Download The chicken lysozyme chromatin domain contains a

© 2002 Oxford University Press
Nucleic Acids Research, 2002, Vol. 30, No. 2
463–467
The chicken lysozyme chromatin domain contains a
second, widely expressed gene
Suyinn Chong, Arthur D. Riggs and Constanze Bonifer1,*
Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA and
1Molecular Medicine Unit, St James’ University Hospital, University of Leeds, Leeds LS9 7TF, UK
Received October 3, 2001; Revised and Accepted November 20, 2001
ABSTRACT
The chicken lysozyme (cLys) locus has been shown to
contain all of the cis-elements necessary for positionindependent and tissue-specific expression entirely
within a 24-kb region defined by general DNase I
sensitivity and flanked by matrix attachment regions.
As such, it has been viewed as an example of a functional chromatin domain, which is structurally and
functionally isolated from neighbouring chromatin.
We report here the identification and characterisation of the chicken glioma-amplified sequence
(cGas41) locus, which though widely expressed, is
contained entirely within the lysozyme chromatin
domain. The cGas41 transcript encodes a putative
transcription factor, starts 207 bp downstream of the
cLys polyadenylation site and is preceded by a CpG
island with proposed dual promoter/origin function.
The location and differential expression of cGas41
compels re-evaluation of the accumulated literature
on the lysozyme domain, and represents an example
of two unrelated, differentially expressed vertebrate
genes coexisting in the same functional chromatin
domain.
INTRODUCTION
Functional chromatin domains in vertebrates have been
defined as extended regions of ‘open’, DNase I-sensitive
chromatin that contain a gene or a related gene cluster with all
the cis-elements necessary for their appropriate expression as
transgenes. This definition also includes that such regions of
general DNase I-sensitive chromatin are flanked by domain
boundary/insulator elements, which act to segregate or protect
against chromosomal position effects (1). Three prominent
examples of such functional domains are the chicken lysozyme
locus, and the chicken and human β-globin loci (reviewed in
1,2). However, the genome-wide relevance of this model has
been questioned, as a number of characterised loci are not
organised into structurally confined regulatory units (reviewed
in 3). Evidence is mounting that a functional gene domain can
be equally well defined by the gene-specific interaction of
cis-elements (reviewed in 4).
DDBJ/EMBL/GenBank accession no. AF410481
Chicken lysozyme is expressed in the oviduct and in myeloid
cells, where expression progressively increases during macrophage differentiation. In these tissues, lysozyme is located in a
24-kb domain of general DNase I sensitivity, whose 5′ and 3′
boundaries are 14 kb upstream and 6 kb downstream, respectively, of the lysozyme transcription start (1,5). The transition
from increased to decreased DNase I sensitivity at each
domain boundary coincides with matrix attachment regions
(MARs) (6). Consequently, the lysozyme chromatin domain
was thought to be structurally isolated. It was hypothesised that
this separation from the chromosomal environment is required
for the appropriate expression of the lysozyme gene (1,6). This
hypothesis was supported by the finding that a reporter gene
was buffered from position-effects when flanked by the 5′ MAR
and stably transfected into cultured cells (7). Several DNase I
hypersensitive sites (DHS) have been identified around the
lysozyme coding region, most of which correspond to discrete
cis-elements that regulate the spatial and temporal expression
of lysozyme, such as enhancers, a silencer, hormone response
element, a promoter (reviewed in 8) and a replication origin
(9).
Bonifer et al. (10) demonstrated that transgenic mice
carrying multiple copies of a 21.4-kb domain fragment
randomly integrated into the genome, expressed lysozyme in a
copy number-dependent manner, leading to the conclusion that
the chicken lysozyme chromatin domain acted as a functional
regulatory unit. Deletion analysis experiments aimed at localising the elements responsible for this feature revealed that
position independence was conferred by the combined effect
of several upstream tissue-specific cis-elements and the
promoter (11). However, questions remain about how the
tissue-specific activity of the lysozyme locus is assured at the
molecular level. Subsequent studies have, for that reason,
focused on the molecular interactions at various cis-elements,
particularly at the level of chromatin fine structure (12) and
non-histone protein–DNA associations (reviewed in 13).
We report here that a BLASTN search of the complete
genomic sequence of the chicken lysozyme domain resulted in
the discovery of a widely expressed second gene, cGas41, that
is highly homologous to a ubiquitously expressed human gene
of unknown function, but which is often amplified in gliomal
tumors (glioma-amplified sequence; GAS41). We also report
the preliminary characterisation of cGas41 and discuss the
*To whom correspondence should be addressed. Tel: +44 113 206 5676; Fax: +44 113 244 4475; Email: [email protected]
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Nucleic Acids Research, 2002, Vol. 30, No. 2
implications of its proximity to cLys, as well as its general
location within the lysozyme chromatin domain.
MATERIALS AND METHODS
Cell lines, tissues and RNA preparation
Cell lines MEP, HD37, DT40, MSB1, BM2 and HD11 were all
grown in Iscove’s modified Dulbecco’s medium containing
L-glutamine (Gibco BRL), 8% foetal calf serum, 2% chicken
serum, 100 U/ml penicillin, 100 mg/ml streptomycin and
0.15 mM monothioglycerol. Mouse macrophages carrying cLys/
cGas41 were obtained by the differentiation in vitro of bone
marrow progenitor cells obtained from adult transgenic mice
as described previously (10). Total RNA was prepared from
cultured cells and tissues with Trizol Reagent (Gibco BRL),
according to the manufacturer’s instructions.
RT–PCR
A 5–10 µg sample of intact total RNA was treated with 1 U/µg
RQ1 RNase-free DNase (Promega) in a total volume of 50–100 µl
for 10 min at 37°C followed by heat inactivation of the enzyme
at 65°C for 10 min. cDNA was prepared from the RNA pellets
with M-MLV reverse transcriptase (Gibco BRL) as recommended by the manufacturer, using 250 ng of random primers
(Gibco BRL). As a result of its GC-richness, PCR of cGas41
was performed with HotStarTaq DNA polymerase and Q-solution (Qiagen). cLys and control (chicken β-actin, mouse
GAPDH) PCR reactions were all performed as described
previously (12). All PCR reactions, containing ∼5% of each
cDNA mixture in a total volume of 30 µl, were amplified for
30 (cGas41), 30 (cLys), 25 (chicken β-actin) or 20 cycles
(mouse GAPDH). The cGas41 PCR primers are identical to
the gene-specific, forward inner primer and a reverse outer
primer used for 5′ RACE below. RT–PCR products were
resolved on 1.5–2.5% agarose gels.
5′ and 3′ RNA ligase-mediated rapid amplification of
cDNA ends
RNA ligase-mediated rapid amplification of cDNA ends
(RLM-RACE) was performed with Ambion’s FirstChoice
RLM-RACE kit using either DT40 (5′ RACE) or MSB1
(3′ RACE) total RNA according to the manufacturer’s instructions, except that the 5′ RACE inner adapter primer was not
used due to mispriming problems. Additional gene-specific
PCR primers used for 5′ RACE included a reverse outer
primer, 5′-ACCGTCCACTGATGCGTGTG-3′, as well as an
inner primer set, forward 5′-ATGTTCAAGAGAATGGCTGAG-3′ and reverse 5′-ATTGGTACCTACACTACCGGCTTCACGAT-3′. Similarly, gene-specific primers designed for
3′ RACE included a forward outer primer, 5′-AAACTCGAAGAGGATGATCAGTC-3′, and inner primer, 5′-CGGGGTACCAAAGATATGTGATGAGTGTTG-3′. KpnI restriction
sites incorporated into PCR primers are underlined. RACE
products were either sequenced directly (5′ RACE) or
subcloned into pBluescript II KS+ and then sequenced
(3′ RACE).
RESULTS
We recently completed the sequence of a fragment carrying the
chicken lysozyme chromatin domain (10; submitted to the
GenBank database under accession no. AF410481). This
sequence was used in a BLASTN search of the National Center
for Biotechnology Information (NCBI) non-redundant nucleotide
database. To our surprise, high percentage identity to human
glioma-amplified sequence (GAS41, GenBank accession
no. NM_006530) was observed at several discrete sequences
in the 3′ half of the lysozyme domain. The finding that each
region of homology corresponded to individual human GAS41
exons suggested that we had identified the chicken orthologue
of this gene. As can be seen in Figure 1A, the first sequence
with homology to human GAS41 (exon 1, 92% identity over
38 bp) is located immediately 3′ to the chicken lysozyme
coding region, in a CpG island reported previously to function
as an origin of replication (9). Other sequences in the lysozyme
domain exhibiting homology to human GAS41 are located
further downstream (exon 4, 88% identity over 50 bp; exon 5,
86% identity over 93 bp; exon 7, 84% identity over 151 bp)
and extend into the 3′ MAR (Fig. 1A).
Human GAS41 was initially identified as part of a multigene
region at 12q13.15 that was amplified and expressed in
gliomas (14). As a result of its homology to human AF-9 and
ENL genes, it is believed to be a transcription factor (15,16).
The chicken Gas41 genomic locus spans >2.8 kb and consists
of seven exons and six introns. All of the exon/intron junctions
except one, at the 3′ end of intron 4, conform to the GT/AG
rule. cGas41 encodes either a 223 or 227 amino acid protein,
depending on which of two possible translation start sites are
used, and a multiple sequence alignment using the Pileup and
Pretty programs from the Genetics Computer Group sequence
analysis software package illustrates that GAS41 is highly
conserved, with cGas41 exhibiting 97.4 and 96.5% identity to
human and mouse Gas41, respectively, at the amino acid level
(Fig. 1B). Interestingly, the proximity of lysozyme and GAS41
is conserved in humans, where they are located 5.53 kb apart
on 12q14.3.
Figure 2 details the results of RT–PCR analyses of endogenous cGas41 expression in various chicken tissues and cell
lines. The forward and reverse PCR primers were designed to
anneal to exons 1 and 2, respectively, so that PCR products
containing the intervening intron 1 region could be easily identified. In Figure 2A, several chick and adult hen tissue samples
were tested, all of which were positive for cGas41 expression.
The two bands obtained for cGas41 correspond to spliced and
unspliced transcripts. No such PCR products were obtained in
control reactions done without reverse transcriptase (data not
shown). In contrast, cLys is only expressed in adult hen oviduct
tissues. Faint bands corresponding to lysozyme expression in
all the chick tissues were most likely due to the inevitable
contamination of tissues with peripheral blood (and therefore
macrophages) at harvest. This was confirmed by analysis of six
chicken cell lines grown as pure cultures, corresponding to
multipotent myeloid progenitor cells (HD50 MEP), erythroblasts (HD37), B cells (DT40), T cells (MSB1), monocytes
(BM2) and macrophages (HD11) (Fig. 2B). cGas41 is clearly
expressed to a similar extent in all the chicken cell lines tested,
whilst cLys exhibits its characteristic tissue and developmental
Nucleic Acids Research, 2002, Vol. 30, No. 2
Figure 1. (A) Summary of important features in the cLys chromatin domain.
The open sharp-ended box at top represents the region of general DNase I sensitivity. Below this, MARs and the CpG island are indicated by black and
hatched boxes, respectively. Vertical arrows indicate sites of DNase I hypersensitivity, and the orientation of cLys and cGas41 are also shown. At the bottom,
cGas41 has been expanded to show individual exons and introns; filled boxes
represent those exons initially identified by homology to human GAS41 in the
BLASTN search. (B) Multiple alignments of GAS41 amino acid sequences.
The complete amino acid sequence of human GAS41 is shown, with asterisks
designating the two possible translational start sites. The mouse and chicken
gas41 sequences are shown below. A consensus mouse gas41 amino acid
sequence was generated from multiple entries in the GenBank database (accession nos BE448250, BF022066, AW210316, AW412092, AW476549 and
BF011985). Only those amino acids that differed from the human are shown,
those that are identical to human are marked by a dash (–).
stage-specific expression profile, with a low level of expression in
monocytes and a high level of expression in macrophages. In
summary, endogenous cGas41 is widely expressed and, whilst
the range of tissues tested was not exhaustive, a cGas41 nonexpressing tissue was not identified. Expression of chicken
Gas41 thus seems to be similar to the ubiquitous expression
seen for human GAS41.
RLM-RACE was used to determine the 5′ and 3′ ends of the
cGas41 transcript and the results are shown in Figure 3. The
full-length cGas41 transcript is comprised of 1125 nt, and is
contained entirely within the chicken lysozyme chromatin
domain. More specifically, cGas41 transcription is in the same
orientation as cLys and starts 207 bp downstream of the
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Figure 2. Expression analysis of endogenous chicken gas41 and cLys in (A) a
range of chicken tissues, (B) cell lines and (C) tissues of transgenic mice as
well as in transfected (Tr) or non-transfected (nTr) mouse embryonic stem
(ES) cells. Tissues: Ov, Oviduct; Ov.m, oviduct magnum; Br.m, breast muscle;
H, heart; L, liver; K, kidney; I, intestine; T, testis; B, brain; Sk.M, skeletal muscle; U, uterus; O, ovar; Mac, macrophages. Cell lines: MEP, HD50 MEP
multipotent precursor cells; HD37, erythroblasts; DT40, B cells; MSB, T cells;
BM2, monocytes; HD11, macrophages. RT–PCR was carried out described in
Materials and Methods. M, markers; *, unspliced transcript. Unspliced cGas41
was not detected in chicken cell lines. Chicken β-actin or mouse GAPDH, was
used as a control, respectively.
lysozyme polyadenylation site, in a previously characterized
unmethylated CpG island (9). As such, cGas41 appears to have
a CpG island promoter typical of constitutively expressed
genes, including a 26-bp GC-box with perfect dyad symmetry
(17). At the 3′ end, cGas41 exons 5–7 and the 3′ untranslated
region are all either partially or fully localized within a previously characterized MAR (6). In accordance with its widespread expression, the CpG island promoter of cGas41
displays a DHS in all tissues tested so far (18,19). These earlier
DHS mapping studies as well as experiments performed in our
laboratory studies also demonstrated that probes covering most
of cGas41 do not cross-hybridise with any other gene in the
chicken genome (M.Huber and C.Bonifer, unpublished observation).
A transgenic mouse line, TgH(cLys)3, carrying a single copy
of the 21.4-kb cLys/cGas41 chromatin domain targeted to the
X-linked mouse Hprt locus, and created to further examine
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Nucleic Acids Research, 2002, Vol. 30, No. 2
Figure 3. Summary of results from 5′ and 3′ RLM-RACE. The 2.8-kb cGas41
transcript is shown at top, with each of the seven exons indicated by a black
box. Expanded sections of the 5′ and 3′ ends are shown below. Asterisks designate the two possible ATG initiation codons at the 5′ end. At the 3′ end, a
region matching the consensus polyadenylation signal (AATAAA) is underlined, followed by A(n) at the polyadenylation site. The novel sequence data
reported in this manuscript have been submitted to the GenBank database
under accession no. AF410481.
chicken lysozyme regulation, was used to determine whether
cGas41 expression could be reproduced in the mouse genome
(S.Chong, J.Kontaraki, C.Bonifer and A.Riggs, manuscript in
preparation). Previous studies revealed expression of the
chicken lysozyme transgene only in the brain and macrophages
(10,11; data not shown). In contrast, the results in Figure 2C
show that the expression of the cGas41 transgene is detectable
in all tested mouse tissues. Control experiments with the
embryonic stem (ES) cell lines used to generate the transgenic
mice and non-transgenic ES cells demonstrate that cGas41
expression originates exclusively from the transgene.
DISCUSSION
cGas41 was identified when a BLASTN comparison of the
21.4-kb chicken lysozyme chromatin domain to the NCBI
nucleotide database revealed high homology to human GAS41
cDNA. The cGas41 genomic locus is at least 2.8 kb and
consists of seven exons. A 1.1-kb cGas41 mRNA is produced
in the same orientation as cLys transcription, and encodes a
223–227 amino acid protein, which exhibits at least 96% identity
to human and mouse Gas41 at the amino acid level. Accordingly,
an avian example can be added to the growing list of highly
conserved GAS41 proteins of fungal, yeast, plant and mammalian
origin (16). Human GAS41 is a nuclear protein that has a
proposed role as a transcription factor, but no apparent
DNA-binding domain (15,16).
Human GAS41 produces a single transcript of ∼1.7 kb,
which is present in a wide range of human tissues (16). Likewise, RT–PCR analysis of endogenous cGas41 revealed its
constitutive expression in a range of adult hen and chick tissues
as well as chicken cell lines. Initiation of cGas41 transcription
occurs 207 bp downstream of the cLys polyadenylation site.
This is intriguing for a number of reasons. First, the 5′ end of
cGas41 coincides with a region characterised previously both
as a CpG island and replication origin (9). Unmethylated CpG
islands are commonly associated with the promoters of housekeeping genes (20), and this report establishes that the CpG
island at the 3′ end of cLys is, similarly, associated with the
promoter of the widely expressed cGas41. It has also been
shown that many CpG islands are origins of replication
(21,22). Correspondingly, the GC-rich region between cLys
and cGas41 represents a specific example of a CpG island with
dual promoter/origin function. Moreover, as it was reported
that replication at this site is bidirectional and initiates early in
S phase in both lysozyme-expressing and non-expressing cells
(9), the question arises whether in this case a common replication
origin is used for both a constitutive gene and a tissue-specific
gene.
The most important result from this study is our finding that
a highly expressed, tissue-specific gene and a widely expressed
gene with a housekeeping promoter coexist in close proximity
on the same structurally defined chromatin domain. Moreover,
the expression in several tissues of a cLys/cGas41 transgene in the
mouse genome suggests that all of the required cis-regulatory
elements may be present on the transgene and thus in the
chromatin domain. Support for this notion comes from earlier
DHS mapping experiments (18,19). A second, constitutive
DHS is located 4 kb downstream of the cGas41 transcription
start site and is contained within the transgene. No other DHS
is found within the next 15 kb downstream of this site. Our
results also invite a re-evaluation of the literature regarding the
definition of a domain of general DNase I sensitivity. The
example presented here suggests a strong link between gene
expression levels and the degree of nuclease sensitivity across
the entire chromatin domain, as in cLys/cGas41 expressing
cells a significant difference in nuclease sensitivity could be
detected as compared with cells expressing cGas41 only (1,5).
A similar link between promoter activity and general DNase I
sensitivity is also apparent in the β-globin locus where intergenic
transcripts were identified that may be implicated in the generation of open chromatin in this locus (23).
Our data provide an example of two unrelated, differentially
expressed vertebrate genes coexisting in the same functional
chromatin domain. The implications to chicken lysozyme
regulation, in particular, and functional chromatin domains, in
general, illustrate the power of genomics to both alter established view points and to open up new avenues of research.
ACKNOWLEDGEMENTS
The authors thank Gerd Pfeiffer for valuable insight. The
authors also thank Dr Helen Sang, Roslin Institute, for preparation of chicken and hen tissues. This work was funded by a
grant from the National Insitutes of Health (GM50575) to
A.D.R. and grants from the Wellcome Trust, the BBSRC and
the Leukaemia Research Fund to C.B.
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