labial
labial contains a TATA-box deficient (TATA-less) promoter. Such promoters have a conserved sequence motif, A/GGA/TCGTG, termed the downstream promoter element (DPE), which is located about 30 nucleotides downstream of the RNA start site of many TATA-less promoters, including labial. DNase I footprinting of the binding of epitope-tagged TFIID to TATA-less promoters reveals that the factor protects a region that extends from the initiation site sequence (about +1) to about 35 nucleotides downstream of the RNA start site. There is no such downstream DNase I protection induced by TFIID in promoters with TATA motifs. This suggests that the DPE acts in conjunction with the initiation site sequence to provide a binding site for TFIID in the absence of a TATA box to mediate transcription of TATA-less promoters (Burke, 1996).
labial is induced to high levels of localised
expression in the endoderm of Drosophila embryos by the action of DPP. Dissection of LAB 5' flanking sequences reveals two types of response
elements. One of these mediateslab dependent activity, providing evidence that lab induction in the
endoderm is autoregulatory. The other element, to a large extent independent of lab function,
responds to DPP (Tremml, 1992).
Examination of the Ultrabithorax midgut enhancer, for which the Dpp response element has been localized to the 95 bp DI-DII interval, and the labial endoderm enhancer reveals a Mothers against Dpp binding-site consensus of GCCGnCGC. The two sites of highest affinity match the MAD consensus perfectly, and three lower-affinity sites contain mismatches in one or as many as three positions (Kim, 1997).
The homeotic proteins encoded by the genes of the Drosophila HOM and the vertebrate HOX
complexes do not bind divergent DNA sequences with a high selectivity. In vitro, HOM (HOX)
specificity can be increased by the formation of heterodimers with Extradenticle (Exd) or PBX
homeodomain proteins. A single essential Labial (Lab)/Exd-binding site has been identified in a
Decapentaplegic (Dpp)-responsive enhancer of the homeotic gene lab, which drives expression in the
developing midgut. Lab and Exd bind cooperatively to the site in vitro, and the
expression of the enhancer in vivo requires exd and lab function. In addition, point mutations in either
the Exd or the Lab subsite compromise enhancer function, strongly suggesting that Exd and Lab
bind to this site in vivo. Interestingly, the activity of the enhancer is only significantly stimulated by
Dpp signaling upon the binding of Lab and Exd. Thus, the enhancer appears to integrate
positional information via the homeotic gene lab, and spatiotemporal information via Dpp signaling; only
when these inputs act in concert in an endodermal cell is the enhancer fully active. These results illustrate
how a tissue-specific response to Dpp can be generated through synergistic effects on an enhancer
carrying both Dpp- and HOX-responsive sequences (Grieder, 1997).
How does Dpp signaling affect the expression of the lab550 enhancer? The enhancer has been reported as a Dpp
response element based on the fact that all of its activity in the midgut requires dpp function, but very little of it is
due to lab function; in addition, it drives expression one or two cells more posterior than the endogenous lab
gene, thus apparently displaying lab-independent expression. Although these
conclusions somewhat contrast with those presented in the current paper, further analysis has suggested that several cyclic AMP response elements (CREs) in the enhancer mediate the
Dpp responsiveness, directly or indirectly. More recently, in vitro binding sites for
Drosophila Mad (a member of the SMAD protein family) and thought to act as co-activators of
transcription in the Dpp signaling pathway, have also been identified in the lab
enhancer. In addition, there are numerous binding sites for Schnurri, a putative
transcription factor that is required for endodermal cells to respond to Dpp (Grieder, 1997 and references).
Despite the presence of all these putative target sequences of the Dpp signaling pathway, the current paper demonstrates
that the expression of the lab enhancer strongly depends on the presence of a 48 bp region that harbors an
Exd/Lab site; in exd and lab mutant embryos, expression of the element is strongly reduced. Since it recently
has been shown that the translocation of Exd from the cytoplasm to the nucleus in the endoderm is controlled by
Dpp and Wingless (Wg) , and since the endogenous lab gene is strongly
induced by Dpp, it is likely that part of the Dpp regulation of the lab enhancer enters through the Exd/Lab site.
This is consistent with the current findings and with the previous observation that in the absence of lab function,
dpp-mediated induction is hardly working or working with reduced efficiency (Grieder, 1997 and references).
What is the role of the additional Dpp response elements in the lab enhancer? Clearly, the activity of the
enhancer also depends on the presence of CRE sites; similar to observations concerning the Lab/Exd site,
mutations in the four CRE sites result in weaker expression of the enhancer. This suggests
that on the full-length enhancer, the additional (direct or indirect) Dpp input via the CRE and other sites is
required in concert with the input through the Lab/Exd site, and that the absence of either input impairs expression of the enhancer. Two elements appear to act synergistically with respect to their response to Dpp; only on the addition of a weak Dpp response element to the Lab/Exd-containing element is a cis-acting element generated which is strongly Dpp inducible (and Lab/Exd dependent). It is proposed that the strong Dpp responsiveness of the enhancer is limited, through synergistic interactions, to those cells in which the Lab/Exd site is occupied. The function of this dual requirement might be to insure that the enhancer activates transcription exclusively in cells located in the central portion of the midgut endoderm where low levels of Lab in the tip of the two endoderm primordia coincide with Dpp, secreted from the visceral mesoderm parasegment 7 (Grieder, 1997).
To regulate their target genes, the Hox proteins of
Drosophila often bind to DNA as heterodimers with the
homeodomain protein Extradenticle (Exd). For Exd to
bind DNA, it must be in the nucleus, and its nuclear
localization requires a third homeodomain protein,
Homothorax (Hth). A conserved N-terminal
domain of Hth directly binds to Exd in vitro,
and is sufficient to induce the nuclear localization of Exd
in vivo. However, mutating a key DNA binding residue in
the Hth homeodomain abolishes many of its in vivo
functions. Hth binds to DNA as part of a Hth/Hox/Exd
trimeric complex, and this complex is
essential for the activation of a natural Hox target
enhancer. Using a dominant negative form of Hth evidence is provided that similar complexes are important for
several Hox- and exd-mediated functions in vivo. These
data suggest that Hox proteins often function as part of a
multiprotein complex, composed of Hth, Hox, and Exd
proteins, bound to DNA (Ryoo, 1999).
The tight interaction between Hth and Exd proteins, together
with the requirement for the Hth homeodomain for many of
Hths functions, suggested that Hth might be binding to the
same target enhancers as Hox/Exd heterodimers. One well
characterized Hox/Exd target is an autoregulatory enhancer
from the labial (lab) gene, called lab550. A 48 bp fragment of lab550,
lab48/95, is necessary for lab550 activity and, in one copy, is
sufficient to direct a labial- and exd-dependent pattern of
expression in endodermal cells. In lab48/95 there is a single Lab/Exd heterodimer
binding site, TGATGGATTG; this binding site is necessary
for the activity of lab550. Also in
lab48/95 is a binding site that resembles a high affinity
site for MEIS1: GACTGTCA, a murine Hth homolog. To test if this
site is a bona fide Hth binding site, band shift
experiments were performed with Lab, Hth, and Exd proteins on the wild-type
lab48/95 oligo, and on an oligo with point mutations in
the putative Hth binding site, GACTtatA (lab48/95 hth). Neither Lab, Exd, nor Hth are able to bind
lab48/95 on their own. The combination
of Exd plus Hth is able to weakly bind this DNA. Because binding is diminished on lab48/95 hth, these data suggest that Exd and Hth exhibit
weak cooperative binding to lab48/95, consistent with previous
studies with MEIS1 and PBX1.
Lab cooperatively binds with Exd to lab48/95 and the binding of this heterodimer requires both the
Exd and Lab half sites. In contrast, no
complex formation is observed when Hth and Lab are
combined. However, when increasing
amounts of Hth are added to a constant amount of Lab plus
Exd, the Lab/Exd band disappears and in its place a
Hth/Lab/Exd trimeric complex is observed. The Hth/Lab/Exd band is more intense than the
Lab/Exd band, suggesting that
Hth contributes to the DNA binding affinity of the trimeric
complex. Additonal tests show that the Hth/Lab/Exd complex
requires the putative Hth binding site; use of truncated proteins show that protein-protein interaction between Hth and
Exd is necessary for the formation of the Hth/LAB/Exd
complex, but that DNA binding by the Hth homeodomain
contributes to the stability of this complex. Also, the Hth binding site is required for lab48/95 activity
in embryos. Thus a DNA bound
Hth/LAB/Exd triple complex is capable of activating lab48/95-lacZ in
vivo. This was confirmed by interfering
with the stable assembly of this complex by expressing the HM
domain, which binds to Exd and therefore competes
with the interaction between Exd and Hth (Ryoo, 1999).
During embryonic development of the Drosophila brain, the Hox gene
labial is required for the regionalized specification of the
tritocerebral neuromere. In order to gain further insight into the mechanisms of
Hox gene action in the CNS, the molecular and genetic basis of
cross-regulatory interactions between labial and other more posterior Hox
genes were examined using the GAL4/UAS system for targeted misexpression. Misexpression of
posterior Hox genes in the embryonic neuroectoderm results in a labial
loss-of function phenotype and a corresponding lack of Labial protein expression
in the tritocerebrum. This is due to repression of labial gene
transcription in the embryonic brain. Enhancer analysis suggests that this
transcriptional repression operates on a 3.65 kb brain-specific
labial-enhancer element. A functional analysis of Antennapedia and
Ultrabithorax protein domains shows that the transcriptional repression of
labial requires homeodomain-DNA interactions but is not dependent
on a functional hexapeptide (a conserved stretch of aminoacids
that is found in many Hox proteins and is involved in interactions between
Hox proteins and Exd).
The repressive activity of a Hox protein on
labial expression in the tritocerebrum can, however, be abolished by
concomitant misexpression of a Hox protein and the cofactors Homothorax and
nuclear-targeted Extradenticle. Taken together, these results provide novel and
detailed insight into the cross-regulatory interactions of Hox genes in
embryonic brain development and suggest that specification of tritocerebral
neuronal identity requires equilibrated levels of a Hox protein and Hth and nuclear-targeted Exd (n-Exd) cofactors (Sprecher, 2004).
The main function of Hox genes is to assign positional identities along the
embryonic body axis in animals ranging from arthropods to vertebrates.
Several mechanistic paradigms
have been proposed to describe Hox gene action, two of which are the concepts of
cross-regulation among Hox genes, and of co-operative interactions between Hox
genes and protein cofactors.
In the developing CNS of Drosophila, loss-of-function studies
have shown that Hox genes expressed in more posterior regions act as negative
regulators of Hox genes that are expressed in more anterior regions of the CNS.
For example Antp is primarily expressed in Parasegment (PS) 4 and PS5 of
the CNS, but it is also expressed at lower levels in PS6-13.
In embryos that lack the Bithorax-complex
genes, Antp expression is high in PS4-13, suggesting that
BX-C gene action keeps
Antp expression low in PS6-13. Similarly, BX-C genes that are expressed
and function in more posterior abdominal segments keep Ubx expression low
in PS7-13. In the absence of the abdominal BX-C genes, Ubx products are
found at high levels in PS6-13.
In addition, recent gain-of-function experiments have shown that
ectopic Ubx and Abd-A are able to repress lab and Scr in the CNS
in a timing dependent manner while otherwise overlapping expression of other Hox
genes is tolerated (Sprecher, 2004).
In this analysis, focus was placed on lab,
the Hox gene specifically expressed in
the tritocerebral neuromere. Genetic analyses have shown that lab is
essential for the acquisition of neuronal identity in its tritocerebral
expression domain, and lab loss-of-function mutations leads to severe
defects in the establishment of the tritocerebral neuromere.
The action of lab in this domain
can be eliminated by targeted misexpression of posterior Hox genes through the
sca::Gal4 driver, resulting in a lab loss-of-function phenotype in
the brain. This suppression of lab action has a number of features that
are characteristic of the type of cross-regulatory Hox gene interactions that
have been demonstrated in developing epidermal structures.
(1) The suppression of lab in
the tritocerebrum appears to be time dependent. While early misexpression of
posterior Hox genes during neuroectoderm specification and neuroblast formation
at embryonic stage 9 reliably results in lab suppression in the
tritocerebrum, later misexpression, after embryonic stage 10/11, does not.
(2) lab suppression by misexpression of posterior Hox genes is tissue
specific. Thus, while Hox gene misexpression via the sca::Gal4 driver
suppresses lab expression in the tritocerebrum, it augments lab
expression in the endodermal cells of the midgut. (3) Misexpression of
posterior Hox genes leads to a loss of Lab protein in the affected domain, and
this lack of Lab is in accordance with the observed phenocopy of a lab
loss-of-function mutation observed in this domain (Sprecher, 2004).
In several respects these
experiments extend insights into cross-regulatory interactions beyond the
observations made on developing epidermal structures. Evidence suggests that
the suppression of lab by a posterior Hox gene like Ubx is due to
transcriptional repression. Thus, in sca::Gal4/UAS::Ubx embryos,
lab transcripts disappear and are absent in the developing tritocerebrum
from stage 10/11 onward. This tritocerebrum-specific repression appears to be
mediated through a 3.65 kb enhancer element upstream of the lab gene
transcriptional start site. Moreover, the results imply that suppression of
lab in the developing tritocerebrum by posterior Hox genes requires a
functional homeodomain; mutations of the homeodomain in the Antp gene
abolish the repressive activity of this Hox gene. In addition, the findings
indicate that the suppressive cross-regulatory action of a posterior Hox gene
like Ubx is not dependent on a functional hexapeptide. Thus,
misexpression of a UAS::UbxYAAA transgene in which the critical YPWM
motif of Ubx was mutated to the sequence YAAA,
still results in complete suppression of lab in the developing
tritocerebrum. Finally, evidence is provided that concomitant misexpression of
Ubx, nuclear-targeted Exd and Hth is able to completely rescue the
lab loss-of-function mutant phenotype. This implies that the Exd and Hth
cofactors can switch Ubx protein action between different functional states in
which Exd and Hth are required for Hox protein transcriptional activation
functions whereas they are dispensable for Hox transcriptional repression
functions. Moreover, these findings
can be explained by models in which the hexapeptide is involved in the
regulation of Hox protein activity, and
may also reflect a requirement for equilibrated levels of a Hox gene product and
the Hth and n-Exd cofactors in the specification of tritocerebral
identity (Sprecher, 2004).
Many developmental control genes contain stalled RNA Polymerase II (Pol II) in the early Drosophila embryo, including four of the eight Hox genes. Evidence is presented that the stalled Hox promoters possess an intrinsic insulator activity. The enhancer-blocking activities of these promoters are dependent on general transcription factors that inhibit Pol II elongation, including components of the DSIF (Spt4, and Spt5) and NELF complexes. The activities of conventional insulators are also impaired in embryos containing reduced levels of DSIF and NELF. Thus, promoter-proximal stalling factors might help promote insulator-promoter interactions. It is proposed that stalled promoters help organize gene complexes within chromosomal loop domains (Chopra, 2009b).
Hox genes are responsible for the anterior-posterior patterning of most metazoan embryos. They are typically organized in gene complexes containing a series of cis-regulatory DNAs, including enhancers, silencers, and insulator DNAs . In Drosophila, the eight Hox genes are contained within two gene complexes: the Antennapedia complex (ANT-C), which controls the patterning of anterior regions, and the Bithorax complex (BX-C), which controls posterior regions. The proper spatiotemporal transcription of Hox genes is achieved by the coordinated action of linked cis-regulatory DNAs that are organized in a colinear fashion across the ANT-C and BX-C complexes (Chopra, 2009b).
Chromosomal boundary elements, or insulators, are essential for the orderly regulation of Hox gene expression. They are thought to ensure proper cis-regulatory 'trafficking,' whereby the correct enhancers interact with the appropriate target promoters. Insulators might also help control the levels of transcription by attenuating enhancer-promoter interactions. Insulators are sometimes associated with promoter targeting sequences (PTS), which can facilitate enhancer-promoter interactions by modulating the activities of neighboring insulators (Chopra, 2009b).
Recently, long-range cis-regulatory interactions have been mapped in Drosophila Hox complexes using the DamID technique, chromosomal conformation capture (3C) assays, and transgenic approaches. These studies suggest that the Fab7 and Fab8 insulators are associated with the Abd-B promoter under repressed conditions, even though they map >30-50 kb downstream from the promoter. These long-range interactions depend on the CTCF boundary-binding protein, thereby raising the possibility that insulators interact with one another and organize Abd-B cis-regulatory DNAs within chromosomal loop domains. Similarly, the prototypic insulators flanking the heat-shock puff locus, scs and scs', have also been shown to interact with one another. Additional insulator-insulator loops have also been documented. These loops are thought to facilitate the interactions of remote enhancers and silencers with appropriate target promoters. This study presents evidence that Hox promoters with stalled RNA Polymerase II (Pol II) possess an intrinsic insulator activity, which might help foster the formation of insulator-promoter chromosomal loop domains (Chopra, 2009b).
Four of the eight Hox genes contained in the ANT-C and BX-C contain stalled Pol II. Interestingly, all four stalled genes map at the boundaries of the two Hox complexes. In contrast, internal Hox genes (pb, Dfd, and Scr within the ANT-C, and abd-A within the BX-C) lack stalled Pol II. This arrangement of stalled Hox genes raises the possibility that stalling contributes to the chromosomal organization of Hox complexes. All four stalled Hox genes (lab, Antp, Ubx, and Abd-B) were tested for enhancer-blocking activity in transgenic embryos, along with the promoter regions of two nonstalled genes (Scr and abd-A). Test promoters were placed 5' of lacZ and inserted between a divergent white reporter gene and 3' iab-5 enhancer (IAB5) (Chopra, 2009b).
IAB5 regulates Abd-B expression in posterior regions of the early embryo, corresponding to the primordia for parasegments 10-14. IAB5 is a robust enhancer, and can activate lacZ and white even when positioned far from the reporter genes. This assay was used to reveal an intrinsic enhancer-blocking activity of the eve promoter region. eve/lacZ fusion genes block the ability of IAB5 to activate a distal CAT reporter gene. However, mutagenized eve promoter sequences lacking a critical proximal GAGA element failed to block IAB5-white interactions. Similarly, the Abd-B proximal promoter (Abd-Bm) and Ubx promoter regions block activation of distal white expression, whereas the abd-A promoter does not interfere with the activation of white expression in the presumptive abdomen by the IAB5 enhancer (Chopra, 2009b).
These results suggest that the stalled Abd-B proximal promoter and Ubx promoters possess an enhancer-blocking activity, whereas abd-A does not. A similar trend was observed for Hox promoter sequences from the ANT-C. The Antp and lab promoters block IAB5-white interactions, whereas the Scr promoter (which lacks stalled Pol II) does not interfere with the activation of white expression in the presumptive abdomen. Stalled genes from the tinman complex (Tin-C), which encode NK homeobox proteins responsible for patterning mesodermal lineages, were also examined. All of the stalled promoters from the Tin-C contain insulator activities. In contrast, nonstalled promoters from lbl and C15 lack such activities when tested in similar transgenic assays. Even the Hsp70 promoter, the classic example of Pol II pausing, displayed insulator activity when tested in similar enhancer-blocking transgenic assays (Chopra, 2009b).
The preceding experiments suggest that stalled Hox gene promoters contain enhancer-blocking activities. However, an alternative possibility is that stalled promoters are 'stronger' than the white promoter, and are able to sequester the shared IAB5 enhancer. To distinguish between competition and insulator activities, the IAB5 enhancer was placed between the divergently transcribed white and lacZ reporter genes. When the white promoter sequence was placed 5' of the lacZ reporter gene, the shared IAB5 enhancer worked equally well to activate both white and lacZ expression. Similar results were obtained when the leftward lacZ reporter gene was placed under the control of either the stalled Abd-B or Ubx promoters. In all of these cases, both white and lacZ are expressed equally well in the presumptive abdomen. These results suggest that stalled promoters do not block enhancer-promoter interactions by a competition mechanism. Rather, they work like insulators and block such interactions only when positioned between the distal enhancer and target promoter (Chopra, 2009b).
To determine whether stalled Pol II is important for the enhancer-blocking activities of Ubx and Abd-B, mutant embryos were examined with reduced levels of critical Pol II elongation factors. Ubx and Abd-B were selected for further studies since optimal expression of both genes depends on the Pol II elongation factors Cdk9 (pTEFb) and Elo-A (Chopra, 2009a). It was reasoned that destabilization of stalled Pol II might reduce the enhancer-blocking activities of the Ubx and Abd-B promoter regions. However, reductions in Cdk9 and Elo-A are expected to stabilize, not destabilize, Pol II stalling since both are positive factors that promote elongation (Saunders, 2006). Indeed, reductions in Cdk9 or Elo-A activity do not alter the enhancer-blocking activities of the Ubx and Abd-B promoters (Chopra, 2009b).
To investigate the link between Pol II stalling and enhancer blocking, two negative elongation factors were examined: NELF (Lee, 2008) and DSIF (Wada, 1998; Yamaguchi, 1998; Kaplan, 2000). The NELF-E protein binds to the short nascent transcripts protruding from the active site of Pol II after transcription initiation and promoter clearance, and thereby inhibits Pol II elongation (Wu, 2005; Lee, 2008). Both NELF and DSIF are thought to help stabilize Pol II at the pause site, typically 20-50 base pairs (bp) downstream from the transcription start site (Saunders, 2006; Gilchrist, 2008
). Since Pol II elongation factors are encoded by essential genes, it is not possible to examine the lacZ/white reporter genes in homozygous mutant embryos. Instead, the transgenes were expressed in embryos derived from heterozygous females, and thereby contain half the normal levels of NELF and DSIF (Spt) subunits. Reductions in Nelf-E, Nelf-A, Spt4, and Spt5 cause clear disruptions in the enhancer-blocking activities of both the Ubx and Abd-B promoters, as seen by the strong activation of the distal white reporter gene. In contrast, white expression is blocked when the same transgenes are expressed in a wild-type background. The simplest interpretation of these results is that reduced levels of the NELF and DSIF inhibitory complexes destabilize stalled Pol II at the pause site. Reduced pausing results in diminished enhancer-blocking activities. There is a similar loss in the enhancer-blocking activities of the eve promoter and Fab7 insulator when the transgenes are expressed in embryos containing reduced levels of the GAGA factor, Trl. It is conceivable that the GAGA factor also contributes to the enhancer-blocking activity of the Ubx promoter since Trl/+ embryos display augmented expression of white (Chopra, 2009b).
In principle, the augmented expression of the white reporter gene might not result from the impaired function of the stalled insulators, but might arise from enhanced activity of the white promoter. To investigate this issue, Pol II chromatin immunoprecipitation (ChIP) assays were performed, coupled with quantitative PCR (qPCR) assays. In DSIF and NELF mutant embryos, there is no increase in Pol II levels at either the white promoter or intronic regions as compared with wild-type embryos. These results suggest that augmented expression of white is due to diminished insulator activities of stalled promoters in embryos containing reduced levels of negative Pol II elongation factors (Chopra, 2009b).
It has been suggested that insulators might work, at least in part, via promoter mimicry. To explore this issue, the impact of reductions in NELF and DSIF on the activities of two known insulators, Fab7 and Fab8, from the BX-C, were examined. Previously published transgenic lines were used that contain Fab7 or Fab8 inserted between the IAB5 and 2XPE (twist) enhancers attached to a leftward lacZ reporter gene and rightward white reporter. In wild-type embryos, the reporter genes are activated only by the proximal enhancer. Thus, white is activated solely in the mesoderm by the 2XPE enhancer, while lacZ is activated in the presumptive abdomen by IAB5. The distal enhancers are blocked by the Fab7 or Fab8 insulators. Consequently, IAB5 fails to activate white and the 2XPE enhancer fails to activate lacZ (Chopra, 2009b).
Very different results are observed when the transgenes are crossed into mutant embryos containing reduced levels of NELF or DSIF (Spt) subunits. There is a loss in the enhancer-blocking activities of the Fab7 and Fab8 insulators and, as a result, white and lacZ display composite patterns of expression in the mesoderm and abdomen since they are now activated by both enhancers. These results suggest that negative Pol II elongation factors are required for the enhancer-blocking activities of the Fab7 and Fab8 insulators (Chopra, 2009b).
It is proposed that insulators interact with stalled promoters to form higher-order chromatin loop domains, similar to those created by insulator-insulator interactions. Perhaps proteins that bind insulators interact with components of the Pol II complex at stalled genes. Indeed, the recent documentation that the BEAF insulator protein binds to many of the same sites as NELF is consistent with a physical link between stalled Pol II and insulators (Jiang, 2009). The resulting chromatin loops can prevent the inappropriate activation of stalled genes by enhancers associated with neighboring loci. As discussed earlier, stalled Hox genes are located at the boundaries of the ANT-C and BX-C. This arrangement might help ensure that cis-regulatory sequences located outside the complexes do not fortuitously interact with genes contained inside the complex and vice versa. The demonstration that stalled Hox promoters possess an intrinsic insulator activity adds to the intricacy of the chromosomal landscapes that control Hox gene expression in both arthropods and vertebrates (Chopra, 2009b).
Stalled Hox promoters may help promote higher-order chromatin organization within the Hox loci (see illustration). These results suggest that the stalled promoters contain intrinsic insulator activity that requires NELF and DSIF proteins, and this may help define higher-order loops within gene complexes such as the Hox complex. The stalled Pol II along with the NELF and DSIF complex may interact with putative insulator sequences, as seen for the Abd-B promoter and the Fab7. These experiments also suggest that that putative insulator sequences also require NELF and DSIF proteins, and this could be due to sharing of these proteins via the formation of higher-order loops. Such loop domains may help in proper regulation of genes and prevent any aberrant activation from neighboring enhancers, thus favoring proper gene regulations at the higher-order level (Chopra, 2009b).
Continued: Regulation part 2/3 | part 3/3
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