Genome-wide Regulation of
Gene Expression and
Transcription Factor Binding
during Human Hematopoeisis
Hematopoeisis
•
Every functionally specialized mature blood cell develops from a common stem cell. A
hematopoietic stem cell is pluripotent, able to differentiate along a number of different
pathways and thereby generate erythrocytes, granulocytes, monocytes, mast cells,
lymphocytes, and megakaryocytes.
– Few in number – approximately one stem cell per 104 bone marrow cells
•
Early in hematopoiesis, a pluripotent stem cell differentiates along one of two
pathways: lymphoid stem cell or a myeloid stem cell.
– Each type of stem cell can further differentiate into progenitor cells, which have
lost the capacity to for self-renewal and a committed to a specific cell lineage.
– Lymphoid stem cells generate B and T cell progenitors
– Myeloid stem cells generate progenitor cells for:
 Red blood cells – erythrocytes
 White blood cells – neutrophils, eosinophils, basophil, monocytes, and
mast cells.
 Platelets
•
Progenitor commitment depends on responsiveness to particular growth factors.
When the appropriate signal is present, progenitor cells proliferate and differentiate.
HL60
Vitamin D2
phorbol myristic acid (PMA/TPA)
M-CSF
All-trans Retinoic Acid (ATRA)
9-cis-Retinoic Acid
DMSO
HL60 cell-line
• Acute myeloid Leukemia
• No PML-RAR translocation
• Like most AML cells, fail to express secondary
granule protein genes late in differentiation
• RA treatment
• VitD Treatment
] Clinical trials in patients with AML
Experimental Setup
•
HL60 cells are treated with ATRA and harvested at 0, 2, 8, and 32
hours.
•
For each time point:
– Cells are phenotyped by:
• CD11b cell surface expression
• NBT Reduction (phagocytic activity potential)
– Cells are counted and tested for viability (Trypan Blue exclusion)
– Cells are harvested for RNA
– Cells are crosslinked and processed for chromatin preparation.
CD11b Expression: A hallmark cell surface marker
indicating differentiation toward granulocyte lineages
Kansas et al. 1990 Blood 9(12):2483-92
CD11b Expression: A hallmark cell surface marker
indicating differentiation toward granulocyte lineages
•
Cell differentiation analyzed by Flow Cytometry using a Alexa-Fluor 488 conjugated aCD11b antibodies and an Alexa-Fluor 488 isotype specific control antibody.
•
Samples prepared in triplicate for each time point and 10,000 events are counted for
each time point.
CD11b Expression: A hallmark cell surface marker
indicating differentiation toward granulocyte lineages
Nitroblue Tetrazolium Chloride Reduction Assay
Superoxide
O2-
Nitroblue Tetrazolium Chloride Reduction Assay
NBT Reduction Assay
50
45
% Cells Positive
40
35
30
25
20
15
10
5
0
0
2
8
Time After ATRA treatment
32
HL60 differentiation was performed in triplicate and both
chromatin and RNA were prepared from each time point
Replicate #1
60
40
NBT
30
CD11b
Replicate #2
20
60
10
50
0
2
8
40
Time after ATRA treatment
32
NBT
30
CD11b
Replicate #3
20
60
10
50
0
0
2
8
40
Time after ATRA treatment
% Cells Positive
0
% cells positive
% Cells Postive
50
32
NBT
30
CD11b
20
10
0
0
2
8
Time after ATRA treatment
32
Average HL60 differentiation over all replicates
60
% Positive Cells
50
40
NBT
30
CD11b
20
10
0
0
2
8
Time after ATRA treatment
32
Chromatin IPs for ENCODE Arrays
• Retinoic Acid Receptor a: Cellular receptor protein for retinoic acid ligands.
Binds to retinoic acid response elements (RARE) in target regions to activate and
some times repress transcription. Although several isoforms of RAR are present in
mammalian systems, RARa appears to have the central role in RA induced
granulopoeisis.
• Ezh2: Homo sapiens enhancer of zeste homolog 2 (Drosophila). A member of the
polycomb family and found in a complex called HPC2. Shown to be the methylase
component of polycomb complexes and to be associated primarily with H3K27 trimethylation but also with H3K9 tri-methylation to some extent.
• SirT1: Human homolog of the S. cerevisiae Sir2 protein known to be involved in cell
aging and in the response to DNA damage, binds and deacetylates the p53 protein
with a specificity for its C-terminal Lys382 residue, modification of which has been
implicated in the activation of p53 as a transcription factor
Chromatin IPs for ENCODE Arrays
• RNA polymerase II
• Acetylated histone H4: associated with gene activation
• Histone H3K9 di-methylated: associated with repressed genes
• Histone H3K9 tri-methylated: associated with repressed and
silenced genes (i.e. Suv39H1 and polycomb)
•
Histone H3K27 di-methylated: associated with repressed genes
•
Histone H3K27 tri-methylated: associated with repressed and
silenced genes (i.e. polycomb)
• Histone H4K20 di-methylated: associated with repressed and
silenced genes
• HA: Negative Control
• RPL10: ribosomal protein L10
– This gene encodes a ribosomal protein that is a component of the 60S subunit. The
protein belongs to the L10E family of ribosomal proteins. It is located in the
cytoplasm.
– In vitro studies have shown that the chicken protein can bind to c-Jun and can
repress c-Jun-mediated transcriptional activation, but these activities have not been
demonstrated in vivo.
– This gene has been referred to as 'laminin receptor homolog' because a chimeric
transcript consisting of sequence from this gene and sequence from the laminin
receptor gene was isolated; however, it is not believed that this gene encodes a
laminin receptor.
– Transcript variants utilizing alternative polyA signals exist. The variant with the
longest 3' UTR overlaps the deoxyribonuclease I-like 1 gene on the opposite strand.
This gene is co-transcribed with the small nucleolar RNA gene U70, which is located
in its fifth intron. As is typical for genes encoding ribosomal proteins, there are
multiple processed pseudogenes of this gene dispersed through the genome
– Down regulated during adipocyte, kidney and heart differentiation.
– Not well expressed in non-differentiated HL60 cells as determined by prior array
analysis.
RLP10 Gene Chr. X
0 hour RNA
2hour RNA
8 hour RNA
32 hour RNA
0 hour RNAP II
32 hour RNAP II
0 hour H4-Ac
32 hour H4-Ac
• PSMB4: proteasome (prosome, macropain) subunit, beta type, 4
– The proteasome is a multicatalytic proteinase complex with a highly ordered ringshaped 20S core structure.
– The core structure is composed of 4 rings of 28 non-identical subunits; 2 rings are
composed of 7 alpha subunits and 2 rings are composed of 7 beta subunits.
– Proteasomes are distributed throughout eukaryotic cells at a high concentration and
cleave peptides in an ATP/ubiquitin-dependent process in a non-lysosomal pathway.
– An essential function of a modified proteasome, the immunoproteasome, is the
processing of class I MHC peptides. This gene encodes a member of the
proteasome B-type family, also known as the T1B family, that is a 20S core beta
subunit.
– Expressed in HL60 by prior array analysis.
PMSB4 Chr.1
0 hour RNA
2hour RNA
8 hour RNA
32 hour RNA
0 hour RNAP II
32 hour RNAP II
0 hour H4-Ac
32 hour H4-Ac
• EEF1A1: Eukaryotic translation elongation factor 1 alpha 1.
– This gene encodes an isoform of the alpha subunit of the elongation factor-1
complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the
ribosome.
– This isoform (alpha 1) is expressed in brain, placenta, lung, liver, kidney, and
pancreas, and the other isoform (alpha 2) is expressed in brain, heart and skeletal
muscle.
– This isoform is identified as an autoantigen in 66% of patients with Felty's syndrome.
– This gene has been found to have multiple copies on many chromosomes, some of
which, if not all, represent different pseudogenes.
– Exhibits moderate expression in undifferentiated HL60 cells by prior microarray
analysis.
EEF1A1 Gene Chr. 6
0 hour RNA
2hour RNA
8 hour RNA
32 hour RNA
0 hour RNAP II
32 hour RNAP II
0 hour H4-Ac
32 hour H4-Ac
Representative gene for Retinoic Acid
Receptor a association.
• IRF-1: interferon regulatory factor 1
– IRF1 encodes interferon regulatory factor 1, a member of the interferon
regulatory transcription factor (IRF) family.
– IRF1 serves as an activator of interferons alpha and beta transcription, and in
mouse it has been shown to be required for double-stranded RNA induction of
these genes.
– specifically binds to the upstream regulatory region of type I IFN and IFNinducible MHC class I genes (the interferon consensus sequence (ICS)) and
activates those genes.
– IRF1 also functions as a transcription activator of genes induced by interferons
alpha, beta, and gamma.
– Further, IRF1 has been shown to play roles in regulating apoptosis and tumorsuppression.
– Induced by viruses, IFN and Retinoic Acid.
– Deletion or rearrangement of irf1 are a cause of preleukemic myelodysplastic
syndrome (MDS) and of acute myelogenous leukemia (AML).
– Not well expressed in uninduced HL60 cells as shown in prior array analysis.
IRF-1 Gene Chr. 5
0 hour RNA
2hour RNA
8 hour RNA
32 hour RNA
0 hour RNAP II
32 hour RNAP II
0 hour H4-Ac
32 hour H4-Ac
0 hour RARa
32 hour RARa
• IRAK1: Interleukin-1 receptor-associated kinase 1
–
IRAK1 encodes the interleukin-1 receptor-associated kinase 1, one of two
putative serine/threonine kinases that become associated with the
interleukin-1 receptor (IL1R) upon stimulation.
– IRAK1 is partially responsible for IL1-induced upregulation of the
transcription factor NF-kappa B.
– Expressed well in HL60 cells as determined by prior array analysis.
IRAK1 Chr.X
0 hour RNA
2hour RNA
8 hour RNA
32 hour RNA
0 hour RNAP II
32 hour RNAP II
0 hour H4-Ac
32 hour H4-Ac
Examples of SirT1 potential targets as
determined by ENCODE array analysis.
• RFX5:
– A lack of MHC-II expression results in a severe immunodeficiency
syndrome called MHC-II deficiency, or the bare lymphocyte
syndrome.
– At least 4 complementation groups have been identified in B-cell
lines established from patients with BLS.
– The molecular defects in complementation groups B, C, and D all
lead to a deficiency in RFX, a nuclear protein complex that binds to
the X box of MHC-II promoters.
– The lack of RFX binding activity in complementation group C
results from mutations in the RFX5 gene encoding the 75-kD
subunit of RFX. RFX5 is the fifth member of the growing family of
DNA-binding proteins sharing a novel and highly characteristic
DNA-binding domain called the RFX motif.
– Not great expression in undiffernitiated HL60s by previous array
analysis.
RFX5 Gene, Chr.1
RNA 0 hr
RNA 2 hr
RNA 8 hr
RNA 32 hr
RNA polII 0hr
RNA polII 32hr
H4 tetraAc 0hr
H4 tetraAc 32hr
SIRT1 0hr
SIRT1 32 hr
Examples of Polycomb association
• GMMPA: GDP-mannose pyrophosphorylase A.
– This gene is thought to encode a GDP-mannose pyrophosphorylase.
– This enzyme catalyzes the reaction which converts mannose-1-phosphate
and GTP to GDP-mannose which is involved in the production of N-linked
oligosaccharides.
• MDFI: MyoD family inhibitor
– This protein is a transcription factor that negatively regulates other
myogenic family proteins.
– Inhibits the transactivation activity of the Myod family of myogenic factors
and represses myogenesis. Acts by associating with Myod family members
and retaining them in the cytoplasm by masking their nuclear localization
signals. Can also interfere with the DNA-binding activity of Myod family
members. Plays an important role in trophoblast and chondrogenic
differentiation
– Knockout mouse studies show defects in the formation of vertebrae and ribs
that also involve cartilage formation in these structures.
– Moderately expressed in HL60 cells as determined by prior array analysis.
GMPPA Gene Chr.2
0 hour RNA
32 hour RNA
0 hour H3K27 di-Meth
32 hour H3K27 di-Meth
0 hour RNAP II
32 hour RNAP II
0 hour H4-Ac
32 hour H4-Ac
0 hour Ezh2
32 hour Ezh2
0 hour H3K27 tri-Meth
32 hour H3K27 tri-Meth
Histone H3K9 di-methylation
• PIPK15A:68 kDa type I phosphatidylinositol-4-phosphate 5-kinase
alpha
– kinase activity: 1-phosphatidylinositol-4-phosphate 5-kinase activity
– transferase activity
– glycerophospholipid metabolism
– signal transduction
– Not great expression in undifferentiated HL60 cells by prior array
analysis.
PIP5K1A Gene Chr. 1
0 hour H3K9 di-Meth
32 hour H3K9 di-Meth
0 hour H3K9 tri-Meth
32 hour H3K9 tri-Meth
0 hour H4K20 di-Meth
2 hour H4K20 di-Meth
0 hour SirT1
32 hour SirT1
0 hour RAR-a
32 hour RAR-a
Histone H3K9 tri-methylation?
Histone H4K20 di-methylation?
Future Work
•
Finish processing RNA for biological replicates #2 and #3.
•
Repeat ChIP-Chip for RNAP II, H4-Ac, and H3K37 di-methylation for all
time points in all three biological replicates.
• Hybridize to ENCODE
• Hybridize to Whole Genome Array.
•
Potentially pursue the Ezh2 and SirT1 antibodies for Whole Genome
Array.
•
Do Real-Time PCR at several genes to determine whether H3K9
methylation and H4K20 methlyation ChIPs are actually working.
•
•
•
•
•
CCAAT/Enhancer Binding protein a (C/EBPa)
– within haematopoeisis, a, b, d are primarily expressed in granulocute, monocytes and
and eosinophil lineages. C/EBPa is the isoform primarily expressed in immature
granulocytes.
– Target Genes: MPO (myeloperoxidase), M-CSF receptor, neutrophil elastase (ELA2),
Lactoferrin (LF), human neutrophil defensins (HNP), G-CSF receptor, PU.1, MBN
(serine protease), Azurocidin (serine protease)
C/EBPb
– Expressed in a wide variety of cells including hepatocytes and haematopoeitic cells.
Decrease in a correlates with increase in b. Low expression in immature cells and
highest in neutrophils and macrophages.
– Target genes: ELA2 and IL-6
C/EBPg
– Dominant inhibitory due to ability to dimerize with other C/EBPs and lack intact basic
regions. Ubiquitously expressed but highest expression level is in B cells. Currently
no role described in myeolopoeisis.
C/EBPd
– Shows a similar expression pattern to b but is a stronger activator.
– Target Genes: ELA2
C/EBPe
– Found in later stage granulocytes and T cells. Has both an activation and repression
domain. In knock out, neutrophils do not develop secondary granules.
– Target Genes: LF and HNP.
•
•
•
•
CCAAT displacement factor (CDP)
– Antagonizes C/EBP by competing for binding sites. Should be inactivated as cells go to
terminal differentiation
– Target Genes: Gp91-phox, C/EBPe, HNP, LF
Hox10A
– Homoebox domain protein which binds as a dimmer with PBX.
– May play a role analogous to CDP in granulopoesis. Preferentially expressed in myeloid
cell lines but not present in mature neutrophils or monocytes. -/- mice have increased
numbers of monocytes.
– Target Genes: Gp91-phox, p21
Retinoic acid receptor (RAR a, b, g)
– All RARs (especially a) are expressed in haematopoetic cells. Dominant negative mutant of
a result in inability of myeloid precursor cells to undergo neutrophil or monocytes
differentiation
– Target Genes: IRF-1, CEBPe, Folate Receptor b
c-Myb
– Expressed in immature myeloid, lymphoid, and erythroid cells. Myb -/- mice lack all of
these cell lineages. Expression patterns indicate that the gene has a primary role in
haematopoeisis. CD34+ progenitor cells have the highest levels but c-myb is not
expressed in mature granulocytes.
– Target Genes: MPO, ELA2, MBN, Azurocidin, CD11b, CD34, c-myc, cdc2, c-myb, mim-1.
•
PU.1
–
–
–
–
Ets family transcription factor with glutamine rich transactivation domain.
Interaction with GATA1 and inhibits activation of gene expression.
Expression levels increase during granulocytic and monocytic differentiation.
Specifically represses erythroid genes to push cells toward myeloid lineages. Expressed in B,
granulocytic, and monocytic cells.
– Target Genes: M-CSF receptor, ELA2, MPO, MBN (serine protease), azurocidin (serine
protease), Gp91-phox, C/EBPe, HNP, and LF
•
GATA1
– Lowered levels after cell lineage commitment signal neutrophil/eosinophil development rather
than erythroid. Critical regulator of erythroid, megakaryocytic, eosinophil, and mast cell
differentation.
– Target Genes: GATA1, GATA2, a-globin
•
GATA2
– Essential for haematopoeitic stem and progenitor function cells function.
– GATA1 and 2 seem to be reciprocally expressed in haematopoeitic cells.
– Target Genes: GATA1, GATA2, a-globin
•
AML/CBFA
– Large family defined by homology to a runt domain. Heterodimerize with CBFb to bind to DNA.
Interact with Ets-1 and C/EBP.
– Deletion of AML1 results in embryonic death as a result of haematopoetic defects. Target for
chromosomal translocations in many acute myeloid leukemias. Expression is largely restricted to
myeloid and lymphoid cells that are CD34 positive. Stimulates the G1 to S transition in myeloid
cells.
– Target Genes: MPO, ELA2, NF3 (Defensin ), M-CSF receptor,