttranscriptional control mechanisms, including control at the level of translation initiation factors and the role of RNA binding proteins, affect hematopoiesis. The clinical relevance of these mechanisms in haematological disorders indicates clearly the potential therapeutic implications and the need of molecular tools that allow measurement at the level of translational control. Although the importance of miRNAs in translation control is well recognised and studied extensively, this paper will exclude detailed account of this level of control
Molecular characterization of translocation (6;9) in acute nonlymphocytic leukemia
Specific chromosomal translocations are one of the defects associated
with leukemia. Isolation and characterization of genes affected by these
translocations may give insight into the processes of both leukemogenesis
and normal hematopoiesis. When the experiments described in this thesis
were started, several genes involved in translocations in lymphoid
leukemia were isolated. These genes were all translocated into T-cell
receptor and Immunoglobulin loci, which deregulated their expression. In
myeloid leukemia only translocation (9;22) was characterized molecularly
and the resulting bcr-abl gene was the only fusion gene known. Chapter 1
gives an overview of what is known to date about genes involved in
leukemogenesis. To extend the research on the molecular characterization
of translocations in myeloid leukemia, we decided to clone and
characterize the translocation breakpoints of t(6;9) that characterizes a
subtype of acute myeloid leukemia. Chapter 2 gives an introduction to
t(6;9) AML and reports the results of our investigations. Chapter 3
discusses these results in relation to our current understanding of
leukemogenesi
Erythropoiesis and Megakaryopoiesis in a Dish
Erythrocytes and platelets are the major cellular components of blood. Several hereditary diseases affect the production/stability of red blood cells (RBCs) and platelets (Plts) resulting in anemia or bleeding, respectively. Patients with such disorders may require recurrent transfusions, which bear a risk to develop alloantibodies and ultimately may result in transfusion product refractoriness. Cell culture models enable to unravel disease mechanisms, and to screen for alternative therapeutic products. Besides these applications, the ultimate goal is the large-scale production of blood effector cells for transfusion. Cultured RBCs that lack many of the common blood group antigens and Plts-lacking HLA expression would improve transfusion practice. Large numbers of RBCs and Plts can already be generated using hematopoietic stem cells derived from fetal liver, cord blood, peripheral blood, and bone marrow as starting material for cell culture. The recent advances to generate blood cells from induced pluripotent stem cells provide a donor-independent, immortal primary source for cell culture models. This enables us to study developmental switches during erythropoiesis/megakaryopoiesis and provides potential future therapeutic applications. In this review, we will discuss how erythropoiesis and megakaryopoiesis are mimicked in culture systems and how these models relate to the in vivo process
The Shape Shifting Story of Reticulocyte Maturation
The final steps of erythropoiesis involve unique cellular processes including enucleation and reorganization of membrane proteins and the cytoskeleton to produce biconcave erythrocytes. Surprisingly this process is still poorly understood. In vitro erythropoiesis protocols currently produce reticulocytes rather than biconcave erythrocytes. In addition, immortalized lines and iPSC-derived erythroid cell suffer from low enucleation and suboptimal final maturation potential. In light of the increasing prospect to use in vitro produced erythrocytes as (personalized) transfusion products or as therapeutic delivery agents, the mechanisms driving this last step of erythropoiesis are in dire need of resolving. Here we review the elusive last steps of reticulocyte maturation with an emphasis on protein sorting during the defining steps of reticulocyte formation during enucleation and maturation
EZH2-dependent chromatin looping controls INK4a and INK4b, but not ARF, during human progenitor cell differentiation and cellular senescence
<p>Abstract</p> <p>Background</p> <p>The <it>INK4b-ARF-INK4a </it>tumour suppressor locus controls the balance between progenitor cell renewal and cancer. In this study, we investigated how higher-order chromatin structure modulates differential expression of the human <it>INK4b-ARF-INK4a </it>locus during progenitor cell differentiation, cellular ageing and senescence of cancer cells.</p> <p>Results</p> <p>We found that <it>INK4b </it>and <it>INK4a</it>, but not <it>ARF</it>, are upregulated following the differentiation of haematopoietic progenitor cells, in ageing fibroblasts and in senescing malignant rhabdoid tumour cells. To investigate the underlying molecular mechanism we analysed binding of polycomb group (PcG) repressive complexes (PRCs) and the spatial organization of the <it>INK4b-ARF-INK4a </it>locus. In agreement with differential derepression, PcG protein binding across the locus is discontinuous. As we described earlier, PcG repressors bind the INK4a promoter, but not ARF. Here, we identified a second peak of PcG binding that is located ~3 kb upstream of the <it>INK4b </it>promoter. During progenitor cell differentiation and ageing, PcG silencer EZH2 attenuates, causing loss of PRC binding and transcriptional activation of <it>INK4b </it>and <it>INK4a</it>. The expression pattern of the locus is reflected by its organization in space. In the repressed state, the PRC-binding regions are in close proximity, while the intervening chromatin harbouring <it>ARF </it>loops out. Down regulation of EZH2 causes release of the ~35 kb repressive chromatin loop and induction of both <it>INK4a </it>and <it>INK4b</it>, whereas <it>ARF </it>expression remains unaltered.</p> <p>Conclusion</p> <p>PcG silencers bind and coordinately regulate <it>INK4b </it>and <it>INK4a</it>, but not <it>ARF</it>, during a variety of physiological processes. Developmentally regulated EZH2 levels are one of the factors that can determine the higher order chromatin structure and expression pattern of the <it>INK4b-ARF-INK4a </it>locus, coupling human progenitor cell differentiation to proliferation control. Our results revealed a chromatin looping mechanism of long-range control and argue against models involving homogeneous spreading of PcG silencers across the <it>INK4b-ARF-INK4a </it>locus.</p
Igbp1 is part of a positive feedback loop in stem cell factor–dependent, selective mRNAtranslation initiation inhibiting erythroid differentiation
The authors thank Dr Victor de Jager for assistance with the Rosetta
Resolver software; Dr Ivo Touw for many fruitful discussions and
critical reading of the manuscript; Liu Wing for technical assistance;
Drs Peter Seither, Andreas Weith (Boehringer Ingelheim,
Biberach, Germany), Helmuth Dolznig, Thomas Waerner, and
Sandra Pilat (IMP, Vienna, Austria) for mRNA profiling of
erythroblasts, of which the complete data will be published
elsewhere; Dr Bart Aarts (Erasmus MC, Rotterdam, The Netherlands)
for assistance in confocal scanning microscopy; Dr David Brautigan
(University of Virginia, Charlottesville) for anti-Igbp1 antibodies;
Dr Manfred Boehm (National Institutes of Health/National
Heart, Lung, and Blood Institute, Bethesda, MD) for anti-Uhmk1
antibodies; and Ortho-Biotech (Tilburg, The Netherlands) for their
kind gift of Eprex (erythropoietin).Stem cell factor (SCF)–induced activation
of phosphoinositide-3-kinase (PI3K) is required
for transient amplification of the
erythroblast compartment. PI3K stimulates
the activation of mTOR (target of
rapamycin) and subsequent release of
the cap-binding translation initiation factor
4E (eIF4E) from the 4E-binding protein
4EBP, which controls the recruitment of
structured mRNAs to polysomes. Enhanced
expression of eIF4E renders proliferation
of erythroblasts independent of
PI3K. To investigate which mRNAs are
selectively recruited to polysomes, we
compared SCF-dependent gene expression
between total and polysome-bound
mRNA. This identified 111 genes primarily
subject to translational regulation. For
8 of 9 genes studied in more detail, the
SCF-induced polysome recruitment of
transcripts exceeded 5-fold regulation and
was PI3K-dependent and eIF4E-sensitive,
whereas total mRNA was not affected by
signal transduction. One of the targets,
Immunoglobulin binding protein 1 (Igbp1),
is a regulatory subunit of protein phosphatase
2A (Pp2a) sustaining mTOR signaling.
Constitutive expression of Igbp1
impaired erythroid differentiation, maintained
4EBP and p70S6k phosphorylation,
and enhanced polysome recruitment
of multiple eIF4E-sensitive mRNAs.
Thus, PI3K-dependent polysome recruitment
of Igbp1 acts as a positive feedback
mechanism on translation initiation underscoring
the important regulatory role of
selectivemRNArecruitment to polysomes
in the balance between proliferation and
maturation of erythroblasts. (Blood. 2008;
112:2750-2760)peer-reviewe
The potential role of the homeobox gene, Hhex in haematopoietic progenitor expansion
Background: The decision of an erythroid progenitor
to proliferate or differentiate is regulated at the level of (i)
transcription; (ii) recruitment of transcripts to polysomes
for protein synthesis and (iii) signal transduction activating
functional effectors. We utilized a factor sensitive erythroid
progenitor cell model to study the gene expression profile of
cells under proliferative signals. We have shown previously
that translation control is an extremely important level of
regulation that controls the balance between proliferation
and differentiation of erythroid progenitors. This led us to
investigate those transcripts that are shifted to polysomes in
cells stimulated by erythropoietin (Epo) or stem cell factor
(SCF).peer-reviewe
Loss of Ercc1 Results in a Time- and Dose-Dependent Reduction of Proliferating Early Hematopoietic Progenitors
The endonuclease complex Ercc1/Xpf is involved in interstrand crosslink repair and functions downstream of the Fanconi pathway. Loss of Ercc1 causes hematopoietic defects similar to those seen in Fanconi Anemia. Ercc1−/− mice die 3-4 weeks after birth, which prevents long-term follow up of the hematopoietic compartment. We used alternative Ercc1 mouse models to examine the effect of low or absent Ercc1 activity on hematopoiesis. Tie2-Cre-driven deletion of a floxed Ercc1 allele was efficient (>80%) in fetal liver hematopoietic cells. Hematopoietic stem and progenitor cells (HSPCs) with a deleted allele were maintained in mice up to 1 year of age when harboring a wt allele, but were progressively outcompeted when the deleted allele was combined with a knockout allele. Mice with a minimal Ercc1 activity expressed by 1 or 2 hypomorphic Ercc1 alleles have an extended life expectancy, which allows analysis of HSPCs at 10 and 20 weeks of age. The HSPC compartment was affected in all Ercc1-deficient models. Actively proliferating multipotent progenitors were most affected as were myeloid and erythroid clonogenic progenitors. In conclusion, lack of Ercc1 results in a severe competitive disadvantage of HSPCs and is most deleterious in proliferating progenitor cells
Stem cell factor induces phosphatidylinositol 3'-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells
Stem cell factor (SCF) has an important role in the proliferation,
differentiation, survival, and migration of hematopoietic cells. SCF
exerts its effects by binding to cKit, a receptor with intrinsic tyrosine
kinase activity. Activation of phosphatidylinositol 3'-kinase (PI3-K) by
cKit was previously shown to contribute to many SCF-induced cellular
responses. Therefore, PI3-K-dependent signaling pathways activated by SCF
were investigated. The PI3-K-dependent activation and phosphorylation of
the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported.
The study shows that Tec and Dok-1 form a stable complex with Lyn and 2
unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and
the SH2 domain of Tec were identified as being required for the
interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the
association with Tec. In addition, Tec and Lyn were shown to phosphorylate
Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2
domains of several signaling molecules activated by SCF, including Abl,
CrkL, SHIP, and PLCgamma-1, but not those of Vav and Shc. These findings
suggest that p62Dok-1 may function as an important scaffold molecule in
cKit-mediated signaling
FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1
Erythropoiesis requires tight control of expansion, maturation, and survival of erythroid progenitors. Because activation of phosphatidylinositol-3-kinase (PI3K) is required for erythropoietin/stem cell factor–induced expansion of erythroid progenitors, we examined the role of the PI3K-controlled Forkhead box, class O (FoxO) subfamily of Forkhead transcription factors. FoxO3a expression and nuclear accumulation increased during erythroid differentiation, whereas untimely induction of FoxO3a activity accelerated differentiation of erythroid progenitors to erythrocytes. We identified B cell translocation gene 1 (BTG1)/antiproliferative protein 2 as a FoxO3a target gene in erythroid progenitors. Promoter studies indicated BTG1 as a direct target of FoxO3a. Expression of BTG1 in primary mouse bone marrow cells blocked the outgrowth of erythroid colonies, which required a domain of BTG1 that binds protein arginine methyl transferase 1. During erythroid differentiation, increased arginine methylation coincided with BTG1 expression. Concordantly, inhibition of methyl transferase activity blocked erythroid maturation without affecting expansion of progenitor cells. We propose FoxO3a-controlled expression of BTG1 and subsequent regulation of protein arginine methyl transferase activity as a novel mechanism controlling erythroid expansion and differentiation
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