Granulopoieis (continued from Part 1)
In addition to GATA consider…. (specifically note as an example of how the system
is integrated miR-196 which we can then bring back up when note GIF-1
downstream)
Self-renewal and early stem cell/progenitor differentiation related microRNAs
1. miR-125 family is crucial in balance of self-renewal and differentiation
balance and are highly expressed in HSPC populations and decreases with
increased differentiation  but exact targets are unknown. Data suggests
that they may target proapoptotic genes and p53-related genes (blockage of
which by the miR-125 family thus inhibits apoptosis)
2. miR-196 family found within the HOX gene (TF proteins with a role in
hematopoiesis). The expression of miR-196 genes and HOX genes are
correlated and both increased in HSPC populations. HOX genes are
upregulated during myeloid differentiation. MiR-196b appears to regulate
genes involved in cell survival and proliferation while maintaining cells in the
undifferentiated state by suppressing differentiation-related gene
expression, such as HOX. GFI-1 (a TF protein which acts to repress
transcription , ie of PU.1) which is required for downstream myeloid
differentiation toward granulopoiesis also down-regulates miR-196b
expression effectively promoting myeloid differentiation
3. miR-17-92 cluster is transcribed as one non-coding RNA that is then
processed into seven different mi-RNAs  four of them (miR-19a, miR-19b,
miR-20a, and miR-20b if overexpressed lead to stem cell expansion.
Under the influence of the combination of factors such as IL-3 (derived from
activated T-lymphocytes) and GM-CSF (derived from marrow stromal cells),
the pleuripotent stem cell proliferates and differentiates toward the common
myeloid progenitor cell.
The common myeloid progenitor cell (aka CFU-GEMM) is as the name suggests
committed to the myeloid lineage. However, it still has the potential to differentiate
in any number of ways (red cells, platelets, monocytes, neutrophils, eosinophils, or
basophils). The only door closed at this point technically is that to the lymphoid
lineage. Under added influence of G-CSF differentiation continues toward the
combined granulocyte-monocyte committed progenitor (CFU-GM), i.e. even less
potential options for differentiation.
The process is incredibly complex in terms of the orchestration of the continued
proliferation and differentiation under the combined influences of growth factors,
microRNA, and transcription factors. The relative contribution of each of these is
unclear though they are no doubt interrelated.
In terms of the transcription factors keep the following in mind throughout…
1. Those that influence the HSC and are involved in the differentiation
toward any/all lineages, such as
a. Stem cell factor (SCL)
b. GATA2 (see above)
c. RUNX-1 [aka AML-1 or core binding factor- (CBF-)]
2. Those that are promoting lineage-specific gene expression and
differentiation BUT ALSO suppressing alternative lineage pathways
a. GATA-1
b. PU.1
c. CEBP-
From Hematology: Basic Principles and Practice (6th Edition)
With the added influence of G-CSF, expression of RUNX1 increases which in turn
leads to the increased expression of PU.1 and together the increased expression of
M-CSF receptors. Similarly, expression of CEBP-a increases. CEBP-a acting in
collaboration with PU.1 promotes the differentiation of the CFU-GEMM to the CFUGM concurrently with the further increased expression of GM-CSF, M-CSF, and GCSF receptors.
NOTE: A counter balancing cross talk is operating between two major TFs in
myeloid differentiation—PU.1 and GATA-1. Both inhibit the transcriptional activity
and expression of the other. How this balance plays out is dependent upon the
relative balance existing between the two.
But, if GATA-1 expression is relatively increased in relation to the PU.1 expression,
the GATA-1 exerts an inhibitory effect on further PU.1 expression. Similarly, if GIF-1
expression is increased, GIF-1 also represses further PU.1 expression. Additionally,
increased CEBP-a expression represses c-JUN expression. With relatively decreased
PU.1 expression and decreased c-JUN expression in the CFU-GM, the monocyte
differentiation genes are repressed (i.e. the genes appear to require higher presence
of PU.1 for their transcription activation). By contrast, the granulocyte
differentiation genes appear to be regulated at lesser levels of PU.1 activity. Thus,
with relatively decreased PU.1 expression differentiation and maturation continues
toward the neutrophil.
However, if GATA-1 expression is relatively decreased in relation to the levels of
PU.1 expression, the PU.1 exerts and inhibitory effect on further GATA-1 expression.
The increased PU.1 combined with RUNX-1 activates and promotes more PU.1 gene
expression along with the further expression of M-CSF receptors. With increased
expression of Egr/Nab transcription factors, the expression of GIF-1 and CEBP-a is
repressed. This allows for a further increase in the expression of PU.1. Collectively,
the result is an increasing expression of PU.1 in the differentiating cell. The higher
relative concentrations of PU.1 promotes the expression of the monocyte
differentiation and maturation genes. Thus, under these conditions the CFU-GM
differentiates along the terminal monocyte pathways.
From Hematology: Basic Principles and Practice (6th Edition)
Let’s consider further one of the critical TF proteins in hematopoiesis, PU.1
Epigenetic Control of Hematopoiesis: The PU.1 Chromatin Connection
PU.1 = Purine-rich box 1 is the product of the SPI-1 human gene (on chromosome
11) and is a TF protein which plays an essential role in development of most
hematopoietic cell lines (as well as in leukemia suppression).
Structure
1. Member of the ETS TF family (share and ETS-domain that facilitates
binding to the core GGAA motif)
2. Next to the ETS domain is a winged helix domain that mediates binding
to an extended consensus longer then the ETS-motif
3. Activation domain
4. PEST domain (protein-protein interactions)
Functions:
1. Dysregulation of PU.1 leads to functional HSC-depletion in mouse
models.
2. It is involved in myeloid vs. lymphoid differentiation branch point
decisions.
3. It also is involved in maturation of macrophages, B-cells, early T-cell
progenitors and T-helper 9 cells.
4. Suppression of myeloid leukemia development
Molecular Functions of PU.1
PU.1 can both activate and repress gene transcription. In PU.1 knock-out model in HSC
 225 genes were up-regulated and 97 were down-regulated! How? Works via complex
networks of protein partners.
PU.1 is a key factor for initiating and maintaining transcriptionally permissive chromatin.
But only ~1% of possible binding sites are occupied at any one time. How does the
system regulate/select for binding among eligible sites? Not clearly understood but
current thinking guided by some observations…
1. Sites with the highest affinity for PU.1 binding tend to be located in gene
deserts lacking obvious binding motifs for other TF proteins and typically in
less conserved areas of the genome—the biological significance of this fact is
uncertain.
2. The functional binding sites for PU.1 located in enhancers or promoters are
generally of lower binding affinity and always flanked by binding site for 1 or
more other TF proteins.
Thus, functional binding site selection seems to rely on collaborative lower affinity
combination/complex binding. This likely serves to (a) stabilize PU.1 binding to DNA
and/or (b) enhance transcription activating functions. In many cases it takes the total
collaborative complex effort of the TF proteins to maintain the open chromatin structure.
This may relate also to the means by which the PU.1 penetrates the chromatin at different
binding sites to reach the DNA?
1. In higher affinity sites, it appears that PU.1 probably just competes with
nucleosomes for DNA binding
2. But at lower affinity binding sites, PU.1 probably cannot access the site alone
which appear to be opened first
Thus companion TF proteins influence PU.1 ability to penetrate the chromatin and bind
DNA…
1. RUNX-1 and CEBP enhance PU.1 binding to DNA (appear to prime and open
the chromatin before PU.1 binds)
2. NF-kB also enhances PU.1 binding to DNA
PU.1 expression level also contributes  more binding sites and especially more lower
affinity binding sites are occupied with increasing PU.1 concentrations.
Post-translational modifications of PU.1 (acetylation, etc) may also influence PU.1
binding?
In in vitro studies… thus unclear which if any exist in vivo.
1. TATA-binding protein (TBP).
a. Through TBP, PU.1 recruits basal transcriptions machinery to its
binding sites at promoters and enhancers
b. Many PU.1 target genes lack TATA box sequences thus cannot
themselves bind TBP and rely on PU.1 to do so.
2. Histone acetyltransferases such as CREB-binding protein (CBP) and p300
a. Acetylation of histone tails is thought to open chromatin  allows
other proteins to bind to the DNA and activate transcription
3. Histone deacetylase complex [a complex of histone deacetylase 1 (HDAC-1)
and mammalian Sin3a (mSIN3a)]
a. PU.1 combined with HDAC1-mSIN3a can also bind MeCP2 (methyl
CpG-binding protein 2) to repress genes
4. DNA methyltransferases (Dnmt3a and Dnmt3b) believed to site-specifically
methylate DNA leading to repression of transcription
a. PU.1 with Dnmt3b interactions appear to occur in monocyte to
osteoclast differentiation.
PU.1 cross-talks with other lineage specific TF proteins in blood development.
1. Early stages, i.e. HSC and multipotent progenitor cells
a. CCAAT-enhancer binding proteins (CEBP)
i. CEBP- together with PU.1 regulates genes encoding for M-CSF
receptor and GM-CSF receptor-.
b. Runt-related transcription factor 1 (aka RUNX1, AML-1, or CBF-) is
important in the development of the HSC pool
i. Runx1 together with PU.1 leads to activation of the Csf1r gene
encoding for M-CSF.
ii. Runx1 together with PU.1 leads to activation of the Sfpi1 gene
encoding for PU.1 itself
2. In myeloid specific cells, PU.1 is required for guiding differentiation of early
progenitors and with dynamically changing partner proteins helps
orchestrate the step-wise process.
a. CCAAT-enhancer binding proteins (CEBP)
i. CEBP- together with PU.1 is required for the transition from
CMP (common myeloid progenitor) to GMP (granulocyte
macrophage progenitor) but NOT thereafter
ii. CEBP- together with PU.1 is involved with terminal myeloid
differentiation
iii. CEBP- or CEBP- together with high levels of PU.1 expression
can convert other lineages to macrophages?
b. Growth Factor Independent-1 (GFI-1, a TF protein which in complex
with PU.1 acts as a repressor of transcription)
i. GFI-1 represses macrophage-specific PU.1 target genes BUT
NOT PU.1 granulocyte-specific target genes. This is thought to
be related to the fact that higher PU.1 expression levels are
needed for macrophage differentiation.
c. GATA-binding factor 1 (GATA-1) expressed in erythroid and
megakaryocytic lineages.
i. GATA-1 is key driver of erythroid lineage differentiation.
ii. GATA-1 ectopic expression in GMPs can even to a point
reprogram GM progenitors toward erythroid cells.
iii. PU.1 binds GATA-1 and physically prevents GATA-1 from being
able to bind DNA
iv. PU.1 expression is down-regulated at the erythroblast
stage of differentiation and failure to do so leads to
erythroid maturation arrest and subsequently erythroid
leukemia.
1. Thus terminal differentiation of erythroblasts requires
PU.1 transcriptional activity be repressed by GATA-1
a. Inhibit binding of co-activators of PU.1
b. Block transcription of the Sfpi-1 gene  thus
down-regulating PU.1 expression
3. In lymphoid lineage, PU.1 is also involved with maturation-differentiation
a. NOTCH represses PU.1 activity in early T-cells (like GATA-1 does in
erythroid cells), which allows the thymocytes to be further pushed
into maturation.
b. Runx1 binding to a silencer element (located between the Sfpi-1
promoter and its upstream enhancer elements) also contributes to the
repression of PU.1 in T-cells
PU.1 and other TF (such as RUNX1) appear to be important in DNA looping, a
process which allows the distal regulatory sequences (enhancers, insulators, and
silencers) to loop back to the proximal promoters despite the long genomic distances
between the regions. Not clear how but this may be a more global function of PU.1 and
may be dependent upon the TF protein partnerships, as well.
Granulopoiesis Regulating microRNAs
1. miR-223. CEPB is a very potent activator of miR-223 expression. Thus leads
to repression o f the erythroid TF protein, NFI-A.
Erythropoiesis
Erythropoiesis is the process by which red blood cell differentiation and maturation
occurs. As with all of the hematopoietic cells, the erythrocytes derive form the
pleuripotent hematopoietic stem cell. As seen yesterday with granulopoiesis, under
the influence of the combination of factors such as IL-3 (derived from activated Tlymphocytes) and GM-CSF (derived from marrow stromal cells), the pleuripotent
stem cell proliferates and differentiates toward the common myeloid progenitor cell
(CFU-GEMM).
There is no doubt that the process is incredibly complex in terms of the orchestration of
the continued proliferation and differentiation under the combined influences of growth
factors, microRNA, and transcription factors. The relative contribution of each of these is
unclear though they are no doubt interrelated. But, the exact driver(s) of erythropoiesis at
the level of the committed progenitor remains unknown.
In granulopoiesis, the influence of G-CSF contributes to an increase expression of TFs
that lead toward the granulocyte-monocyte committed progenitor. In erythropoiesis, an
increased expression of GATA-1 in conjunction with its cofactor FOG1 (a.k.a. friend of
GATA) and without a concurrently increased CEBP- expression contributes to
differentiation toward the erythroid-megakaryocyte committed progenitor. Additionally,
GATA-1 represses the expression of PU.1—a transcription factor that plays a critical role
in myeloid gene expression and differentiation.
There is a growing body of evidence looking at the role of microRNAs in influencing
cellular differentiation and development. As an example, mi-RNA150 expression
influences the differentiation of the combined megakaryocyte-erythroid precursor
toward one or the other committed lineage pathways. Mi-RNA150 binds to the
3’UTR region of Myb mRNA, which in turn leads to decreased Myb protein
expression. Decreased Myb expression leads to increased megakaryocyte
differentiation (and decreased erythroid differentiation). Conversely, decreased miRNA150 binding leads to increased Myb protein expression and decreased
megakaryocyte differentiation (and increased erythroid differentiation). There are
many other miRNAs that have been identified that play a variety of roles in
erythropoiesis, globin gene expression, RBC enucleation, etc.
Erythropoietin
A major humoral regulator of erythropoiesis is erythropoietin (EPO). Though
erythropoietin receptors are found on blast forming units – erythroid (BFU-E), these
cells are more reliant on GM-CSF, IL-3, IL-6, IGF-1 and glucocorticoids for their
growth. Colony forming units—erythroid (CFU-E) have EPO receptors, and it is at
the CFU-E stage and beyond that EPO exerts its influence. Thus, EPO influences only
the final roughly two days of erythroid development from a CFU-E toward a mature
erythrocyte. As such, at times of significant strain on the erythroid system, there
may be an inadequate number of CFU-E to respond to the needs even under high
EPO conditions. How the body regulated the response to generate more CFU-E is not
clear.
During the final two days of erythroid maturation under the influence of
erythropoietin, the erythroid cells completes several developmental steps including
the following:
1. The erythroid cells undergo a series of rapid cellular divisions and express
surface transferrin receptors (allowing the uptake of iron necessary for final
hemoglobin production).
2. The erythrocyte loses its EPO-receptors. Thus, the cell is no longer under the
influence of erythropoietin but rather directed by interactions between
fibronectin and the erythrocyte’s surface a4b1 integrin.
3. Finally, the erythrocyte represses all further gene transcription and
condenses its chromatin before the red cell is enucleated.
Erythropoietin Regulation
EPO is primarily released from the kidney. It should be noted that the body does not
regulate erythropoiesis by monitoring the actual red blood cell mass. There is no
means to actually count the number of cells coursing through the body. Thus,
erythropoiesis is not specifically increased in response to anemia (too few red blood
cells) nor decreased in response to erythrocytosis a.k.a. polycythemia (too many red
blood cells).
Instead, erythropoietin production by the kidney is in response to oxygen delivery,
specifically to hypoxia. If the kidneys sense decreased oxygen delivery, production
of erythropoietin increases resulting in increased erythropoiesis within the bone
marrow (see figure below). Thus, when one is anemic and tissue oxygen delivery is
diminished. This regulatory mechanism works nicely to try to restore the red blood
cell mass. But, how does hypoxemia regulate the production of erythropoietin?
Hypoxia Inducible Factor-
The production of erythropoietin in response to hypoxia is regulated by hypoxia
inducible factor alpha (HIF-). Under normal oxygen conditions, HIF- undergoes
hydoxylation by prolyl hydoxylases (PHDs). Hydroxylated HIF- is then bound to
the von Hippel-Lindau tumor suppressor protein (VH), which is the substratebinding unit of an E3 ubiquitin ligase complex. Ubiquitinization leads to rapid decay
of the HIF protein by the ubiquitin proteasome pathway.
Conversely, under hypoxic conditions, HIF- escapes the hydoxylation and is thus
stabilized and transported to the nucleus where it dimerizes with ARNT (aryl
hydrocarbon receptor nuclear translocator a.k.a. HIF-1). In conjunction with
additional transcriptional co-activators, such as CBP/p300, the complex then
induces transcription of the hypoxia-response element (HRE)-related genes. The
HRE-related genes include the genes for erythropoietin, as well as vascular
endothelial growth factor (VEGF), the VEGF-receptor, the glucose transporter, and
glycolytic enzymes (see figure below).
NOTE: Note the differences by which the body is regulating the production of
platelets and red cells. Megakaryopoiesis is trying to maintain a consistent platelet
mass. Erythropoiesis is trying to maintain tissue oxygenation.
The Erythropoietin Receptor
Erythropoietin (EPO) exerts its effect via the EPO-receptors located on the cell
surface of erythroid precursors. Associated with the EPO receptor’s proximal
cytoplasmic portion is a positive regulatory domain that interacts with the Janus 2
tyrosine kinase (JAK2). Upon binding of EPO, the EPO receptor dimerizes and
undergoes conformational changes. These changes lead to immediate selfphosphorylation of JAK2 and the EPO receptor itself. Activated JAK2 then
phosphorylates STAT-5, which is transported to the nucleus where it serves as a
transcription activator for genes which lead to stimulation of erythroid mitosis and
differentiation; to stimulation of the expression of globin, spectrin and ankyrin; and
to inhibition of erythroid precursor apoptosis.
The more distal c-terminus portion of the erythropoietin receptor (the cytoplasmic
portion) is a negative regulatory domain. This domain interacts with phosphatases
such as SH protein tyrosine phosphatase (SHP-1 and SH-PATP1). Within about 30
minutes of the receptor being activated, these tyrosine phophatases
dephosphorylate the EPO receptor and JAK2, which allows their degradation via the
ubiquitin proteasome pathways. Thus, the c-terminus and SHP-1 are responsible
for downregulating the signal transduction otherwise induced by the activated EPO
receptor (see figure below).
Megakaryopoiesis and Platelet Phase Hemostasis
Platelet Phase Hemostasis
The coagulation system is an amazing process characterized by many checks and
balances. The principle requirements of normal hemostasis
•Must stop blood loss.
•Must work at any point in the vascular bed.
•Must be rapid.
•Must be confined to the point of injury.
•Must be removable when hemostasis is re-established.
Megakaryopoiesis
Platelets arise from the megakaryocyte. Like the other hematopoietic elements,
megakaryopoiesis begins with the pluripotent stem cell which gives rise to the
common myeloid progenitor. Transcription factor expression influences cellular
differentiation in megakaryocyte development. One major transcription factor
involved is GATA1, which in conjunction with its cofactor FOG1 (a.k.a. friend of
GATA) without a concurrently increased CEBP-a expression contributes to
differentiation toward the combined megakaryocyte-erythroid precursor.
Additionally, GATA-1 represses the expression of PU.1—a transcription factor that
plays a critical role in myeloid gene expression and differentiation.
The combined megakaryocyte-erythroid precursor may then differentiate to either
committed stem progenitors of the erythroid or megakaryocytic lineages. There is a
body of evidence looking at the role of microRNAs in influencing cellular
differentiation and development. As an example, mi-RNA150 expression influences
the differentiation of the combined megakaryocyte-erythroid precursor toward one
or the other committed lineage pathway. Mi-RNA150 binds to the 3’UTR region of
Myb mRNA, which in turn leads to decreased Myb protein expression. Decreased
Myb expression leads to increased megakaryocyte differentiation (and decreased
erythroid differentiation). Conversely, decreased mi-RNA150 binding leads to
increased Myb protein expression and decreased megakaryocyte differentiation
(and increased erythroid differentiation).
Platelet Formation
Pleuripotent
Stem Cell
Myeloidcommitted
stem cell
CFU-Mega
Megakarocyte
Platelets
THROMBOPOIETIN
Megakaryocyte Development
Megakaryocyte colony-forming units (CFU-Meg) represent the first committed
progenitor of megakaryocytic differentiation.
Though the humeral regulation is not fully understood, thrombopoietin is a major
humeral regulator of megakaryocyte, and thus platelet, development. This
predominantly hepatic-derived substance works via the c-MPL receptor (aka
thrombopoietin receptor). Thrombopoietin stimulates CFU-Meg division and
megakaryocyte development. It affects the rate of endomitosis and inhibits
apoptosis of megakaryocytes during their maturation. Thrombopoietin is certainly
not the only regulator of this development process. Additional factors with evidence
supporting their role in megakaryocyte development include IL-3 and IL-11, which
along with thrombopoietin are involved with the early differentiation of the CFUMeg to early megakaryocyte forms.
The earliest megakaryocyte has a normal (diploid) DNA content, but the nuclear and
cytoplasmic mass, and DNA content rapidly increases as the cell undergoes a series
of mitotic amplifications and nuclear endoreduplications (mitosis without cell
division). Mature, polyploid (4N to 64N) megakaryocytes are the largest
hematopoietic cells, with a volume of approximately 15,000 fL (20 - 50 mm3).
Intracellular factors influence the process of endoreduplication. Increased
expression of cyclin D3 and decreased expression of cyclin B1 may play a role in
endomitosis by allowing mitosis to be aborted without entering cytokinesis (i.e. for
the nuclear material to duplicate and divide without the same occurring in the
cytoplasm).
As a result of their size, megakaryocytes are, in general, confined to the bone
marrow.
Platelet Formation
Platelets are anuclear fragments derived from the cytoplasm of the megakaryocyte.
As the megakaryocyte matures, its nuclear material begins to degenerate; granules
appear in the cytoplasm; and an intricate series of demarcation membranes
separate the cytoplasm into a series of small fields surrounded by membrane
material identical to the megakaryocyte plasma membrane. When cell maturation is
complete, the demarcation membranes separate and cytoplasm fragmentation
occurs leading to the production of 1,000 to 8,000 platelets, each with a volume of 7
to 9 fL.
This fragmentation of the megakaryocyte, i.e. the final stage of platelet development,
is not thrombopoietin dependent. The final processes involved in platelet release
remain unclear. Pseudopod projections from the megakaryocytes have been noted
to extend through the endothelial cells lining the marrow sinusoids. However, it is
unclear if the platelets are released from these pseudopods into the marrow
sinusoids or, as some evidence suggests, if the megakaryocyte migrates out of the
marrow to the lung where platelet release subsequently occurs. Either way,
platelets are shed from the megakaryocytes and enter the peripheral circulation.
In total, the time interval from differentiation of the stem cell to platelet production
is on average 7-10 days.
Platelet Homeostasis
How precisely does the body regulate the number of platelets?
As platelets are merely fragments of the megakaryocyte, the only means by which
platelet numbers can be increased is by increasing the number of megakaryocytes.
It has been observed that the platelet count and the mean platelet volume (MPV) are
inversely related. Thus, it appears the body may not actually regulate the number of
platelets but rather the total platelet mass.
Thrombopoietin is produced by the liver at a fairly constant rate and most is bound
by c-MPL receptors on the platelets (stored and circulating platelets). Remember,
as fragments of megakaryocytes, the platelet membrane (and thus platelet
membrane receptors) is identical to the mature megakaryocyte membrane. It is
only that portion of thrombopoietin that is not bound to the platelets that can affect
megakaryocyte development in the marrow. This fact may explain the mechanism
by which the system defends the total platelet mass and not the platelet number.