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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3578-3584
RED CELLS
FKLF-2: a novel Krüppel-like transcriptional factor that
activates globin and other erythroid lineage genes
Haruhiko Asano,
Xi Susan Li, and
George Stamatoyannopoulos
From the Division of Medical Genetics, University of Washington,
Seattle, WA.
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Abstract |
FKLF-2, a novel Krüppel-type zinc finger protein, was cloned
from murine yolk sac. The deduced polypeptide sequence of 289 amino
acids has 3 contiguous zinc fingers at the near carboxyl-terminal end,
an amino-terminal domain characterized by its high content of alanine
and proline residues and a carboxyl-terminal domain rich in serine
residues. By Northern blot hybridization, the human homologue of FKLF-2
is expressed in the bone marrow and striated muscles and not in 12 other human tissues analyzed. FKLF-2 is constitutively expressed in
established cell lines with an erythroid phenotype, but it is
inconsistently expressed in cell lines with myeloid or lymphoid
phenotypes. The expression of FKLF-2 messenger RNA (mRNA) is
up-regulated after induction of mouse erythroleukemia cells. In
luciferase assays, FKLF-2 activates predominantly the  , and to a
lesser degree, the  and  globin gene promoters. The activation of
gene promoter does not depend on the presence of an HS2 enhancer.
FKLF-2 activates the promoter predominantly by interacting with the
CACCC box, and to a lesser degree through interaction with the TATA
box or its surrounding DNA sequences. FKLF-2 also activated all the
other erythroid specific promoters we tested (GATA-1, glycophorin B,
ferrochelatase, porphobilinogen deaminase, and 5-aminolevulinate
synthase). These results suggest that in addition to globin, FKLF-2 may
be involved in activation of transcription of a wide range of genes in
the cells of the erythroid lineage.
(Blood. 2000;95:3578-3584)
© 2000 by The American Society of Hematology.
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Introduction |
There has been a considerable effort to identify
transcriptional factors that provide developmental specificity in
globin gene expression. The first such factor to be identified was
EKLF.1 This is a member of the KLF/Sp1 family of
transcriptional factors and it is characterized by specificity of
expression in the erythroid lineage and its interaction with the CACCC
box of the globin gene promoter. There are considerable differences
in the sequence of CACCC boxes of the and gene
promoters2 and there is clear-cut evidence that EKLF fails
to activate the promoter.3 EKLF-deficient homozygous
mice die from severe anemia due to globin chain deficiency when the
definitive erythropoiesis is established in the fetal
liver.4,5 When a human transgene is transferred into
the background of the EKLF-deficient animals, human gene
transcription is increased, providing direct in vivo evidence that EKLF
does not interact with the gene promoter.6,7
It is reasonable to assume that, on the model of EKLF, transcriptional
factors that can activate the embryonic  or the fetal globin
genes exist. Because the and genes have CACCC box motifs in
their promoters, it is also reasonable to assume that transcriptional
factors of the KLF/Sp1 family may participate in the
developmental control of these genes. We have previously described the
cloning and characterization of a factor, designated as
FKLF,8 that activates the expression predominantly of the , and to a lesser degree, of the globin genes. We have shown that gene expression is activated through the interaction of FKLF
with the gene CACCC box and that this transcriptional factor fails
to activate other erythroid genes that contain CACCC or GC
motifs.8 In this paper, we describe the cloning and
characterization of a new transcriptional factor belonging to the
KLF/Sp1 family, which we designate as FKLF-2. This factor
increases gene expression more than 100-fold in transient
expression assays. FKLF-2 is not a specific activator of the gene
expression because it activates to a lesser extent the and the globin genes. This transcriptional factor also activated all other
erythroid genes we used in our assays, although to a much lower degree,
compared with the globin gene.
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Materials and methods |
Induction of erythroid cells and RNA extractions
Cells were induced as follows: K562 with 50 µmol/L hemin for 3 days; MEL with 3 mmol/L N, N'-hexamethylenebisacetamide (HMBA) and 10 µmol/L hemin for 3 days; and HEL with 500 µmol/L
 -aminolevulinic acid (-ALA) for 5 days. Total RNA extraction and
processing are described elsewhere.8
Polymerase chain reaction screening for zinc finger motifs
Screening of mouse yolk sac cell cDNA for zinc finger motifs was
performed by polymerase chain reaction (PCR) essentially as described
previously.8 Random hexamer-primed cDNA of 1 µg of
poly(A)+RNA from day-10 mouse yolk sac cells was subjected to
degenerate primer PCR. The expected 210 base pairs (bp) band was cut
out, and the extracted DNA was ligated into a plasmid vector (T vector,
Promega, Madison, WI). Plasmid DNA was analyzed by
restriction enzyme digestion, and clones containing an insert were
subjected to PCR to exclude EKLF clones. Clones that were not excluded
were sequenced using a kit (Cyclist; Stratagene, La Jolla, CA).
Complementary DNA cloning
The 3' and 5' unknown complementary DNA (cDNA) sequences
were amplified by the RACE (rapid amplification of cDNA ends)-PCR using
a kit (Marathon cDNA amplification kit; Clontech, Palo Alto, CA), and a cDNA sequence composed of 1378 bp nucleotides
was determined. On the basis of this sequence, a 917 bp open reading
frame (ORF) predicted to encode the FKLF-2 protein was amplified from
random hexamer-primed cDNA using Pfu DNA polymerase (Stratagene). The PCR fragment was inserted into T-vector after dA addition to the 3' ends of the PCR fragment, according to the manufacturer's
instruction. A clone (pGEM/FKLF-2) without mutation was used for
further plasmid construction.
Plasmid constructions
Transactivator plasmid of FKLF-2 was prepared as follows: FKLF-2
cDNA was cut out as a blunted SpeI-NcoI fragment from the pGEM/FKLF-2
and was inserted into the pSG5DD vector at blunted EcoRI and
BamHI sites (pSG5/FKLF-2).
Reporter plasmids, pHS2Luc and pHS2Luc, pHS2Luc and pLuc,
and pHS2( CAC)Luc were already described elsewhere.8
p(CAC)Luc, in which a 9-bp CACCC sequence was deleted from the
promoter, was constructed by insertion of
ApaI-HindIII fragment of pHS2(CAC)Luc into
ApaI- and HindIII-digested pLuc. Stage selector
element (SSE)9 was deleted from pLuc and p(CAC)Luc
by in vitro mutagenesis (Altered Sites II in vitro Mutagenesis Systems,
Promega). KpnI-HindIII fragments of pLuc and
p(CAC)Luc were subcloned into KpnI- and HindIII-digested pALTER-1 vector (Promega). The SSE was deleted using a 5' phosphorylated oligonucleotide;
5'-pTGAGGCCAGGGGCCAAGAA- TAAAAGGAAGCA-3', which lacks
nucleotides  35 to 52 relative to the cap site of gene. Constructs generated by in vitro mutagenesis were verified by
sequencing. Reporter constructs with truncated promoters were
constructed by inserting ApaI (blunted)-HindIII, NcoI (blunted)-HindIII, EcoNI
(blunted)-HindIII, and NaeI-HindIII fragments
from pLuc into KpnI (blunted)/HindIII sites of the pGL2-Basic vector (Promega), resulting in
p 201Luc, p141Luc,
p103Luc, and p52Luc, respectively.
DNA of promoters of GATA-1, porphobilinogen deaminase (PBGD),
glycophorin B (GPB), ferrochelatase (FC), and 5-aminolevulinate synthase (ALAS) were obtained from pHS2-GATA-Luc, pHS2-PBGD-Luc, pHS2-GPB-Luc, pHS2-FC-Luc, and pHS2-ALAS-Luc8 as
BglII fragments (GATA, PBGD, GPB, and FC) or
BglII-PvuII fragment (ALAS). Subsequently, they were inserted
into a BglII site or BglII/HindIII (blunted) sites of pGL2Basic vector, resulting in pGATA-Luc, pPBGD-Luc, pGPB-Luc,
pFC-Luc, and pALAS-Luc.
Transactivation analysis, Northern blotting, and messenger RNA
detection by polymerase chain reaction
Transient transfections of K562 cells, Northern blottings, and
semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to study gene expression were performed as described previously.8 Primer information and PCR conditions used in this study will be provided on request.
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Results |
Cloning of FKLF-2 complementary DNA
For cloning cDNAs encoding EKLF- or Sp1-type zinc finger proteins by
PCR, a set of degenerate primers was designed on the basis of the amino
acid homology of the zinc finger region of Sp1 and EKLF family
proteins. The upstream primer corresponded to the conserved amino acid
sequence GCGKVY, whereas the downstream primer to the conserved
sequence F(SM)RSDEL (Figure 1A). One
hundred eighty-nine individual clones were isolated from a day-10 mouse yolk sac cDNA and sequenced. One hundred six clones encoded
Cys2-His2 type zinc finger motifs: 69 encoded
EKLF; 31 encoded BKLF;10 2 encoded the murine equivalent of
Sp311,12; 1 encoded the murine equivalent of
CPBP/Zf913,14; 2 encoded the murine equivalent of
UKLF15 ;and 1 encoded a novel cDNA. The human homologue of
this novel cDNA was also cloned from human fetal liver erythroid cells
using a similar method. On the basis of its structural similarity to the previously described FKLF,8 the novel gene was
designated as FKLF-2.
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| Fig 1.
FKLF-2.
(A) Deduced polypeptide sequence of murine FKLF-2 (mFKLF-2, Genebank
accession number: AF251796). Proline residues are shown with a bold
typeface, and alanine residues are highlighted with a shaded box. The
sequence of the 3 zinc fingers is underlined, and the conserved
polypeptide regions used for the preparation of degenerate primers are
indicated by boxes with gray stripes. Polypeptide sequence of human
FKLF-2 (hFKLF-2) is also shown with an italic typeface below the
equivalent mFKLF-2 sequence. (B) Schematic representation of structure
of deduced FKLF-2 protein. Alanine and proline-rich in serine residues
are shown by shaded boxes. Three zinc fingers are shown by striped
boxes. Numbers above the boxes represent amino acid positions from the
first methionine. (C) Comparison of amino-terminal polypeptide
sequences between mFKLF-2 and mBTEB1.22 Identical amino
acid residues are shown by a bold typeface and marked with a symbol
(:).
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The 1378-bp cDNA of murine FKLF-2 includes an ORF that encodes 289 amino acids with a 31.1 kd molecular mass (Figure 1A). The deduced
polypeptide sequence of the human FKLF-2 is also shown in
Figure 1A. The human and murine sequences differ by a single amino acid change (glutamic acid at 204 of mFKLF-2  aspartic acid).
FKLF-2: a new Krüppel-type zinc finger protein of the
FKLF/TIEG family
The deduced polypeptide sequence of the FKLF-2 contains a zinc
finger domain composed of 3 contiguous fingers present near the
carboxyl-terminal end, a long amino-terminal domain, and a short
carboxyl-terminal domain (Figure 1A and 1B). The structure of the zinc
finger
(C-X4-C-X12-H-X3-H-X7-C-X4-C-12-H-X3-H-X7-C-X2-C-X12-H-X3-H, where X represents any amino acid residue), is similar to that of Sp1,
EKLF, and other proteins of their families.12,16,17 The
zinc finger motif of FKLF-2 has an intermediate homology to the
proteins of both the Sp1 family (Sp1, Sp2,12 Sp3, and
Sp411) and the proteins of the EKLF family (EKLF,
BTEB2/IKLF,18,19 LKLF,16 BKLF,
GKLF/EZF,20,21 CPBP/Zf9, UKLF, and
AP-2rep22) (Table 1).
FKLF-2 thus belongs to the third group of Sp1/EKLF proteins, composed
of BTEB1,23 TIEG-1,24 and
FKLF/TIEG-2.8,25
The amino-terminus of FKLF-2 has significant homology to BTEB1; 29 of
72 initial amino acid residues (40%) are identical (Figure 1C). No
homologies to other known proteins were detected in the amino- or
the carboxyl-terminal domains. The amino-terminal domain is
characterized by a high content of proline and alanine residues, whereas the carboxyl-terminal domain is rich in serine residues (Figure
1A and 1B). The respective percentages of alanine, proline, and serine
residues are 21%, 17%, and 6% in the amino-terminal domain, and 8%,
10%, and 26% in the carboxyl-terminal domain. The FKLF-2 protein is
basically charged (isoelectric point, 10.4). Basic residues (arginines
and lysines) are accumulated at amino acids 149 to 167, making a net
charge of this region +6 (Figure 1B). Neither the amino- nor the
carboxyl-terminal domains contain acidic regions.
Expression in human tissues
To examine the tissue-specificity of FKLF-2 mRNA expression,
Northern blot hybridization using a human FKLF-2-specific RNA probe
(encompassing the carboxyl-terminal coding through 3' UTR) was
performed using a commercially available membrane (MTN Blots Human I
and III, Clontech), which contains poly(A)+RNA extracted from adult
human stomach, thyroid gland, spinal cord, lymph node, trachea, adrenal
gland, bone marrow, heart, brain, placenta, lung, liver, skeletal
muscle, kidney, and pancreas. An intense band was detected at the
1.35-kb position in the bone marrow, the heart, and skeletal muscles
(Figure 2A), indicating that the bone
marrow and striated muscles are the major FKLF-2-expressing tissues.
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| Fig 2.
FKLF-2 mRNA.
(A) FKLF-2 mRNA expression by Northern blotting. Each lane contains 2 µg poly(A)+RNA from adult human tissues. The RNA was blotted with a
specific FKLF-2 probe. A distinct band at about 135 kb was detected in
the bone marrow and the heart and skeletal muscle. Another major band
between 4.4 and 7.5 kb seemed to be a cross hybridization to 28S
ribosomal RNA because of its size. (B) FKFL-2 mRNA expression in
various cell lines by RT-PCR. Three bands in each picture show the
results of amplification in different cycles; ie, from left to right,
34, 32, and 30 cycles for FKLF-2, and 22, 20, and 18 cycles for 28S
rRNA. (C) FKLF-2 mRNA expression in K562 and HEL cells before (ui) and
after (i) induction by Northern blottings. Two micrograms poly(A)+RNA
was blotted with probes indicated. (D) FKLF-2 mRNA expression in MEL
cells before and after induction (Northern blotting). Four micrograms
poly(A)+RNA was blotted with probes indicated. The position of the
FKLF-2 band is approximately 6 kb.
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Expression in established hemopoietic cell lines
Semiquantitative RT-PCR was performed using RNA from human cell
lines with erythroid, myeloid, or lymphoid phenotypes. In this assay
template, cDNAs were appropriately diluted to give similar band
patterns of 28S ribosomal RNA (rRNA) (Figure 2B). FKLF-2 cDNA was
efficiently amplified from all the cell lines with erythroid phenotypes
(K562, HEL, CHRF, MB-02, KU812, and MEG-O1). In myeloid lines, results
were inconsistent; FKLF-2 cDNA was undetectable in KG-1 and HL-60
cells, whereas in EM-3, the FKLF-2 was detected at a level comparable
to that observed in the erythroid lines. In lines with a B-cell
phenotype, FKLF-2 cDNA could not be amplified from Daudi cells, but it
was amplified from EB virus transformed lymphocytes (marked as
"Lymphs" in Figure 2B). Of the 2 lines with a T-cell phenotype,
FKLF-2 cDNA was weakly amplified from Jurkat cells, whereas the
amplifications from CEM cells were much stronger than erythroid cells.
Two monocytic lines (U937 and THP-1) gave weak FKLF-2 amplicons.
Response to inducers of differentiation
To test whether FKLF-2 expression changes on induction of erythroid
differentiation, K562, HEL, and MEL cells were induced by hemin,
-ALA, or hemin + HMBA,26,27 and FKLF-2 mRNA was assessed
with Northern blotting. As shown in Figure 2C and 2D, induction of
erythroid differentiation up-regulated FKLF-2 expression in all 3 cell
lines. FKLF-2 thus differs from EKLF and FKLF, which fail to
be up-regulated by inducers of erythroid
differentiation.1,8
Activation of globin gene transcription
To test the role of FKLF-2 on globin gene regulation, the mammalian
expression vector pSG5DD (pSG5/FKLF-2), which contains FKLF-2 cDNA, was
cotransfected into K562 cells with a reporter construct containing a
luciferase gene that was driven by a globin hypersensitive site
(HS) 2, and an , , or globin gene promoter. As shown in
Figure 3, FKLF-2 functions as
a transcriptional activator of these promoters. In the presence of
the exogenous FKLF-2, the mean luciferase activity obtained from the
promoter construct (pHS2Luc) was 4179% of that without FKLF-2
(considered as 100%) (Figure 3). Mean luciferase activity of the promoter with and without FKLF-2 were 1753% and 71%. The average
luciferase activities of promoter constructs were 1% and 76% in
the absence and presence of exogenous FKLF-2, respectively.
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| Fig 3.
Trans-activation of and and globin gene promoters by FKLF-2.
Reporter constructs containing a luciferase gene driven by HS2 and ,
, or gene promoter were transfected into K562 cells with or
without pSG5/FKLF-2. Luciferase activities were corrected by protein
concentrations, and expressed as relative percentages of luciferase
activity of pHS2Luc that were not cotransfected by pSG5/FKLF-2
(100%). Protein concentration was used to correct the transfection
efficiency, because in our preliminary experiments we could not rule
out that FKLF-2 does not activate the SV-40 promoter/enhancer of the
pSV-Gal control plasmid.3 Data are expressed as mean
(columns) ± SD (error bars) derived from multiple transfections using
2 different plasmid sets.
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Interaction with the CACCC box of the gene promoter
To examine which cis-element of the globin gene promoter
is critical for the FKLF-2 function as a transcriptional activator, we
prepared reporter constructs with a truncated gene promoter (Figure
4). Because preliminary experiments showed
that the gene promoter could also be activated by FKLF-2 in the
absence of HS2 reporter, constructs lacking the HS2 were used. Four
truncations were used, 2 of which have been previously characterized in
transgenic mice. 201 contains all the
cis-elements of the proximal promoter and lacks an upstream
negative element.28 141 destroys the
CACCC box, resulting in a lack of gene expression in transgenic
mice.28 103 lacks the CACCC and the
distal CAAT boxes, but it contains the proximal CAAT box, the stage
selector element (SSE),9 and the TATA box.
52 lacks all gene promoter elements, except
the SSE and the TATA box.
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| Fig 4.
Structure of gene promoter.
Location of cis-elements are indicated by solid
rectangles. Individual truncated gene promoters are shown below the
wild promoter.
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As shown in Figure 5, FKLF-2 is a powerful
activator of the gene promoter, even in the absence of HS2. The
luciferase activity of the wild Luc construct in the presence of
FKLF-2 was 11 237% of the activity in the absence of FKLF-2
(considered as 100%) (P = .013). Strong activation was still
observed in the construct p201Luc (Figure 4):
9381% with FKLF-2 and 81% without FKLF-2 (P < .01). The
luciferase activity dropped remarkably when the construct p-141Luc, which lacks a functional CACCC box was used;
however, FKLF-2 activated the -141 promoter (869% and
33% with and without FKLF-2, respectively, P < .01),
despite the fact that this promoter lacked a functional CACCC motif.
Mean luciferase activities with and without FKLF-2 were 48% and 17%
when the 103 construct was used
(P = .023), and 8% and 1% when the 52
construct (P < .01) was used. Thus, although
progressive reduction of luciferase activities was observed by the
sequential truncations of the gene promoter, FKLF-2 retained its
activity on the truncated promoters. The effect of FKLF-2 on the gene promoter was not ablated by any of these truncations, as is
demonstrated by the fold increase of luciferase activities by the
addition of FKLF-2, as shown in Figure 5. These data suggested that (1)
the CACCC box is the primary cis-element interacting with
FKLF-2; and (2) FKLF-2 may interact with the SSE or it may activate the
globin gene promoter through the TATA box or its surrounding
sequences.
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| Fig 5.
Effect of truncation deletions of gene promoter on
FKLF-2 activity.
Reporter constructs containing a luciferase gene driven by various gene promoters depicted in Figure 4 were tested in K562 cells.
Luciferase activities were expressed as relative percentages of
luciferase activity of pLuc that were not cotransfected by
pSG5/FKLF-2 (100%). Fold increases (expressed as mean ± SD) of
luciferase activities by FKLF-2 compared with those in the absence of
the transactivator are shown below.
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Interaction with other cis elements of the
proximal gene promoter
To further analyze the interaction between FKLF-2 and the
cis elements of the gene promoter, reporter constructs with
an internal deletion of the CACCC (p[CAC]Luc), or the SSE
element (p[SSE]Luc), or both elements (p[CACSSE]Luc)
(Figure 6A) were produced. These reporter
constructs were transiently transfected into K562 cells with and
without FKLF-2. As shown in Figure 6B powerful promoter activation
by FKLF-2 was consistently observed. Similarly to the pattern observed
in 141 truncation (Figure 5), deletion of a 9 bp
CACCC sequence ([CAC]) reduced gene promoter activity
(Figure 6B). However, FKLF-2 still activated the (CAC) promoter:
The mean luciferase activities in the presence and the absence of
exogenous FKLF-2 were 991% and 50%, respectively (the wild-type promoter activity in the absence of FKLF-2 was considered as
100%).
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| Fig 6.
Deletions of gene promoter.
(A) Internal deletions of gene promoter. CACCC and stage selector
(SSE) sequences were deleted from the gene promoter as indicated.
(B) Effect of deletion of CACCC box and SSE on promoter
activation by FKLF-2. Reporter constructs containing a luciferase
gene driven by gene promoters with internal deletions depicted
in Figure 6A were tested in K562 cells.
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Deletion of SSE in the (SSE) construct, in which nucleotides
35 to 52 relative to the cap site were deleted, did not
significantly affect the promoter activity: The mean luciferase
activities with and without FKLF-2 were 9754% and 112%, respectively,
(Figure 6B) indicating that SSE is not required for activation of the gene promoter by FKLF-2.
Deletion of both the CACCC box and the SSE elements in the
([CACSSE]) construct failed to ablate the activation of
gene by the FKLF-2: The average luciferase activity without
exogenous FKLF-2 was 68%, whereas it was 1254% with FKLF-2
(Figure 6B).
The results described above indicate that FKLF-2 interacts with at
least 2 regions of the globin gene promoter: (1) the CACCC element
and (2) the TATA box or its surrounding DNA sequences.
FKLF-2 activates nonglobin erythroid gene promoters
To test whether FKLF-2 functions as a transcriptional activator of
other promoters with CACCC (or GC) motifs, we performed transactivation
assays using FKLF-2 and reporter constructs carrying promoters of 5 genes expressed in erythroid cells, ie, GATA-1, GPB, FC, PBGD, and
ALAS. Because of the high level of activation of the gene promoter
in the absence of HS2, reporter constructs lacking the HS2 enhancer
were used.
Luciferase activities driven by these promoters are shown in Figure
7A. Luciferase activity from pLuc in the
absence of FKLF-2 was considered as 100%. Mean luciferase activities
in the absence and the presence of exogenous FKLF-2 were as follows:
5% and 33% for GATA (P = .059); 3% and 117% for PBGD
(P < .001); 48% and 241% for FC (P = .025);
20% and 536% for GPB (P = .043); and 3% and 17% for ALAS
(P < .005). Therefore, FKLF-2 activated all the erythroid
promoters we used in our assay, although with different intensity. The
globin and the PBGD and GPB genes showed the highest level of
activation, whereas the activation of GATA, FC, and ALAS was low
(Figure 7B). The level of activation of these promoters by FKLF-2 did
not correlate well with the presence of CACCC or GC motifs, or the
number of the motifs. For example, the GPB promoter, which lacks
typical CACCC or GC-rich sequences, showed higher activation than the
GATA-1 promoter, which contains 3 CACCC motifs and 1 GC-box (Figure
7B). This observation suggests that the overall context of the promoter
is more important for FKLF-2 function than the mere presence of a
consensus motif in the DNA sequence. A similar conclusion was
previously reached with our studies on EKLF3 and
FKLF.8
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| Fig 7.
Effect of FKLF-2 on various erythroid promoters.
(A) Reporter constructs containing a luciferase gene driven by either
GATA-1 (GATA), porphobilinogen deaminase (PBGD), ferrochelatase (FC),
glycophorin B (GPB), or 5-aminolevulinate synthase (ALAS) promoter were
cotransfected with or without pSG5/FKLF-2. (B) Fold increase of
luciferase activities by FKLF-2 from the reporter constructs used in
this experiment. Numbers above the promoters are base pair distances
from the cap site (, GPB, FC, PBGD, and ALAS) or from the end of
exon 1 (GATA), and they indicate the upstream ends of the promoter
sequences cloned, and the positions of the CACCC and the GC-rich
sequences (solid rectangles and open ellipses, respectively).
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Discussion |
The Sp1/EKLF multigene family to which FKLF-2 belongs is composed of
3 groups, the EKLF group, the Sp1 group, and a third subgroup composed
of BTEB1, TIEG-1, and FKLF/TIEG-2. This group is characterized by
intermediate amino acid homology to both Sp1 and EKLF
subfamilies8; FKLF-2 is the fourth member of this group. In
addition to sharing highly homologous zinc finger motifs, FKLF-2 and
BTEB1 show a high homology at their amino-terminal end. Homologies in
the amino-terminal domains of 30% to 40% is characteristic of other
Sp1- or EKLF-like proteins (Table 2).
FKLF-2 has a number of features that distinguish it from FKLF. First,
FKLF-2 activates mainly the gene promoter, whereas FKLF activates
mainly the gene promoter. Second, although the activity of FKLF is
totally dependent on the presence of the 140 CACCC box, FKLF-2
activates the gene promoter mainly through the CACCC box and, to a
lesser degree, through the TATA box or a neighboring sequence. Third,
in contrast to FKLF and EKLF, which are not up-regulated after
induction of erythroleukemia lines,1,8 FKLF-2 mRNA
expression is up-regulated after induction of erythroid differentiation. Fourth, in addition to the globin genes, FKLF-2 activated all the erythroid promoters we tested. Perhaps in addition to
its effect on globin activation, FKLF is involved in the molecular
events of erythroid differentiation. Expression studies of adult human
tissues using Northern hybridization showed that FKLF-2 is expressed
only in bone marrow and striated muscle. This is of special interest in
view of the recent evidence29,30 that muscle-origin stem
cells can repopulate the hematopoiesis of sublethally irradiated animals.
The activation of the gene promoter by FKLF-2 is not enhanced when
HS2 is present in the constructs used for the transient expression
assays. Thus, although FKLF-2 increases promoter expression from
62- to 115-fold in the absence of HS2, it increased expression only
42-fold in the presence of HS2. Such results are compatible with the
interpretation that FKLF-2 is a powerful activator of gene
transcription independent of the presence of an enhancer. The results
also indicate that, if FKLF-2 interacts with LCR, the interaction
cannot be mediated by HS2, despite the fact that this HS contains 3 CACCC motifs. As in the case of EKLF, other DNase I hypersensitive
sites, such as HS3,7,31,32 might be important for this interaction.
The murine FKLF-2 was cloned from the yolk sac and its human homologue
from the human fetal liver. By Northern hybridization, the human adult
bone marrow appears to be the adult tissue in which FKLF-2 is
predominantly expressed. Thus, FKLF-2 is expressed in embryonic, fetal,
and adult erythropoiesis. Expression in the adult stage of
erythropoiesis appears to be inconsistent with a role of FKLF-2 in the
regulation of a fetal gene such as the gene. However, it is well
established that there is gene expression in the adult stage of
erythropoiesis, and it is increased substantially in individuals with
various pathologic conditions.33 Therefore, expression in
adult bone marrow is not incompatible with the possibility that FKLF-2
functions as a gene activator.
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Footnotes |
Submitted September 29, 1999; accepted January 21, 2000.
Reprints: George Stamatoyannopoulos, Division of Medical
Genetics, University of Washington, Box 357720, Seattle, WA 98195;
e-mail: gstam{at}u.washington.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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References |
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Abstract
Introduction