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Intergenic transcription at the abdA-AbdB locus. (A) Summary
of abdA-AbdB locus. The abdA and AbdB transcription start sites are indicated
by leftward arrows. The intergenic region is 100 kb in length. The iab
regions that control expression of the two Hox genes are indicated (IAB2
to IAB8). IAB2, IAB3, and IAB4 (shown in green) regulate expression of abdA.
IAB5, IAB6, IAB7, and IAB8 (shown in yellow) direct AbdB expression. The insulator
DNAs that separate the different iab regions are indicated (black ellipses).
The presumptive Fab6 insulator (gray ellipse) has yet to be identified. Characterized
enhancers within the iab regions are shown as blue rectangles. The locations
of probes used for in situ hybridization analysis in this study are shown
as black bars under the locus. (B–I) Transcription patterns detected with
in situ hybridization probes. Embryos are orientated with anterior to the
left and dorsal up. Probes against the abdA (B and C) and AbdB (F and G)
coding regions detect the expected distribution of transcripts (see text).
An APP probe detects transcription at blastoderm stage 5 (D) and later in
development (E), unlike a BPP probe, which detects transcription in abdominal
segments 8 and 9 only later in embryonic development (H and I).
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Transcription in the iab regions. (A) The location of probes
at the abdA-AbdB locus. (B–M) Patterns of sense transcription detected
by in situ hybridization RNA probes. At stage 5, transcripts from the different
iab regions show distinct distribution patterns along the A–P axis of the
embryo. A probe from the iab3 region detects expression (B) extending farther
toward the anterior of the embryo than iab4 (E). Expression patterns become
increasingly restricted toward the posterior of the embryo in the iab5
region (H) and iab8 region (K). From stage 9 of development, transcription
from all iab regions is restricted to the two most posterior abdominal segments,
8 and 9 (C, F, I, and L). This pattern persists through stage 13 of development
(D, G, J, and M).
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Temporal activation of sense iab transcripts. (A) At late stage
4 in embryos hybridized with probes against engrailed and iab4s transcripts,
expression is detected only in the posterior iab4 domain, before the
appearance of anterior engrailed expression. (B) In early blastoderm
stage 5 embryos, sense transcripts can be detected in the iab4 domain
and the anterior engrailed stripe 2. (C) Colinear distribution of transcripts
in the iab regions. Measure of transcript distribution is shown as a percentage
of embryo length. The anterior tip of the embryo corresponds to 0 and the
posterior tip corresponds to 100. The probes are listed according to their
order on the chromosome, with the exception of probes against the two Hox
genes, Aexon (green) and Bexon (yellow), which are shown at the bottom
and the top of graph, respectively. Colinearity between the chromosomal
order of probes and their transcription patterns is observed. The anterior
limits of transcription for probes from the iab5, iab6, iab7, and iab8
regions are restricted within the AbdB domain of expression (pale yellow),
whereas probes from iab3 and iab4 regions detect patterns extending into
the abdA expression domain (pale green). The distribution of the antisense
transcripts detected in iab4 and iab6 are also colinear with their chromosomal
locations and are shown in black.
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Antisense transcripts in the iab regions. (A–D) Sense transcription
pattern at iab4 region during embryonic development detected by probe 4-5.
No transcript is detectable at stage 5 (A). Transcription is detectable
at stage 8 and, by stage 9, is restricted to abdominal segments 8 and 9.
Transcription persists in the two most posterior abdominal segments through
stage 13 (D). (E–H) The iab4as transcript (23) also is detected by probe
4-5, although the pattern is distinct from that of sense transcription. At
blastoderm stage 5, the antisense transcript is expressed strongly in the
iab4 domain (E) and persists in abdominal segments 1–7 through developmental
stages 8 (F), 9 (G), and 13 (H), but is excluded from the most posterior
segments. The distributions of the iab4as and iab4 sense transcripts appear
to be mutually exclusive. (I and J) A novel antisense transcript is detected
by probe 6-1. At blastoderm stage 5, the transcript is restricted to the iab6
domain (I) and, by stage 9, is expressed strongly in abdominal segments 4,
5, and 6 and more weakly in 7, 8, and 9 (J). The sense transcription detected
by probe 6-1 (data not shown) is mutually exclusive to the iab6as pattern.
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Cellular localization of BX-C transcripts. (A) Summary of abdA-AbdB
locus. The abdA and AbdB transcription start sites are indicated by leftward
arrows. The intergenic region is 100 kb in length. The iab regions that
control expression of the two Hox genes are indicated (IAB2 to IAB8). IAB2,
IAB3, and IAB4 (shown in green) regulate expression of abdA. IAB5, IAB6,
IAB7, and IAB8 (shown in yellow) direct AbdB expression. The insulator DNAs
(black ellipses) separate the different iab regions. The presumptive Fab6
insulator has yet to be identified (gray ellipse). Characterized enhancers
within the iab regions are shown as blue rectangles. The location of probes
used for in situ hybridization analysis in this study are shown as black
bars under the locus. Sense transcripts (s), relative to the direction of
AbdB transcription, are detected by antisense probes, and antisense transcripts
(as) are detected by sense probes. (B and C) iab6as expression pattern at
the surface of blastoderm stage 5 embryos. Hybridization to the transcripts
appears uniform throughout the cells in which they are expressed. (D and
E) Sense transcripts from the IAB8 enhancer sequence are visible as discrete
foci in the cells at the surface of blastoderm stage 5 embryos (D). At higher
magnification, two foci can be seen in each expressing cell at the focal
plane (E). (F and G) AbdB expression in stage 9 embryos visualized by confocal
microscopy. The transcript (red) is excluded predominantly from the nucleus
(green) and distributed throughout the cellular cytoplasm (G). (H and I) IAB8
sense transcripts (red) are localized at the nuclear periphery and not distributed
in the cytoplasm of the cell (I).
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Mcp
genomic region regulates transcription. (A–H) Intergenic transcription
is blocked at insulator DNAs. (A) Genomic Mcp region. The 3-kb deletion
in the Mcp mutant (28) is shown between the iab4 (green) and iab5 (yellow)
regulatory regions. The locations of expected RT-PCR products and in situ
hybridization probes are shown below the line. (B–G) Transcription across
the Mcp insulator sequence detected by in situ hybridization. In blastoderm
stage 5 embryos, sense transcripts can be detected in the iab4 region (4-2;
B) and, in the characterized Mcp sequence (MCP-2; F), with the transcription
pattern at MCP-2 restricted farther toward the posterior of the embryo. No
transcription is detected from probe MCP-4 (D), which maps adjacent to the
characterized Mcp sequence early in development. All three probes detect
expression in the posterior abdominal segments at stage 9 (C, E, and G). (H)
RT-PCR detection of transcription at insulator sequences. PCR amplifications
were performed in parallel on reverse-transcribed total RNA (RT) and genomic
DNA (gDNA) from 0- to 4-h embryos. Transcription could be detected with primer
pairs within the characterized Mcp element (MCP-2 and MCP-3) and the iab4
region (4-1), but not at the intervening MCP-4 sequence. No product was detected
from the RT cDNA sample when amplified from MCP-3 to MCP-4 or from MCP-4
to 4-1, suggesting that the iab4 (4-1) and Mcp (MCP2/3) transcripts are
not joined. However, transcription could be detected with primers from MCP-2
into MCP-3, suggesting that they are part of the same transcription unit.
No product was detected from 6-2 to 5-1, although these sequences are transcribed
independently, indicating that transcription is blocked at the intervening
region. The expected size products were obtained with all primer pairs from
gDNA. Control lanes are RT– (absence of reverse transcriptase) for RT and
water for gDNA (–Control). (I–P) Mcp deletion results in disrupted transcription
in the iab4 region. (I) Mcp mutant phenotype. In contrast to wild-type adult
males, the A4 segment is pigmented in homozygous Mcp mutants (black arrow),
indicating that the fourth abdominal segment is transformed into a more posterior
abdominal segment. (J) PCR amplifications were performed in parallel on reverse-transcribed
total RNA from homozygous Mcp mutant (Mcp) and wild-type yw67 0- to 4-h embryos.
No transcription could be detected in the Mcp mutant with primer pairs
from the iab4 region (4-1 and 4-2), although the iab5 (5-1) and AbdB (Bexon)
transcripts are present. In contrast, transcription in the iab4 region
was readily detectable in wild-type embryos. Negative control lane is RT–.
(K–P) Transcription in Mcp mutant and yw67 embryos detected by in situ
hybridization with engrailed and iab4s probes. In blastoderm stage 5 embryos,
sense transcripts can be detected in the iab4 region and in the anterior
engrailed stripe pattern in yw67 embryos (L). The iab4s transcript is absent
in the Mcp mutant embryos (K). The transcription patterns detected at stages
9 and 13 in Mcp (M and O) and yw67 (N and P) are indistinguishable from
each other.
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Model of intergenic transcription-mediated enhancer activation. At
the very early stages of embryonic development, the cis-regulatory elements
(shown here as an enhancer, but also possible for Polycomb response elements)
at the intergenic iab regions are inactive. A closed chromatin configuration
prevents access to the trans factors (black circles), such as the products
of the gap or segmentation genes, which are necessary to direct expression
of the abdA and AbdB protein-coding genes (Gene X). Within each iab region,
discrete anteroposterior patterns of transcription (black arrow) are
initiated at around late stage 4/early stage 5 in development. RNA polymerase
II (Pol II) and its associated remodeling complex (HAT, histone acetyltransferase)
pass through the enhancer and epigenetically modify the histone tails
(shown here as acetylation, but also may involve deacetylation, methylation,
or demethylation of histone tail residues), creating an open chromatin
configuration. Spreading of transcription from one iab region to the next
is prevented by insulator DNAs (black ellipses) at the boundaries of each
iab region. The newly modified open histones now permit the binding of trans
factors to the enhancer sequence, and this stable interaction is able to
direct segment-specific expression of the neighboring Hox genes from midstage
5 of development onward.
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