The "open for business" model established in (Peifer et al. 1987) made some surprisingly accurate predictions about the elements that should compose each of the cis- regulatory domains of the BX-C:
- Each of these domains must have elements able to "sense" the position of the cell along the A-P axis and, therefore, to determine the active or inactive state of the domain itself.
- It must have cell-specific enhancers that allow for the activation of its target Hox gene in a particular manner, different in each parasegment.
- Finally, the division of the BX-C into "domains" implied the existence of clear limits between each of those regions.
The techniques available at that time had not allowed yet for a fine dissection of each of the domains, but time has proven that indeed those three predictions were inherently correct, and corresponded to discrete elements present in each of the cis-regulatory modules of the BX-C. The fact that in each of the modules we find similar kinds of elements is yet another argument in favor of the proposition made in (Lewis 1978), by which the BX-C arose from events of duplication and divergence. For comprehensive reviews on the subject, see (Maeda and Karch 2006, Maeda and Karch 2015).
27 Initiator sequences
At the beginning of embryonic development, the maternal, gap, and pair-rule genes are differentially expressed along the anterior-posterior axis, creating gradients and unique combinations of gene products that divide the embryo in 14 parasegments. Each of the regulatory domains of the BX-C, in a process that is dependent on these proteins, determines the activation and expression pattern of one Hox from a given parasegment (White and Lehmann 1986, Irish et al. 1989).
But how is this positional information transmitted to each of the domains? Most of the early studies of the cis-regulatory domains were made using as approach a typical enhancer activity assay. In this assays, a piece of DNA is cloned upstream or downstream of a reporter (lac-Z at the time) with a minimal promoter. If the DNA fragment is an enhancer, it will confer a reproducible expression pattern to the lac-Z gene (Simon et al.
1990). This approach allowed for the characterization of the first tissue- and cell- specific enhancers of the BX-C, confirming the existence of these elements in the BX-C regulatory domains that had been predicted in (Peifer et al. 1987).
Surprisingly, certain sequences did not drive transcription of the lac-Z reporter in a tissue-specific manner, but in a restricted parasegment of the embryo. The authors called them "parasegmental control elements" and predicted that they might be responding to the variation in the concentration of gap or pair-rule gene products along the axis (Simon et al. 1990, Muller and Bienz 1992). Later, these sequences received the name of
"initiators". They have been identified in different regulatory domains of the BX-C, and they are indeed composed by discrete binding sequences for gap and pair-rule proteins (Qian et al. 1991, Zhang et al. 1991, Busturia and Bienz 1993, Zhou et al. 1999, Barges et al. 2000, Shimell et al. 2000).
Our laboratory has finally confirmed that the activity of the initiator only depends on the positional information along the A-P axis, and not on the regulatory domain in which it is present. In this way, the ectopic insertion of the initiator of the regulatory domain iab-5 (which activates this domain in PS10) into the iab-6 domain (normally active from PS11)
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leads to the activation of the iab-6 domain in PS10, and the subsequent transformation of PS10 into PS11 (Iampietro et al. 2010).
Boundaries
The model proposed in (Peifer et al. 1987) about the organization of the BX-C into domains implied the existence of some kind of boundaries, that delimited each of the regulatory domains to ensure its independence from the neighbor. A first hint on the existence of such elements arose with the localization of the Mcp1 deletion (Karch et al.
1985). The confirmation of this prediction came in 1990, with the discovery of Fab-7, the boundary element between the regulatory domains iab-6 and iab-7 (Gyurkovics et al.
1990), and it was rapidly followed by descriptions of boundaries between the other domains, the latest one being Fub, between bxd and iab-2 (Bender and Lucas 2013), for review see (Maeda and Karch 2015). From a molecular point of view, however, a boundary is a complex of DNA sequences able to recruit insulator proteins that protect the chromatin located on one side from the effects of enhancers and other sequences located in the other. In the BX-C, these proteins are mainly the insulator CTCF (Smith et al. 2009) and, in the case of the Fab-7 boundary, the GAGA factor (Wolle et al. 2015). In fact, the boundaries act as limits for the waves of chromatin derepression caused by the initiators. In accordance, the deletion of a boundary has as an effect the premature activation of a domain (par example, the deletion of Fab-7 causes the ectopic activation of the iab-7 domain in PS11, therefore causing a transformation of PS11 towards PS12).
This boundary activity is not parasegment specific, and due to this, a boundary of the BX-C can nearly totally substitute for the activity of another (Gyurkovics et al. 1990, Iampietro et al. 2008).
Maintenance elements
In the reporter experiments described in (Simon et al. 1990) that first proposed the existence of the initiators, it was observed that, although the lac-Z reporter was activated in the early embryo following a very restricted pattern, this restriction was lost in later embryonic stages. This is due to the loss of positional information that the gap and pair-rules initially provide at the beginning of development. However, the expression pattern
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of Hox genes is not lost, due to the activity of the so called "maintenance elements".
These sequences recruit proteins from the Polycomb family or the Trithorax family, which in turn maintain the active or inactive state of one Hox gene in one parasegment, which had been previously determined during the initiation phase (Simon et al. 1993, Simon 1995, Gross and McGinnis 1996, Strutt et al. 1997, Brock and van Lohuizen 2001, Schuettengruber et al. 2017). This is done via chromatin modifications of different types, which include, but are not restricted to, histone methylation, acetylation, and nucleosome remodeling. Most of these mechanisms are reviewed in (Simon and Tamkun 2002).
Intergenic RNAs in the Bithorax Complex
Intergenic transcription is a common feature of eukaryotic genomes. Noncoding transcription has been shown to perform diverse and important functions (Amaral and Mattick 2008), and the Hox clusters are no exception. During the past years, several noncoding RNAs have been identified in Hox clusters; one of the best known is the human HOTAIR (Rinn et al. 2007), involved in Polycomb-mediated repression of several HoxD genes (Li et al. 2013).
The early domain-specific transcription
Before the Hox genes begin to be expressed, each domain of the BX-C generates transcripts in a specific pattern (iab-2 in PS7, iab-3 in PS8, etc.). This segment-specific early transcription was first described in (Sanchez-Herrero and Akam 1989), and more thoroughly studied in (Bae et al. 2002). Although much is not known about these early transcripts, they seem to originate from the initiator elements present in the regulatory domains (S. Galletti, unpublished). Some authors have suggested that this transcription could intervene in the initial activation of the domains, by displacing Polycomb proteins from the chromosome (Bender and Fitzgerald 2002, Hogga and Karch 2002, Rank et al. 2002).
30 The noncoding RNA bithoraxoid (bxd)
The bxd/pbx regulatory domain that activate Ubx expression from PS6 is also giving rise to a noncoding RNA described for the first time in (Lipshitz et al. 1987). This ncRNA is also transcribed from PS6 but with a complementary expression pattern relative to Ubx inside each parasegment. It has been proposed to repress Ubx by transcriptional interference (Petruk et al. 2006).
The iab-4 noncoding RNA
A promoter located in the iab-3 regulatory region, very close to the boundary with iab-4, is responsible for the transcription of a 6-8Kb noncoding RNA, that gets spliced and polyadenylated. This RNA was first described in 1990, where it received the name of the iab-4 ncRNA. In accordance to the location of its promoter, the iab-4 ncRNA is expressed from PS8 to PS12 (Cumberledge et al. 1990, Garaulet et al. 2014). It is the template of the mir-iab-4 microRNA (Aravin et al. 2003). This microRNA has been shown to be a repressor of Ubx, and it is analogous to mir-196, a vertebrate microRNA that targets the Hox gene HOXB8 (Yekta et al. 2004, Ronshaugen et al. 2005).
The iab-8 noncoding RNA
The transcription of the region between abd-A abd Abd-B had already been hinted by previous studies, by the detection of a distal-to-proximal transcript (Abd-B to abd-A) that could be detected by in situ hybridization in PS13-14 of the embryo using probes against this region. This transcript originates from a promoter located downstream from Abd-B, in the iab-8 regulatory domain (Sanchez-Herrero and Akam 1989, Zhou et al. 1999, Bender and Fitzgerald 2002, Rank et al. 2002).
In 2008 it became evident that this transcription corresponded to the expression of a long noncoding RNA, and that it was responsible for the generation of mir-iab-8, the complementary microRNA from the same locus as the mir-iab-4 hairpin (Bender 2008, Tyler et al. 2008).
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The iab-8 ncRNA is a 92Kb transcript, spliced and polyadenylated. Its main spliced product is composed of 8 exons, respectively derived from each of the cis-regulatory domains. It is unclear whether this splicing pattern responds to a function. Interestingly, the sequences surrounding the splice junctions delimiting each exon seem to be conserved between Drosophila species, as if it was the act of splicing that played a role.
Some minor isoforms of the iab-8 ncRNA skip exon 8, and splice into the transcriptional unit of abd-A (Gummalla et al. 2012).
Figure IV. The iab-8 ncRNA
Schematic representation of the main spliced product of the iab-8 ncRNA.
The iab-8 promoter is located in the iab-8 regulatory region, very close to the Fab-8 boundary. Therefore, it could potentially trap the enhancers that drive the expression of Abd-B in PS13. In agreement, the iab-8 ncRNA is only expressed in PS13-14 (Figure V).
It can be detected by in situ hybridization against its terminal exon already at blastoderm stage, after cellularization. This expression restricted to PS13-14 is constant throughout embryonic development, first in the ectoderm and later in the central nervous system in late embryos and larval brain (Gummalla et al. 2012, Garaulet et al. 2014, Gummalla et al. 2014). Recent observations made in our laboratory have confirmed its expression even in adult brain (Y. Frei, personal communication).
32 Figure V. Expression pattern of the iab-8 ncRNA
In situ hybridization against the iab-6 region in embryos in blastoderm stage (A), germband extension stage (B) and germ band retraction (C), showing the expression pattern of the iab-8 ncRNA. Modified from (Gummalla et al. 2014).
The msa transcript
A transcriptome analysis throughout development identified a new lncRNA derived from the region between Abd-B and abd-A, but transcribed from a promoter located in the iab-6 region. This RNA was predominantly detected in adult males, fact from which it received the name male specific abdominal (msa) (Graveley et al. 2011).
Strikingly, both transcripts, the iab-8 ncRNA and msa, share the same splicing pattern, and they only differ by the first two exons of the iab-8 ncRNA, absent in msa. Their expression pattern, however, is completely different. While the iab-8 ncRNA is expressed from embryonic stages, msa is only expressed in the secondary cells of the accessory gland, a male-specific reproductive structure. It is the result from the transcription of an Abd-B enhancer located in the iab-6 regulatory domain of the BX-C (Gligorov et al. 2013, Maeda et al. 2018).
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A recent publication from our laboratory show that msa is also template for mir-iab-8, which apparently regulates genes in the secondary cells of the male accessory gland.
These genes are essential to elicit a proper post-mating response in females (Maeda et al.
2018).
Furthermore, recent studies performed in our laboratory have shown that msa is associated with polysomes. Indeed a short ORF located in exon 8 of the iab-8 ncRNA (exon 7 of msa) produces a micropeptide, which seems to be involved in sperm competition (C. Immarigeon, in preparation).
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