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Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3730-3736
Changes in Chromatin Organization at the Neutrophil Elastase Locus
Associated With Myeloid Cell Differentiation
By
Edmond T.L. Wong,
Dieter E. Jenne,
Michael Zimmer,
Susan D. Porter, and
C. Blake Gilks
From the Department of Pathology and Laboratory Medicine, University
of British Columbia, Vancouver, British Columbia, Canada; and the
Department of Neuroimmunology, Max-Planck Institute of Neurobiology,
the Department of Neuroimmunology, Planegg-Martinsried, Germany.
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ABSTRACT |
Neutrophil elastase, proteinase-3, and azurocidin are primary
components of neutrophil azurophilic granules and are encoded by
closely linked genes (gene symbols ELA2, PRTN3, and AZU1, respectively) in a region of approximately 50 kb. These genes are coordinately expressed in a granulocyte-specific fashion, but the mechanisms defining this pattern of expression are unknown. To understand the role
of chromatin organization in governing the expression of ELA2, PRTN3,
and AZU1, we mapped this region of chromosome 19 and identified the
adipsin (complement factor D) gene in proximity to the 3' end of ELA2.
We then examined the changes in chromatin structure at the locus which
accompany myeloid cell differentiation and identified 17 DNase I
hypersensitive sites (DHS 1 to 17) in U-937 cells, an early
myelomonocytic cell line expressing high levels of neutrophil elastase.
Chemically induced differentiation and concomitant downregulation of
AZU1, PRTN3, and ELA2 transcription in U-937 cells is not accompanied
by changes in the DHS-pattern. Mature neutrophils, however, do not
carry any of these hypersensitive sites, indicating a large degree of
chromatin remodeling at this locus accompanying terminal granulocytic
differentiation. Sixteen of the 17 DHS identified in U-937 cells are
also present in the HL-60 myelomonocytic cell line. Hematopoietic cell
lines representing the early erythroid and lymphocyte lineages, and a
nonhematopoietic cell line display a subset of the hypersensitive
sites. The altered chromatin structure specific to cells that actively
transcribe the AZU1-PRTN3-ELA2 genes suggests that chromatin
reorganization is an important mechanism regulating the
myeloid-specific transcription of this gene cluster.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HUMAN NEUTROPHIL elastase (also known as
leukocyte elastase and medullasin), proteinase 3 (also known as
myeloblastin and AGP-7), and azurocidin (also known as CAP37) are major
components of the azurophilic (primary) granules of neutrophils.
Neutrophil elastase possesses a broad proteolytic spectrum, functioning
primarily as an antimicrobial agent against gram-negative
bacteria1; it is also capable of degrading many
extracellular matrix components and has thus been extensively studied
as a potential agent of tissue destruction in a number of diseases,
particularly those accompanied by evidence of inflammation and
connective tissue damage such as pulmonary emphysema,2-4
rheumatoid arthritis,5 cystic fibrosis,6 and
glomerulonephritis.7 Proteinase 3 has proteolytic
properties similar to neutrophil elastase and is believed to affect
myeloid cell differentiation8-10; it has been most widely
studied for its role as the autoantigen in Wegener's granulomatosis.11 Azurocidin is a serine protease homolog
with no proteolytic activity, but possesses bactericidal and
chemotactic properties.12
Transcription of the human neutrophil elastase, proteinase 3, and
azurocidin genes (gene symbols ELA2, PRTN3, and AZU1, respectively) is
restricted to the promyelocyte stage of granulocytic differentiation; expression ceases with differentiation to mature granulocytes, and is
coordinately downregulated on phorbol ester induction of U-937
myelomonocytic cells.13,14 AZU1, PRTN3, and ELA2 are structurally related, belonging to class 6 of the trypsin superfamily of serine protease genes, and are closely linked in a region of approximately 50 kb (the ELA2 locus).15 The mechanisms
governing the high-level of transcription of AZU1, PRTN3, and ELA2
specifically in myeloid cells that are committed to granulocytic
differentiation are poorly understood. Previous studies of ELA2 and the
mouse neutrophil elastase homolog have shown minimal 5' proximal
promoter regions, which appear to have myeloid specific, but not
necessarily stage-restricted activity in transient transfection
assays.16,17 The ELA2 promoter and mouse neutrophil
elastase promoter share a notable degree of homology, containing
consensus sequences for c-myb, C/EBP, and PU.1/GABP, which are
prevalent in many myeloid-specific promoters.18,19 An
enhancer of transcription has also been identified approximately 1 kb
upstream of the transcription initiation site of ELA2 with activity in
both myeloid and heterologous cell types.20
Cis-acting elements important in regulation of expression of
PRTN3 and AZU1 are less well characterized than those of ELA2; however,
the physical proximity of the 3 genes, their similar gene structure,
and coordinate pattern of expression suggest that the transcription of
these genes may be similarly regulated and perhaps under the influence
of common regulatory elements.
We have previously produced transgenic mice using a transgene whose
expression is driven by the ELA2 promoter and  1 kb enhancer region.
There was no detectable expression of the transgene in any of the lines
established, indicating that despite the apparent activity of the
proximal ELA2 promoter region in transient transfection assays, it is
not sufficient to reproduce the expression pattern of human neutrophil
elastase when stably integrated in vivo (E.W., unpublished
observations, July 1996). This implied that additional cis-regulatory elements are necessary for directing the high
levels of promyelocyte-specific expression of neutrophil elastase that are normally observed in vivo, when ELA2 is in the context of chromatin.
Chromatin structure has been shown to play an important role in the
transcriptional regulation of eukaryotic genes.21,22 DNase
I hypersensitive sites (DHS) in particular, indicate regions of
chromatin that are preferentially devoid of regular nucleosomal arrangement, and in many cases have been shown to have functional significance, often coinciding with active cis-transcriptional regulatory elements, origins of replication, matrix attachment sites,
and recombinational hot-spots.23 In the present study, we
characterize the genomic regions flanking the AZU1-PRTN3-ELA2 gene
cluster and identify a fourth member of the serine protease gene
family, adipsin (complement factor D, properdin factor D; gene symbol,
ADN) in close proximity to the 3' end of ELA2. We then identify regions
of open chromatin structure within and surrounding the
AZU1-PRTN3-ELA2-ADN (APEA) cluster of genes that are specific to cell
types that actively transcribe ELA2, PRTN3, and AZU1, indicating a
potentially complex mode of gene control, and identifying regions of
putative transcriptional regulatory activity.
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MATERIALS AND METHODS |
Cells.
U-937 (American Type Culture Collection [ATCC] CRL1593),
HL-60 (ATCC CCL240), HUT-78 (ATCC TIB161), Jurkat (ATCC TIB152), and
K-562 (ATCC CCL243) cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum (GIBCO-BRL, Gaithersburg, MD) and maintained at 37°C and 5% CO2; COLO 201 cells (ATCC CCL224) were
grown in RPMI 1640 medium supplemented with 20% fetal calf serum and
maintained under the same conditions. For induction of differentiation,
U-937 cells were cultured for 72 hours in the presence of 25 ng/mL TPA (12-O-tetradecanoyl-phorbol-13-acetate, also phorbol 12-myristate 13-acetate) (GIBCO-BRL). Polymorphonuclear granulocytes were isolated from heparinized donor blood from a healthy donor using Histopaque-1119 and -1077 (Sigma Diagnostics, St Louis, MO) double density
gradient centrifugation according to the manufacturer's directions.
DNase I hypersensitive site mapping.
Near-confluent cultures were obtained, and nuclei were isolated and
subjected to incremental DNase I digestion as previously described.24
Southern blotting.
Restriction-digested genomic DNA was separated using electrophoresis in
0.65% to 0.85% agarose, blotted onto Nytran Plus membrane (Schleicher
and Schuell, Keene, NH) and hybridized with [ -32P]dCTP
random primed labeled probes: The probes are derived from AZU1 and
PRTN3 human cDNA clones,13 from the human ELA2 cDNA clone,
a gift from M. Naruto (Kamakura, Japan),25 and through standard polymerase chain reaction (PCR) amplification of human cDNA
fragments as indicated in Fig 3. Hybridized blots were washed at a
final stringency of 0.1 × SSC/ 0.1% SDS for 2 hours and exposed to
Kodak X-AR film.
Isolation of RNA and Northern blot analysis.
Total RNA was isolated from cells with Trizol Reagent (GIBCO-BRL)
according to the manufacturer's directions. For Northern analysis, 10 µg of total RNA was denatured, separated on a 1% agarose-formaldehyde gel, transferred to Nytran Plus membrane, UV
cross-linked and hybridized as above. The same blots were then stripped
and hybridized with a human  -actin cDNA radiolabeled probe as described.
Isolation of genomic clones and restriction site mapping.
Because we suggested the location of ADN on chromosome 19p13.3 in close
vicinity to the AZU1-PRTN3-ELA2 gene cluster, a partial cDNA probe for
human adipsin (pDJ3) was generated by PCR using the primers DJ21
(5'-CTGGGGCATAGTCAACCA-3') and DJ34 (5'-TGTCGATCCAGGCCGCATAG-3) with
cDNA from U937 cells and was used to screen a chromosome 19-specific
cosmid library. Three positive clones, R27353, R32285, and R27805, were
identified and further characterized by restriction site mapping and
Southern blotting using BamHI, EcoRI, Hind III, and Sac I (Fig
1). The latter restriction sites were
mapped after linearization of cosmid DNA with lambda terminase and
partial enzyme digestion. On the basis of cDNA sequence,
oligonuceotides were designed to amplify genomic fragments from cosmid
R27805 using Taq polymerase and Elongase (GIBCO-BRL) to determine the size and location of the introns. Exon-intron boundaries were identified by directing sequencing of purified cosmid DNA using the
same primers.
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| Fig 1.
Restriction enzyme map of the AZU1-PRTN3-ELA2-ADN (APEA)
locus. Cosmids used to identify the relative position of the ADN gene
to the known genes in the locus are shown as thin horizontal lines
above the restriction map. Restriction sites mapped are indicated. (B,
BamHI; N, Not I; R, EcoRI; S, Sal I). Relative positions of the
identified exons are shown for ADN (thick lines), and also for AZU1,
PRTN3, and ELA2.
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RESULTS |
Physical relationship between the ELA2 and ADN genes.
Adipsin and complement factor D were shown to represent orthologous
gene products with identical functions in humans and
mice.26 The structure of the mouse adipsin gene has been
previously reported.27 Examining these exon-intron data we
noticed that the location and phase type of introns in mouse adipsin
was completely identical to that of other neutrophil serine protease
homologs found on human chromosome 19p13.3, but differed in a subtle
manner from other serine protease homologs clustered on 5q11-12 and
14q11-12.15 Moreover, many structural features like the
length of the putative propeptide and the number of cysteine bonds in
complement factor D were shared with known members of the 19p13.3
serine protease gene cluster. Thus we wished to prove our hypothesis
that complement factor D gene is a fourth member of the neutrophil
enzyme cluster on 19p13.3. As expected, screening of a human cosmid
library for chromosome 19 with the pDJ3 partial cDNA probe was
successful. The three cosmids R27805, R32285, and R27353 identified
were shown to contain identical restriction site fragments and
hybridized to the ADN cDNA probe in Southern blots.
Figure 1 shows the composite restriction site map for the 3 ADN-positive cosmids and the PRTN3-positive cosmid, PR3/1, previously reported.13 The latter cosmid appeared to overlap cosmid
R32285 and R27353 by a few kb (Fig 1). To confirm this overlap, both ends of the R32285 insert were sequenced. One end was found to match
the sequence of intron 3 of ELA2. Figure 1 also shows the location of
the 5 ADN exons. Exon-intron junctions of the ADN gene and
transcriptional orientation were the same as for AZU1, PRTN3, and ELA2.
To confirm the 5' end of the ADN cDNA and translational start site as
predicted from genomic sequences, we performed 5' RACE and compared
these cDNA sequences with those obtained from cosmid clones and an ADN
cDNA sequence previously reported (data not shown). Indeed, 7 additional codons including the first methionine codon were found at
the 5' end which, together with the following 12 amino acid residues,
make up the signal peptide of human ADN.
Models for analysis of chromatin structure at the AZU1/PRTN3/ELA2/ADN
(APEA) locus.
We chose to use the U-937 cell line as a model to study the chromatin
structure of the APEA gene locus because they are arrested at an early
stage of myeloid differentiation and express neutrophil elastase at
high levels.28 To determine whether chromatin structure at
the APEA locus is specific to cell lines that actively transcribe ELA2
(the human neutrophil elastase gene), we also studied nonmyeloid hematopoietic cell lines and a representative nonhematopoietic cell
line. HUT-78 is a mature T-cell line and Jurkat cells are lymphoid
cells derived from an acute T-cell leukemia. K-562 cells are derived
from erythromyeloblastic cells arrested at an early stage of
differentiation. COLO 201 cells are a nonhematopoietic, colonic
epithelial cell line. Among these cell lines, ELA2 transcription is
restricted to the myeloid cell type (Fig
2). Treatment of U-937 cells with the
phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA)
results in downregulation of AZU1, PRTN3, and ELA transcription. Although downregulation of ELA2 and PRTN3 in U-937 is complete in 5 days, we observed that the same cells still produce detectable though
greatly reduced levels of AZU1 transcripts. A previous study did not
detect differences in the levels of downregulation between the 3 genes13 and may be because of differences between U-937
sublines, to prolonged TPA exposure, or to differences in culture
conditions. ADN exhibits a less restricted pattern of expression than
ELA2, PRTN3, and AZU1. ADN transcripts are present in all the
hematopoietic cell lines examined and are also downregulated to low
levels in TPA activated U-937 cells like AZU1 transcripts, but are not
detectable in the nonhematopoietic cell line COLO 201. The level of ADN
transcription in TPA-treated U-937 cells is similar to that of other
cultured hematopoietic cell lines. HL-60 cells were examined to confirm
the presence of the DHS identified in U-937 cells. HL-60 cells are also
myelomonocytic cells, which express high levels of NE.28
Mature peripheral blood granulocytes do not express any of the 4 genes
of this cluster (not shown).
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| Fig 2.
Expression of the AZU1-PRTN3-ELA2-ADN cluster of genes in
cell lines studied. Northern analysis was performed on U-937 cells,
U-937 cells treated with 25 ng/mL TPA for 3 days (TPA), COLO 201 (COLO), HUT 78 (HUT), Jurkat, and K-562 cell lines and hybridized to
32P-labeled human ELA2, AZU1, PRTN3, ADN, and human
-actin cDNA probes as indicated for each blot.
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DNase I hypersensitive mapping of the APEA gene cluster in neutrophil
elastase expressing cells.
We performed DHS analyses on a greater than 50 kb region surrounding
the APEA gene cluster. DHS analysis of more distant DNA was not
performed because no serine protease gene sequences were detected
beyond this gene cluster within 50 kb in either direction using PCR
primer pairs for conserved serine protease gene sequences. During the
process of locating genes and ESTs on our cosmid contig, we identified the gene for polypyrimidine tract binding protein (PTB)
on the telomeric side of AZU1 and the human transcript Hs.24441 (Unigene designation) on the centromeric side of ADN (data not shown),
indicating that the region encompassing the 4 serine protease genes
represents a complete multigene locus. An array of distinct DHS are
detected at the APEA locus in U-937 cells, within a region from 15 kb
upstream of the AZU1 gene to approximately 15 kb centromeric to ADN.
Multiple overlapping restriction enzyme series were used to confirm the
location of each DHS (Fig 3). Typical blots
are shown, showing each of the 17 DHS observed (Figs 4 and
5). All DHS
present in U-937 cells were present in HL-60 cells, with the exception
of DHS-16. DHS-12 (Fig 4A) and 14 (Fig 5) appear as doublets or
triplets on some Southern blots; on others they appear as a single
intense band and they have each been considered to be a single DHS.
Although analysis of nuclease hypersensitivity is not a quantitative
technique, differing intensities of each DHS relative to adjacent sites
on the same blot can be recognized. DHS-4, 5, 10, and 12 coincide with
the promoter regions of AZU1, PRTN3, ELA2, and ADN, respectively (Figs
2, 4A, 4B, and 5). Two sets of hypersensitive sites, DHS-1 to -4 and
DHS-16 and -17, flank the locus (Figs 2, 4A and 4C). DHS-6 is located
within the PRTN3 gene (Figs 2 and 4B), and DHS-11 appears in the 3.5 kb
region between ELA2 and ADN (Figs 2, 4D, and 5). DHS-9 is a
particularly prominent site in U-937 and HL-60 cells, appearing
approximately 2 kb upstream of ELA2 (Fig 4B). DHS-13 and -15 are very
weak sites in U-937 cells, appearing stronger in nonexpressing cells
such as COLO 201 (Fig 5).
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| Fig 3.
Scale map of DHS in U-937 cells (vertical arrows)
relative to the AZU1, PRTN3, ELA2, and ADN genes and to restriction
fragments used for DHS mapping (short vertical lines), (A, Hpa I; B,
BamHI; C, Sca I; G, Bgl II; L, Bcl I; M, Mlul; R, EcoRI; X, Xbal, and
Y, BssHII). Horizontal arrows depict orientation of end-labeled
detection of DHS. Exons from the 4 genes used as probes for DHS
analysis are shown (half-boxes) for each restriction fragment used for
DHS mapping. The EcoRI fragment encompassing AZU1 extends 24 kb beyond
the AZU1 gene and the EcoRI fragment at the ADN end of the locus is 31 kb. Multiple analyses using different restriction fragments were used
to confirm the location of the DHS in U-937 and in other cell lines.
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| Fig 4.
Analysis of chromatin structure at the
AZU1-PRTN3-ELA2-ADN (APEA) locus in untreated U-937 cells. Nuclei were
prepared and treated with increasing concentrations of DNase I (0 to 5 µg/mL, increasing from left to right in each case). The location of
molecular weight DNA markers are shown on the left, in kilobases. DHS
are indicated by solid arrowheads, with DHS-1 lying toward the AZU1
portion of the locus and DHS-15 at the ADN end of the locus. (A) DHS-1
to -4. DNase I-treated DNA was digested to completion with EcoRI and
hybridized with a PCR amplified, 32P-labeled AZU1 exon 4/5
cDNA probe. (B) DHS-5 to -10. DNase I-treated DNA was digested to
completion with Mlu I and hybridized with a PCR-amplified ELA2 exon
I/II probe. (C) DHS-16 and -17. DNase I-treated DNA was digested to
completion with Eco RI and hybridized with a 32P-labeled
ADN cDNA probe. (D) DHS-11 and -12. DNase I-treated DNA was digested
to completion with Bam HI and hybridized with a 32P-labeled
ELA2 exon IV/V probe.
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| Fig 5.
Analysis of DHS-11 to -16 in COLO 201, U-937, and HL-60
cells. All lanes are DNase I-treated DNA digested to completion with
Bgl II and hybridized with a 32P-labeled ELA2 exon IV/V
cDNA probe. From left to right: C0, COLO 201 DNA not treated with DNase
I; C1 to C3, COLO 201 DNA from nuclei treated with increasing
concentrations of DNase I; H, HL-60 DNase I treated time-point; U,
U-937 DNase I treated time-point. The locations of the DNA molecular
weight markers used are shown on the left, and the location of DHS-11
to -16 are indicated by arrowheads on the right.
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Differential chromatin structure at the APEA cluster of genes in
cells that do not express neutrophil elastase.
Treatment of U-937 cells with TPA has previously been shown to induce
partial differentiation of these cells.29 Although TPA-induced differentiation is accompanied by downregulation of ELA2,
PRTN3, and AZU1 transcription, the DHS pattern at the APEA locus in
these cells is identical to that of untreated, expressing U-937 cells
in terms of position and relative intensity (Table 1 and not shown), indicating that change in
chromatin structure at this locus is not necessary for transcriptional
downregulation of the ELA2, PRTN3, and AZU1 genes. The DHS observed in
the early myeloid U-937 cell line, however, are not present in mature
peripheral neutrophils (PMN), evidence that chromatin reorganization at
this region accompanies terminal neutrophilic differentiation. In the nonmyeloid hematopoietic cell lines studied, only a subset of the DHS
observed in U-937 cells exist (Table 1): DHS-1 appears only in the
erythroid line, K-562. DHS-2 appears in both K-562 and COLO 201 cells.
DHS-16 appears in COLO 201 and also in HUT-78 cells but is not present
in HL-60 cells. The other sites, DHS-3 to -10, appear exclusively in
the early myeloid cell type. DHS-17 is present in COLO 201 cells and in
all the hematopoietic lineages but not in mature neutrophils and is the
only site common to all the hematopoietic and nonhematopoietic
lineages. The sites located within the ADN gene are present in all cell
lines examined, with the exception of mature neutrophils.
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DISCUSSION |
Studies to date on the regulation of transcription of the human
neutrophil elastase and proteinase 3 genes (ELA2 and PRTN3, respectively) have focused only on the proximal promoter regions of
these genes and the putative trans-acting factors that may interact with these regions. Although the ELA2 and PRTN3 promoters have
functional activity in transient transfection assays, they are unable
to direct expression of a transgene in vivo, indicating that additional
cis-regulatory elements are necessary to reproduce the
endogenous pattern of expression of neutrophil elastase and proteinase
3 when stably integrated into chromatin. An "active" chromatin
conformation is an important factor in permitting high-level, cell-type
specific, and position-independent expression of stably integrated
genes.30-32
To show changes in chromatin structure at the ELA2 locus with probes
from the flanking regions, we extended the map beyond the 3' UTR of the
ELA2 gene and identified an additional member of the serine protease
gene family, which encodes human adipsin (also complement factor D). In
contrast to AZU1, PRTN3, and ELA2, adipsin is not found in granules of
neutrophils, but is present in the circulation at relatively high
levels of 50 µg/mL. The enzyme is mainly synthesized and secreted by
adipocytes, but also by monocytes and macrophages and is already
converted into the mature form before secretion.33
Adipsin/factor D is a key regulator of the alternative pathway of the
complement system with absolute substrate specificity for factor B as a
component of the reversible C3bB complex. It only acquires its
proteolytically active conformation during cleavage of the C3bB
complex. Although adipsin has not been regarded as a genuine member of
the hematopoietic serine protease gene family previously, exon-intron
organization of the adipsin gene (ADN) is completely conserved,
indicating that it is indeed derived from a common ancestor for
hematopoietic serine proteases. In support of this view is the finding
that adipsin is also expressed by monocytes and macrophages, but not by
human or rodent hepatocytes, which synthesize all other components of the complement system. Furthermore, adipsin and the other neutrophil enzymes are still related in their biological functions in that all
these serine protease homologs contribute to bactericidal defense
mechanisms.9,12,34
In the present study, we observed an altered chromatin conformation at
the APEA locus in cells that are actively transcribing the ELA2, PRTN3,
and AZU1 set of genes. The specificity of DHS-3 to -10 to both U-937
and HL-60 cells, which express the ELA2, PRTN3, and AZU1 genes,
indicates that they may be involved in transcriptional upregulation of
these genes; however, TPA-induced downregulation of ELA2, PRTN3, and
AZU1 transcription is not accompanied by a corresponding reorganization
of the active chromatin conformation. These observations indicate that
although active chromatin conformation appears to be an important event
in permitting gene expression of ELA2, PRTN3, and AZU1, it is not
sufficient in itself to result in normal levels of myeloid specific
transcription. This observation contrasts with the myeloperoxidase
(MPO) model, in which chemically induced granulocytic differentiation
and downregulation of expression of MPO is associated with loss of 3 DHS in the region proximal to the 5' end of the gene.35,36
Although there are regions of extensive homology between the promoters
of MPO and ELA2,37 the promoters of ELA2, PRTN3, and AZU1
possess a TATAA consensus sequence, unlike the promoters of MPO and the
majority of other myeloid-specific gene promoters that have been
studied. Furthermore, unlike expression of the AZU1, PRTN3, and ELA2
genes, MPO expression is not specific to the neutrophilic path of
myeloid differentiation but is also expressed during monocytic
differentiation. The significance of the differences between chromatin
remodeling at the ELA2 and MPO loci in relation to potential
similarities or differences between their mechanisms of transcriptional
regulation is presently unknown. DHS-11 to -15 are present in both
myeloid and nonmyeloid cell types, which, together with their location
in or adjacent to the ADN gene, suggests a role in the positive
regulation of this gene.
The absence of observable change in chromatin organization in
TPA-treated U-937 cells suggests that downregulation of the AZU1,
PRTN3, and ELA2 genes induced by TPA is likely to be a consequence of
the effects of TPA on trans-acting factors involved in the transcription of these genes. C/EBP and c-myb are candidate
transcription factors that may be involved in TPA-mediated
downregulation of these genes. Both C/EBP and c-myb are expressed in
early myeloid cells and their levels are decreased on differentiation.
Consensus binding sites for C/EBP factors are present in the promoters
of all 3 genes and c-myb binding sequences are present in the promoters of ELA2 and PRTN3; both C/EBP and c-myb consensus sequences are important in ELA2 and PRTN3 promoter activity, indicating a role for
these factors in mediating transcription of these
genes.38,39 In particular, TPA-induced differentiation in
HL-60 cells has been shown to reduce c-myb levels.40 The
ets factors PU.1 and GABP are also likely to be involved in
transcriptional regulation at the ELA2 locus. Ets binding sites are
present in the promoters of ELA2 and PRTN3, and mutation of this site
decreases promoter function.41 TPA-induced differentiation
of U-937 cells is accompanied by phosphorylation of PU.1, affecting
PU.1 binding activity.42 Treatment with a protein kinase C
inhibitor antagonizes TPA-induced differentiation, indicating that
decrease in ELA2 and PRTN3 gene expression after TPA treatment in U-937
cells may be mediated in part by alteration of PU.1 activity. Hence,
though transcription at the locus may require an active chromatin
structure, regulation by trans-acting factors alone may be
sufficient for mediating the initial downregulation of transcription in
myeloid cells accompanying differentiation of the promyelocyte. The
broad similarity in the pattern of nuclease hypersensitivity between
HL-60 and U-937 cells in comparison to nonmyeloid cells, however,
suggests that this pattern of chromatin organization is likely to be
important in regulating expression of the genes at this locus.
Although the AZU1, PRTN3, and ELA2 genes can be downregulated in
myeloid cells in the presence of an active chromatin conformation, it
is possible that an "inactive" chromatin conformation
characterized by the absence of DHS seen in nonmyeloid cell types may
be important in preventing aberrant expression of these genes. This
would allow transcriptional silencing of the AZU1, PRTN3, and ELA2
genes even in cell types where C/EBP, c-myb, PU.1, or other
trans-acting factors involved in transcription of these genes
are present. Because TPA-induced transcriptional downregulation in
U-937 cells is not accompanied by change in the active chromatin
conformation of the APEA locus, loss of the DHS accompanying terminal
neutrophil differentiation occurs subsequent to transcriptional
downregulation and may possibly serve as an additional barrier against
aberrent expression of these genes. The biological significance of such a stringent control of transcription of these genes is substantiated by
the destructive nature of neutrophil elastase and proteinase 3 and
their potential contribution to inflammatory-related disease. Specific
downregulation of expression at the APEA locus mediated by inactivation
of transcriptionally permissive chromatin structure is a potential
means through which this repression may be implemented.
The physical proximity, common spatial and temporal pattern of
expression, homologous gene structure, and coordinate downregulation of
AZU1, PRTN3, and ELA2 suggest that the mechanisms governing the
transcriptional regulation of these genes are related and exist in a
common transcriptional regulatory domain. If regulation of
transcription of these genes is indeed governed by common
cis-acting elements, then it is conceivable that an insulatory
element exists between the ELA2 and ADN gene, as ADN exhibits a pattern
of expression that is distinct from the other genes in the region.
Studies in other model systems have shown the existence of such
insulatory elements that can serve to delimit transcriptional
units.43 The most distal 3' DHS, DHS-17, is present in all
the cell types examined except mature neutrophils. It may perhaps serve
an analogous role to the chicken -globin constitutive DHS at the 5'
domain border, which insulates the domain from neighboring chromatin and transcriptional regulatory influences.44 Functional
characterization of the DHS at this locus both in vitro and in
transgenic animals will show their associated functions, some of which
are likely to be involved in mediating the stringent, high-level
expression of ELA2, PRTN3, and AZU1 in granulocytic precursors in vivo.
The cathepsin G cluster of genes on chromosome 14 shares structural
similarities with the ELA2 group of genes, consisting of the granzyme
B, granzyme H, cathepsin G, and mast cell chymase genes. Cathepsin G is
expressed concomitantly with ELA2.45 A 6-kb genomic
fragment encompassing the cathepsin G gene with 2.7 kb of 5' flanking
DNA has been shown to direct myeloid-specific expression when used as a
transgene46 whereas a 1.2-kb 5' flanking fragment of
granzyme H was shown to direct expression to lymphokine-activated natural killer cells.47 Further functional analysis of the
DHS at the ELA2 locus or chromatin structural analysis of the cathepsin G locus may show potential similarities or differences in how these 2 structurally similar loci are regulated.
| |
ACKNOWLEDGMENT |
We are grateful to Heike Reimann for excellent technical assistance and
to Dr H. Mohrenweiser, Lawrence Livermore National Laboratory,
Livermore, CA, for screening their chromosome 19 library with pDJ3.
| |
FOOTNOTES |
Submitted November 5, 1998; accepted July 22, 1999.
Supported by grants from the British Columbia Health Research
Foundation, Medical Research Council of Canada, and
Sonderforschungsbereich 469.
Address reprint requests to C. Blake Gilks, MD, ACU
Building, G227-2211 Wesbrook Mall, Vancouver, BC, Canada, V6T
2B5.
| |
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