University of Groningen
The functional characterization of microRNA-125 in hematopoiesis
Wójtowicz, Edyta Ewa
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Wójtowicz, E. E. (2016). The functional characterization of microRNA-125 in hematopoiesis [Groningen]:
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1
GENERAL INTRODUCTION
AND THE OUTLINE OF THE THESIS
HEMATOPOIESIS AND HEMATOPOIETIC STEM CELLS
Hematopoiesis is the hierarchical process of blood cell formation and development. It assures constant replenishment of differentiated cell types such as
erythrocytes, platelets, monocytes and macrophages, B and T-cells, that collectively contribute to organismal functioning. The continuous production
of blood cells throughout the lifetime of an organism is sustained by a small
population of hematopoietic stem cells (HSCs) that appear at the apex of the
hematopoietic hierarchy.
The field of hematopoietic stem cell biology became amenable to experimental perturbations thanks to pioneering work of Till and McCulloch, who
developed quantitative assays (Till and McCulloch, 1961). Their experiments
provided the first empirical proof of the activity and potential of -single- hematopoietic stem cells. They described stem cells as rare cells characterized by two
unique features: their ability to undergo self-renewal divisions to maintain their
numbers, and the potential to give rise to all types of blood and immune cells
during a step-wise process of differentiation.
Still it took another three decades to prospectively isolate a population
of long-term reconstituting stem cell using flow cytometry (Spangrude et al.,
1989; Visser et al., 1984), which paved the way to develop several alternative
strategies for HSC purification (discussed in more detail in the next Chapter).
To gain insight in the functional activity of hematopoietic stem cells, several in vitro and in vivo assays have been established to assess their properties. Transplantation of stem cells into irradiated recipients is the gold standard assay for the analysis of self-renewal and differentiation potential of HSC
(LORENZ et al., 1951). Usually these experiments last 6 months or longer to
reach steady state hematopoiesis and multilineage reconstitution Therefore, in
vitro approaches including colony forming unit cell (CFU) assays, cobblestone-area forming cell (CAFC) assays or long-term culture-initiating (LTC-IC) assays have been established to estimate the frequency and clonogenic potential of
hematopoietic stem and progenitor cells (HSPCs) (Bradley and Metcalf, 1966;
Ploemacher et al., 1991; van Os et al., 2008a). These are convenient and broadly used methods for screening and testing the effects of different drugs, agents,
or genetic modifications of HSPCs.
HEMATOPOIESIS AND HEMATOPOIETIC STEM CELLS   |
11
SMALL, NON-CODING RNAS - MICRORNA
It has become clear that only the small population of long-term hematopoietic
stem cells (LT-HSCs) contains the ability to sustain blood cell formation. Hematopoietic progenitors, that are thought to be only several cell divisions ‘away’
from their stem cell ancestors, have lost this unique potential. Thus, key gene
expression patterns must be operating in stem cells and their control mechanisms must be cell type specific. One of such mechanisms that regulates the
abundance of proteins is governed by small (~22 nucleotides long), non-coding
RNAs called microRNA (miRNA) (Bartel, 2004). The work described in this
thesis concerns experiments that aimed to elucidate the role of one specific
microRNA, microRNA-125, in controlling the functioning of hematopoietic
stem cells.
The first described miRNA - lin-4 in C. elegans (lin-4) was shown to temporarily decrease the expression of a target gene, lin-14, via extensive antisense
RNA-RNA interactions that regulate protein translation and affect development of the worm (Lee et al., 1993). Since then many groups studied the
process of miRNA biogenesis. MiRNAs are produced in a two-step process;
they are firstly transcribed by RNA polymerase II or III as a primary-miRNA
transcript that is gradually cleaved into a precursor-miRNA (pre-miRNA)
by Drosha, exported to the cytoplasm, and further processed by Dicer to
produce a double stranded miRNA. In the next step one miRNA strands is
incorporated into the miRNA Induced Silencing Complex (miRISC) providing the specificity to recognize and target mRNA transcripts (Fabian et al.,
2010). There are over 3700 annotated miRNAs in the human genome and
many others are expected to be discovered by profiling of various tissues, since
the expression of miRNAs is spatiotemporally controlled (Lewis et al., 2005;
Londin et al., 2015). Each miRNA can potentially bind hundreds of coding
mRNA transcripts, generating a network of interacting partners. However,
to date only a small fraction of these predicted miRNA-mRNA interactions
have been confirmed, and many interactions are expected to be highly cell
type dependent (Londin et al., 2015). Experimental data generated throughout the last decade resulted in the establishment of the following canonical
rules of miRNA-target interactions (Bartel, 2009a):
12
|   CHAPTER 1 | GENERAL INTRODUCTION AND THE OUTLINE OF THE THESIS
1. Interactions are mediated by the ‘seed’ region of miRNA, a 6-8 nucleotide long, highly conservative sequence at the 5` end of the miRNA that forms
Watson-Crick and G-U pairs with the target.
2. Pairing between nucleotides outside of the seed region are reported to
stabilize the interaction but do not influence the miRNA efficacy of target repression (Garcia et al., 2011; Grimson et al., 2007a).
3. The majority of functional miRNA target recognition sites are localized within the 3`untranslated region (UTR) of protein coding genes (Grimson
et al., 2007a).
Seed sequence containing targets cover ~80% of the described interactions,
while the remaining are based on non-seed region pairing (Didiano and Hobert,
2008; Elefant et al., 2011) . Nevertheless, around 60% of seed interactions are
non-canonical and contain bulged or mismatched nucleotides (Helwak et al.,
2013). The short motif of the seed region, together with specific, non-seed base
pairing, leads to a high false discovery rates of predictions that are purely based
on bioinformatics (Yue et al., 2009). Therefore, experimental data are required
to decipher targets of miRNAs in various tissues at specific stage of the development.
A profound role for miRNAs in the regulation of stem cells has been shown
for the hematopoietic system. Deletion of the key enzyme involved in miRNA
processing (Dicer) in HSPCs led to increased apoptosis and reduced repopulation potential in serial transplantation experiments (Guo and Scadden, 2010).
In parallel, several laboratories independently performed miRNA profiling
of HSPCs and have identified a conserved cluster of miRNAs consisting of
miR-99b, let-7e and miR-125a that is preferentially expressed in HSPCs while
its abundance decreases in differentiated blood cell types (Gerrits et al., 2012;
Guo and Scadden, 2010; O’Connell et al., 2010). Importantly there are three
members of the miR-125 family, namely miR-125a, -b1 and –b2, located on
mouse chromosomes 17, 9, and 16, respectively. This suggests complex regulation of their expression, which may be dependent on DNA flanking sequences
containing regulatory elements and transcription factor binding sites in the
proximity of miRNA genes (Kim et al., 2012, Zhou et al., 2009) or SNP in
proximity of miR-125a (Chapiro et al., 2010). In recent years accumulating evidence confirms a crucial role for this microRNA cluster in hematopoiesis. HowSMALL, NON-CODING RNAS - MICRORNA   |
13
ever, contradictory reports have claimed opposite functions of miR-125 family
members in hematopoiesis, either increasing self-renewal or rather enhancing
differentiation (Gerrits et al., 2012; Guo and Scadden, 2010; O’Connell et al.,
2010; Ooi et al., 2010). Importantly, miR-125 has been either described as
a tumor suppressor in chronic lymphocytic leukemia (Tili et al., 2012) but also
as an ‘oncomiR’ promoting myeloid leukemia (Bousquet et al., 2010) or B-cell
leukemia (So et al., 2014). In this thesis we aimed to investigate in depth the
role of the miR-125 family in regulating the potential of hematopoietic stem
cells to self-renew or differentiate.
Chapter 2 reviews the current literature on HSC purification methods,
functional and molecular changes that occur upon aging and describes perspectives on the putative role of long or small non-coding RNAs to hematopoietic
stem cell ageing process.
Chapter 3 provides an introduction to the role of miRNAs in hematopoiesis. This Chapter further describes miRNAs that regulate key transcription factors influencing lineage commitment, and reviews how miRNAs can drive oncogenesis, or cooperate with other genes in various hematological malignancies.
Chapter 4 includes experimental data on the role of the various miR-125
family members in hematopoiesis. As stated above, there are three functional
microRNA-125 genes encoded in the human and mouse genome. It has remained unclear what is the respective role of each of these family members. Our
studies described in this Chapter document a similar phenotype of miR-125a,
-b1 and –b2 overexpression (OE) in HSPCs. All three family members provide
a proliferative advantage to hematopoietic cells, skew differentiation towards
the myeloid lineage and in some cases lead to development of myeloproliferative disorders (MPN). Overall these data support the hypothesis that the shared
seed sequence is the main contributor to effects induced by members of the
miR-125 family in HSPCs.
The experiments described in Chapter 5 were aimed to assess whether microRNA-125a can induce self-renewal and multilineage repopulating potential in progenitors, cells that are normally devoid of such activity. Indeed, we
provide evidence that increased miR-125a levels can induce HSC properties
and activity in murine progenitors that normally lack these features. We further show that the activity of microRNA-125a is conserved between mouse
14
|   CHAPTER 1 | GENERAL INTRODUCTION AND THE OUTLINE OF THE THESIS
and human cord blood HSC that ectopically express miR-125a. We performed
multiple proteomics screens to identify miR-125 targets, and identified MAPK
signaling as a one of the most important contributors to the observed effect.
Chapter 6 summarizes the experimental findings described in previous
chapters and discusses their potential impact.
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|   CHAPTER 1 | GENERAL INTRODUCTION AND THE OUTLINE OF THE THESIS