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Blood, 1 February 2002, Vol. 99, No. 3, pp. 1082-1084
BRIEF REPORT
Regulation of fetal versus embryonic gamma globin genes:
appropriate developmental stage expression patterns in the
presence of HS2 of the locus control region
Timothy Yu,
David M. Thomas,
Wei Zhu,
Morris Goodman, and
Deborah L. Gumucio
From the Department of Cell and Developmental Biology,
University of Michigan Medical School, Ann Arbor, and the Department of
Anatomy and Cell Biology, Wayne State Medical School, Detroit, MI.
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Abstract |
The  genes provide the major contribution to beta-like
globin chain production in the fetal liver of humans. However, the expression of genes in the fetus is a recent evolutionary trend seen only in the primate lineage. In a previous study, it was shown
that galago and human genes retain their characteristic stage-specific expression patterns in transgenic mice (galago is
expressed exclusively in the embryo, whereas human is expressed in
the fetus). In that experiment, human and galago genes were linked
to hypersensitive site 3 (HS3) of the locus control region. To rule out
the possibility that HS3 is required for these differential expression
profiles, additional transgenic lines were tested in which human or
galago genes were linked to HS2. Once again, the galago gene
was embryonic and the human gene was fetal, indicating that the
stage specificity of these genes is driven by elements located within
the 4-kb fragments that contain the human and galago genes proper.
(Blood. 2002;99:1082-1084)
© 2002 by The American Society of Hematology.
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Introduction |
Two developmental switches in gene activity
characterize the human beta-like globin cluster. At the end of
embryonic life, the  gene is silenced, and the genes are
up-regulated. A second switch after birth involves the silencing of genes and the activation of  and  genes. The embryonic-to-fetal
(-to-) switch is particularly interesting because it is not seen
in most other mammals. In nonprimate mammals and in nonsimian primates
(such as galago), the gene exhibits an embryonic expression pattern
and is silent in the fetal liver.1,2 Thus, a single globin
switch (from embryonic and expression to adult and expression) is observed in these species. The recruitment of the gene to a fetal expression profile occurred approximately 58 to
40 million years ago (MYA),2 sometime after the
separation of nonsimian and simian primates but before the divergence
of the 2 major groups of simian primates the catarrhines (Old World
Monkeys, apes, and humans) and the platyrrhines (New World Monkeys).
All species of catarrhines and platyrrhines studied exhibit fetal
expression patterns.2
Thus, the galago and the lemur are the species most closely related to
human that still express in the embryonic and not the fetal time
period. Previously, we showed that the distinct characteristic
expression patterns of galago and human genes can be reproduced in
transgenic mice.3 Using constructs in which the
hypersensitive site 3 (HS3) region of the human locus control region
(LCR) was linked to the human gene plus either the human or the
galago genes, we found that the galago gene was expressed at
high levels in the embryonic yolk sac and was silenced in the fetal
liver, whereas the human gene was expressed at high levels in the
fetal liver.3 These results suggest that elements
responsible for the distinct expression patterns of the human and
galago genes are linked to the genes themselves. However, the
question of whether HS3 was required to observe these distinct expression patterns was not addressed.
Here, we confirm that HS2, like HS3, supports the embryonic profile of
the galago gene and the fetal expression pattern of the human gene. Thus, HS2 and HS3 fragments behave in a redundant fashion;
elements responsible for stage-specific expression patterns of are,
therefore, located within the 4-kb fragments themselves.
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Study design |
Generation of transgenic mice
The HS2-hum transgenic construct was described
earlier.4 Here, we generated HS2-gal in
which galago sequences (spanning 10508 to 14995 of GenBank M73981,
the Galago crassicaudatus globin cluster) were
substituted for the human sequences. The HS2 fragment (described
earlier4) was derived from the human globin cluster.
Purified insert DNA fragments were microinjected by the University of
Michigan Transgenic Animal Core team.
DNA analysis
All restriction enzymes (SphI, ClaI,
EcoRV, PstI, BamHI, NheI,
and XbaI) came from New England Biolabs, Beverly, MA.
Polymerase chain reaction genotyping primers,
5'-CAAATTGTTATTATTCCAGGCCACTGAATT (3' end of the HS2 fragment) and
5'-TAGTTATTGTGAATCAAATATTTATCTT-GCAGGTGG (2 kb upstream of the cap
site), detected an 800-base-pair (bp) product in transgenic animals.
For copy number determination, genomic DNA was digested with
SphI, which cuts once within the construct, and Southern
blots were probed with a ClaI-EcoRV fragment from
the human gene.5
RNA analysis
RNA was extracted as described
previously3,4 from 10.5- and 12.5-day yolk sacs and from
fetal liver of 12.5- and 14.5-day F2 concepti. S1 nuclease
protection was used to quantitate mRNA levels. The probe for mouse  was described previously.4 To enable multiplexing, a human
probe was generated from human exon 2 sequence; after
BamHI digestion and end labeling, the probe protects a
159-bp band. The mouse  probe was labeled at a PstI site
within a NheI-BamHI genomic fragment (116-bp
protected fragment).4 The galago probe was a 435-bp
XbaI-BamHI genomic fragment labeled at the
BamHI site in exon 2 (204-bp protected fragment). S1
analyses were quantitated using a Bio-Rad PhosphorImager with
Multi-Analyst software (Bio-Rad, Hercules, CA).
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Results and discussion |
Of 3 founder animals carrying the
HS2-gal transgene, 2 passed the transgene to progeny
and were used in these studies. Transgene copy numbers of 8 (line 1)
and 57 (line 2) were determined by Southern blot analysis; transgenes
were intact (data not shown).
S1 nuclease assays (Figure 1) revealed
that the galago gene was, like the human gene, expressed at
high levels in the embryonic time period and at considerably reduced
levels in the 14.5-day fetal liver, consistent with the known embryonic
expression pattern of the galago gene. This is in contrast to the
clear activation of the human gene in the fetal liver (Figure
1).4
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| Figure 1.
S1 nuclease analysis of transgene
expression.
Tissues were harvested from transgenic embryos at 10.5, 12.5, and
14.5 days after conception. Yolk sac (YS) tissue was examined at 10.5 and 12.5 days, and fetal liver (FL) tissue was tested at 12.5 and 14.5 days. Bands corresponding to the galago (Gal) , human (Hum) ,
human , and mouse(Mus) genes are labeled. Mouse gene
is not shown. Panels Ai and Aii are derived from analysis of transgenic
lines 1 and 2 carrying the HS2--gal transgene
(diagrammed at top). Panel B is derived from analysis of line 187 carrying the HS2--hum construct; these data are taken
from work described earlier.4 Gels were subjected to
PhosphorImager scanning, and the expression of the human and galago
genes was determined as [transgene expression/(mouse expression/2) + (mouse expression/4)]/copy number.
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HS2 and HS3 contain the most powerful enhancers in the
LCR.6,7 Although both can drive high-level expression of
linked globin genes in transgenic mice, possible functional
differences have been noted: (1) HS3, but not HS2, harbors a
dominant chromatin opening activity8; (2) HS2 and HS3 drive
different developmental patterns of and expression7,9; and (3) HS3 may be essential for specific
parts of the switching program. Embryonic expression of (but not
) was disrupted by deletion of the core of HS3 in a human -locus
YAC.10 Because these investigations indicate that HS2 and
HS3 could differ in their ability to interact with the various globin
promoters, it was important to establish whether the distinct
stage-specific patterns of gene expression, observed for galago and
human genes in an earlier study,3 could also be
detected when the genes were linked to HS2. Here we show that this is
indeed the case, confirming that fetal versus embryonic expression of
is attributed to changes in cis elements linked closely
to the genes proper.
Although the constructs we have used in these 2 studies are relatively
small (11-12 kb) and are subject to position effect, our combined
experience (summarized in Figure 2)
provides compelling evidence that the human and galago genes are
indeed regulated differently in the context of the same
trans environment (the mouse fetal liver). Moreover, the
data indicate that HS3 and HS2 are able to activate in the context
of the fetal environment because the human gene is highly expressed
in the 14.5-day fetal liver regardless of whether it is linked to HS2
or HS3. However, the fact that the galago gene cannot form a
productive interaction with the LCR in the fetal stage suggests that
cis elements within the human gene facilitate productive
interaction with the LCR or that cis elements within the
galago gene prohibit productive interaction. In either case,
eventual identification of these elements will provide insights into
developmental regulation and promoter-LCR interactions.
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| Figure 2.
Combined developmental profiles of human ,
human , or galago transgenes driven by HS2 and HS3.
Panels A and B represent expression of the human (A) and galago (B) transgenes in HS2--gal constructs (this study)
and HS3--gal constructs.3 For all these
lines, the human and galago genes were expressed highly in
embryonic life and were silenced in fetal life. Thus, in panels A and
B, transgene expression levels in embryonic life is set to 100% for
each line, and the level seen in fetal life is expressed relative to
the embryonic level for that line. Panels C and D represent expression
of the human (C) and human (D) transgenes in
HS2--hum constructs3 and in
HS3--hum constructs.4 For the gene,
embryonic levels for each line are set at 100%, and fetal levels are
normalized to this level as for panels A and B. For the human gene,
however, expression was consistently highest in the fetal liver. Thus,
the fetal expression level was set at 100%, and the embryonic level in
each line was normalized to this level. Although human gene
expression is variable in embryonic life, the difference in expression
profile between the human gene and the galago gene is
strikingly consistent.
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Acknowledgments |
We thank Christine Babcock for help with maintaining
the mouse colony.
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Footnotes |
Submitted July 24, 2001; accepted September 24, 2001.
Supported by National Institutes of Health grants HL48802
(D.L.G.) and DK56927 (M.G., D.L.G.) and a Hematology Training Grant (NIH T32-HL07622) (D.M.T.).
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.
Reprints: Deborah L. Gumucio, Dept of Cell and Developmental
Biology, University of Michigan, 5704 Medical Science II, Ann Arbor, MI
48109-0616; e-mail: dgumucio{at}umich.edu.
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Abstract
Introduction