BIOLOGICAL OVERVIEW

As recently as 1986, models of the bithorax complex of genes posited not three genes, (abdominal A, Abdominal B and Ultrabithorax) as is currently assumed, but seven or eight, each independently regulated and controlled by the repressor Polycomb. This earlier model of development suggested that the cuticular phenotype of each segment depended on an array of up to eight BX-C substances elaborated in each segment. Only with the cloning of the bithorax complex genes (Regulski, 1985) was the picture more accurately understood. It then became clear that only three genes, not eight, were responsible for all the phenotypes observed.

The reason for the earlier misunderstanding is found in the existence of infra-abdominal (iab) regions; cis-acting regulatory sequences whose activities regulate the transcription of the abdominal-A and Abdominal-B genes. Ultrabithorax had already been recognized as an independent entity (Lewis, 1978). The iab regions, now termed enhancers, or locus control regions, act in cis and in many instances in trans, to regulate the structural genes. Locus control regions are able to act at a distance to enhance or diminish transcription of a structural gene. On the same chromosome, cis-acting regions are linked with their targets, while trans-acting regions are on separate chromosomes.

abdominal-A has effects that are apparent in the pattern of cuticle generated in the ectoderm and the pattern of muscle generated in mesoderm. Understanding of the effects on mesoderm predated the cloning of the homeodomain proteins. Depending on genotype, any segment can change its identity to resemble segments either more anterior or more posterior to itself. Referring to the ectodermal cuticule pattern: "Third thoracic and anterior abdominal segments can approach or achieve almost all segmental levels of development from the second thoracic to third abdominal depending on genotype" (Lewis, 1978).

The same is true for mesodermal derivatives destined to become the muscles of the fly. With ectopic mesodermal abd-A expression, one finds alterations of the mature muscle patterns of the thoracic segments and the first abdominal segment. These include a change to a more abdominal-like identity for the thoracic longitudinal muscles. In some cases, a gain of one muscle is accompanied by the loss of another (Michaelson, 1994).

Changes in regulation of the bithorax complex are found on an evolutionary scale as well. Changes in regulation by infra-abdominal regions determine how many segments, wings, arms and legs an organism develops. Without the homeodomain proteins, and especially without the regulatory changes in infra-abdominal regions, the diversity found throughout the spectrum of evolution from hydra to man would not exist.

Wnt and TGFbeta signals subdivide the AbdA hox domain during Drosophila mesoderm patterning

Hox genes have large expression domains, yet these genes control the formation of fine pattern elements at specific locations. The mechanism underlying subdivision of the abdominal-A (abdA) Hox domain in the visceral mesoderm has been examined. AbdA directs formation of an embryonic midgut constriction at a precise location within the broad and uniform abdA expression domain. The constriction divides the abdA domain of the midgut into two chambers, the anterior one producing the Pointed (Pnt) ETS transcription factor and the posterior one the Odd-paired (Opa) zinc finger protein. pnt expression in the midgut visceral mesoderm (VM) commences at stage 13, in a single patch in the central midgut. This patch is just posterior to the large basophilic cells of the endoderm, which mark the future site of the central constriction. PNT mRNA is also found at the boundaries of the midgut with the foregut and hindgut, and in the visceral branches of the trachea. The spindle shape of VM cells and their separation into four patches located dorso- and ventro-laterally around the midgut clearly distinguish pnt expression in the VM from pnt expression in the trachea. At stage 16, pnt is expressed throughout the VM of the third midgut chamber. There is a slight but detectable gradient of pnt expression: the cells that border the central constriction display higher levels of pnt than the cells that border the posterior constriction. Odd-paired (opa) is produced throughout the VM of the first and fourth midgut chambers. Examination of pnt and opa reveals that the boundaries of expression of these two genes abut, and that no cells express both pnt and opa (Bilder, 1998a).

Transcription of both pnt and opa is activated by abdA but the adjacent non-overlapping patterns are not due to mutual opa-pnt regulation. Near the anterior limit of the abdA domain, two signals, Decapentaplegic (a TGFbeta) and Wingless, are produced, in adjacent non-overlapping patterns, under Hox control in mesoderm cells. The two signals are known to regulate local mesodermal cell fates and to signal to the endoderm. In addition, they precisely subdivide the abdA domain: Wg acts upon anterior abdA domain cells to activate pnt transcription, while Dpp is essential in the same region to prevent abdA from activating opa transcription. pnt activation is required to determine the appropriate numbers of mesodermal cells in the third midgut chamber (Bilder, 1998a).

Early pnt mutant embryos have wild-type anterior-posterior information along the midgut axis, so the small third chamberof pnt mutants must arise from a spatially restricted reduction in VM cell number. Such a defect would change the apparent location of the posterior constriction, while not changing gene expression outside the affected domain. At stage 16, VM nuclei are aligned in four single files located at dorso- and ventro-lateral locations on each side of the midgut. The sizes of midgut chambers were measured by counting VM nuclei along a single file between the constrictions. In pnt mutant embryos, the third chamber is dramatically reduced, containing only 21 VM nuclei, as compared to 36 in wild-type embryos. The first, second and fourth midgut chambers of pnt embryos contain normal numbers of cells. The reduced size of the third midgut chamber is thus due primarily to a reduction of VM cell number in the third chamber. Neighboring chambers do not appear to expand at the expense of the third. Inappropriate cell death is not the cause of reduced VM cell numbers in pnt mutants. It is also concluded that pnt is not required for cell division in the VM. Because defects in cell death or cell proliferation do not occur in the VM of pnt mutants, the loss of third chamber VM cells must be due to a transformation of cell fate to a cell type that does not contribute to the anteroposterior size of the third chamber (Bilder, 1998a).

The following model for morphogenesis of the posterior midgut is proposed. AbdA activates three targets, in distinct subsets of its broad domain of expression: wingless at the anterior boundary of Connectin (Con) patch 7; pnt from anterior Con patch 7 to anterior Con patch 8, and opa, from anterior Con patch 8 through Con patch 11. Decapentaplegic signaling plays a central role in setting these distinct expression domains. The initial activation of wg by AbdA requires dpp. opa is activated in all abdA-expressing cells that do not receive a Dpp signal, defining the site of the posterior constriction. wg, in collaboration with abdA, activates pnt to generate the appropriate number of cells in the third midgut chamber, positioning the posterior constriction at the proper distance from the central constriction and partitioning the posterior midgut appropriately. Fine patterning of the posterior midgut is achieved by the activity of diffusible signals emanating from the central midgut, a remarkably long-range organizing effect (Bilder, 1998a).

Distinct Hox protein sequences determine specificity in different tissues

How Hox proteins achieve tissue-specific functions and why cells lying at equivalent A/P positions but in different germ layers have distinctive responses to the same Hox protein remains to be determined. This question was examined by identifying parts of Hox proteins necessary for Hox function in different tissues. Available genetic markers allow the regulatory effects of two Hox proteins, Abdominal-A (AbdA) and Ultrabithorax (Ubx), to be distinguished in the Drosophila embryonic epidermis and visceral mesoderm (VM). Chimeric Ubx/AbdA proteins were tested in both tissues and used to identify protein sequences that endow AbdA with a different target gene specificity from Ubx. Distinct protein sequences define AbdA, as opposed to Ubx, and they function in the epidermis vs. the VM. These sequences lie mostly outside the homeodomain (HD), emphasizing the importance of non-HD residues for specific Hox activities. Hox tissue specificity is therefore achieved by sensing distinct Hox protein structures in different tissues (Chauvet, 2000).

As a guide to what protein sequences may be most important for AbdA specificity, AbdA sequences from evolutionarily close species within the insect phylum were compared. Sequence conservation extends beyond the HD, including twelve amino acids C-terminal and adjacent to the HD. This region is also well conserved among Ubx proteins. Within this region, the ten first amino acids are mostly conserved between AbdA and Ubx. This sequence will be referred to as CterC, for C-terminal conserved. Only a few residues distinguish AbdA from Ubx in the conserved HD and in the CterC, although the proteins look mostly different in other regions. The distinguishing residues are the HD amino acids 1, 12, 23, and 35 and amino acids 61, 62, 64, and 67 in the CterC (Chauvet, 2000).

Examination of AbdA protein sequences from the evolutionarily distant insects Drosophila melanogaster and Tribolium castaneum shows that the 40 amino acids preceding the HD have been significantly conserved in AbdA. This region, referred to as NterC for N terminal conserved, contains the hexapeptide, a motif required for appropriate Hox-Exd/Pbx interaction. D. melanogaster and T. castaneum AbdA proteins have the same NterC amino acids at 63% of the positions; that number increases to 78% with conservative changes taken into consideration. This score is significantly higher than the identity/similarity score (34%/47%) found in the remaining N-terminal sequences (N-ter). Apart from the hexapeptide, the NterC region of Ubx and AbdA have largely diverged (Chauvet, 2000).

It was reasoned that the few residues that differ between Ubx and AbdA in otherwise evolutionarily conserved sequences would be good candidates for specificity control. To identify sequence elements critical for AbdA character, conserved parts of the AbdA sequence were introduced into the Ubx protein and analyzed for their ability to confer AbdA-like activity. Chimeras A-F contain combinations of point mutations introduced into Ubx to test the functional importance of the HD and CterC residues that are found specifically in AbdA. Chimeras G and H are protein sequence switches addressing the function of the NterC and of the region following the AbdA CterC sequence (C-ter). All of the protein-coding constructs were fused to a heat-inducible promoter and introduced into the fly by using P-mediated germ-line transformation. Eggs from these transgenic lines were immunostained with an antibody directed against the N-terminal sequences of Ubx, allowing detection of chimeras A-H. After heat induction, all chimeric proteins accumulate at similar levels in the nucleus (Chauvet, 2000).

Eight amino acids distinguish Ubx and AbdA within the HD and CterC region. Chimera A consists of a Ubx protein where all eight amino acids have been changed into the corresponding AbdA residues. The effects on cuticle development of ubiquitous expression of chimera A were compared with those of ubiquitous expression of Ubx and abdA. The number, identity, and spatial organization of denticles readily distinguish these two segments. Uniform Ubx expression transforms anterior segments into extra A1 segments, whereas abdA turns them into A2. Chimera A has the same effect as ubiquitous abdA expression. The three thoracic segments (T1-T3) and the first abdominal segment A1 have been transformed toward an A2 identity. In the epidermis, therefore, chimera A has acquired AbdA-like character (Chauvet, 2000).

Ubx and abdA differentially activate the target genes wg and dpp during midgut morphogenesis. In the midgut mesoderm, dpp is expressed in parasegment (PS) 3-4 and PS7, with Ubx activating dpp in PS7. When Ubx is expressed ubiquitously, dpp is ectopically activated in PS anterior to PS7. Ubiquitous abdA expression represses dpp in all of the VM. Ectopic Ubx does not affect wg transcription that continues to occur only in its normal place, PS8. Ubiquitous abdA expression does induce ectopic wg transcription in anterior regions. Chimera A behaves like Ubx, because it still activates dpp while leaving wg expression unaffected. It was noted, however, that the activation of dpp by chimera A does not occur in all VM cells anterior to PS7. This suggests that the chimera is transcriptionally less potent and/or that it is more sensitive to phenotypic suppression by the more anteriorily expressed Scr and Antp genes. In any case, these results unambiguously show that in the VM, chimera A retains Ubx specificity and has not acquired, as in the epidermis, an AbdA-like character (Chauvet, 2000).

The activities of chimeras B-F in the epidermis were studied to assess the individual contributions of the eight amino acids that distinguish AbdA from Ubx in the HD and CterC region. Changing only one residue in the N-terminal arm of the HD, either amino acid 1 (chimera E) or 12 (chimera F), is not sufficient to give the protein AbdA character in epidermal patterning. Both chimeras induce, like Ubx, ectopic A1 metameres. However, simultaneously changing both amino acids 1 and 12 (chimera C) directs the formation of A2-like segments in place of thoracic and A1 segments. The transformation toward an A2 identity is less complete than with chimera A. Such weak A2-like transformations are also observed when the changes in the protein concern the four residues in the CterC region (chimera D). Among chimeras B-F, the only protein that has an efficient AbdA effect is chimera B. This indicates that amino acids 1 and 12 of the HD, as well as amino acids 61, 62, 64, and 67 of the CterC region, are collectively required for full-potency AbdA effects. HD residues 23 and 35 appear to be unnecessary for AbdA-like activity (Chauvet, 2000).

Because chimera A does not have AbdA-like effects in the VM, the possible contributions of two additional protein sequences were investigated. The importance of the AbdA C-ter region, which is significantly longer than the Ubx C-ter region, was tested by using chimera G. In chimera G, the Ubx HD and downstream sequences are replaced by AbdA sequences. Chimera G behaves like a Ubx protein in the VM: it retains the ability to activate dpp while leaving wg expression unaffected. Sequences downstream of the AbdA HD are therefore not sufficient to convey AbdA function in the VM (Chauvet, 2000).

The function of the NterC sequence was analyzed by using chimera H, which has Ubx sequences through amino acid 234 and AbdA sequences thereafter, including the AbdA NterC, HD, and C-tail. Chimera H therefore tests the contribution of the NterC region to AbdA activity in the VM, particularly in comparison to chimera G. The results show that chimera H has AbdA effects but not Ubx effects: it represses dpp expression and induces anterior ectopic expression of wg. In the VM, therefore, the hexapeptide-containing NterC sequence confers AbdA activity, in contrast to the HD and CterC sequences important for AbdA character in the epidermis (Chauvet, 2000).

Is the NterC region on its own sufficient to confer to an otherwise Ubx protein an AbdA activity in the VM? The Ubx^C1 mutation is a chromosome rearrangement that leads to an AbdA/Ubx fusion product. The resulting protein consists of AbdA N-ter and NterC sequences joined to the Ubx HD, CterC, and C-ter. The hybrid gene is expressed in the posterior VM in the abdA expression domain. The mutation can therefore be used to determine whether the AbdA NterC region is sufficient to provide AbdA activity in the VM. In homozygous Ubx^C1 embryos, wg is not activated in VM PS8 by the C1 fusion product. In the same mutant context, expression of dpp in the anterior VM (PS3-4) is never affected, whereas expression in the central part of the midgut is severely reduced or abolished. A minority of mutant embryos display posterior ectopic expression within the expression domain of the fusion protein C1. These observations indicate that the C1 fusion protein has retained some transcriptional activity in the VM and that the level of Dpp signaling is very low in most mutant embryos (Chauvet, 2000).

wg activation by abdA critically depends on Dpp signaling in the central midgut. The absence of wg expression in Ubx^C1 homozygous embryos might thus be the result of reduced Dpp signaling rather than the inability of the C1 fusion protein to activate wg. To discriminate between these possibilities, wg expression was analyzed in homozygous Ubx^C1 embryos, where a high level of Dpp activity was provided by a heat-inducible transgene (HS.dpp). Even in such a context, no wg expression is observed. Taken together, these experiments indicate that the C1 fusion protein is not capable of providing AbdA-like function in the VM. The AbdA NterC region, although necessary to impart AbdA-like function, is not sufficient on its own. Additional AbdA sequences lying in the HD and/or C-terminal sequences are required as well (Chauvet, 2000).

The effects of ectopically expressed Hox or Hox chimeric proteins depend in some instances on ectopic activation of endogenous Hox gene activity. It was tested whether the switches to AbdA-like function obtained with chimera A in the epidermis and chimera H in the VM require the endogenous abdA gene. Embryos uniformly expressing chimera A or chimera H were stained with a probe corresponding to the N-ter sequences of AbdA, allowing the detection of the endogenous abdA gene exclusively. In both cases, no ectopic expression of abdA was observed, either in the epidermis or in the VM. The AbdA-like activities of chimeras A and H therefore do not rely on activation of the endogenous abdA gene. Instead, both chimeras have acquired an AbdA activity (Chauvet, 2000).

Most of the Hox protein specificity information has been found to lie within the HD. Indeed, in all but two of the chimeras already analyzed, the HD must be switched to change a Hox protein's function into that of another Hox protein. Residues in the N-terminal arm of the HD have been shown necessary and sometimes sufficient to define functional specificity. In agreement with these findings, the results presented here indicate that amino acids 1 and 12, in the N-terminal arm, significantly contribute to defining AbdA character (Chauvet, 2000).

For several reasons, the idea that the HD contains most of the specificity information appears overemphasized. (1) The switch of specificity is often only partial. For example, when the Deformed (Dfd) HD is replaced by that of Ubx, the chimeric protein activates the Antp gene, a target of Ubx, whereas failing to carry out a Dfd function, autoactivation of Dfd transcription. The chimeric protein has therefore acquired the target specificity of Ubx. However, the chimera did not have all of the regulatory specificity of Ubx because Ubx normally represses Antp (Chauvet, 2000).

(2) The switch of the HD alone sometimes does not confer an identity switch: replacing the HD of Ubx by that of Antp results in a chimera that behaves like Ubx, promoting A1 identity in the epidermis. Similarly, the effect of a Hox-A4/Hox-C8 chimera on vertebral patterning does not follow the identity of the HD. The Ubx/AbdA chimera data show that, in vivo, switching the HD is not necessary for changing Hox protein specificity. Chimera A activates dpp in the VM and has no effects on wg expression, indicating that it has Ubx character, despite having the AbdA HD (Chauvet, 2000).

(3) Sequences outside the HD have been shown to provide important functions for Hox gene activity. This includes the C-tail for Ubx, Antp, Scr, and Dfd along with an acidic N-region preceding the HD in the Dfd protein. The results presented here demonstrate that two sets of non-HD sequences contribute to Hox specificity. On the C-terminal side, four residues adjacent to the HD are necessary to impart AbdA-like character in the epidermis. This region is evolutionarily conserved; it has been proposed to be part of a coiled-coil structure, and might constitute an interface for interacting proteins. On the N-terminal side, the NterC region is required to define mesodermal specificity (Chauvet, 2000).

The role of the hexapeptide YPWM in the formation of Hox-Exd/Pbx heterodimers in vitro has been extensively studied, yet its in vivo function has not been established. An interesting feature of the NterC region is that it contains the hexapeptide motif. The requirement of Exd for target gene regulation in the VM suggests that the motif is involved in AbdA mesodermal function. Several lines of evidence, however, do not favor the hypothesis that the YPWM motif itself provides the key for AbdA VM specificity. (1) Ubx and AbdA share the YPWM motif, so sequences other than the motif itself must contribute to distinguishing AbdA from Ubx. (2) Exd that contacts the motif is required for Hox protein activity in several tissues. (3) The conservation within NterC regions of AbdA proteins evolutionarily as distant as D. melanogaster and T. castaneum is not restricted to the hexapeptide motif. One would thus expect these evolutionarily conserved sequences in AbdA, that have diverged in Ubx, to provide the VM-specificity information. The NterC domain therefore likely coordinates the contribution of Exd and putative tissue-specific cofactors for accurate AbdA mesodermal activity (Chauvet, 2000).

Steroid-dependent modification of Hox function drives myocyte reprogramming in the Drosophila heart

In the Drosophila larval cardiac tube, aorta and heart differentiation are controlled by the Hox genes Ultrabithorax (Ubx) and abdominal A (abdA), respectively. There is evidence that the cardiac tube undergoes extensive morphological and functional changes during metamorphosis to form the adult organ, but both the origin of adult cardiac tube myocytes and the underlying genetic control have not been established. Using in vivo time-lapse analysis, this study shows that the adult fruit fly cardiac tube is formed during metamorphosis by the reprogramming of differentiated and already functional larval cardiomyocytes, without cell proliferation. The genetic control of the process, which is cell autonomously ensured by the modulation of Ubx expression and AbdA activity, has been characterized. Larval aorta myocytes are remodelled to differentiate into the functional adult heart, in a process that requires the regulation of Ubx expression. Conversely, the shape, polarity, function and molecular characteristics of the surviving larval contractile heart myocytes are profoundly transformed as these cells are reprogrammed to form the adult terminal chamber. This process is mediated by the regulation of AbdA protein function, which is successively required within these persisting myocytes for the acquisition of both larval and adult differentiated states. Importantly, AbdA specificity is switched at metamorphosis to induce a novel genetic program that leads to differentiation of the terminal chamber. Finally, the steroid hormone ecdysone controls cardiac tube remodelling by impinging on both the regulation of Ubx expression and the modification of AbdA function. These results shed light on the genetic control of one in vivo occurring remodelling process, which involves a steroid-dependent modification of Hox expression and function (Monier, 2005).

The morphological transformations of the cardiac tube during metamorphosis were visualised by polymerised actin (F-actin) staining with phalloidin, which reveals myofibril organisation and enables visualisation of the general morphology of the cardiac tube. Major morphological and functional changes accompany the remodelling of the cardiac tube. In order to unambiguously determine the origin of the adult cardiac myocytes, cardiac tube-specific expression of GFP was analyzed throughout metamorphosis. This approach indicated that the adult organ arises by the remodelling of the larval cardiac tube by a continuous and progressive process. Time-lapse analysis of cardiac tube remodelling, performed from 27 to 64 hours after puparium formation (APF), demonstrates that adult myocytes directly derive from larval myocytes without cell proliferation. In addition, cell tracing experiments show that the adult heart is formed by the myocytes that constituted the larval posterior aorta, and by segment A5 myocytes, which are a part of the larval heart and which constitute the posterior tip of the adult heart (the terminal chamber). Both approaches thus indicate that the adult heart is formed by persisting larval myocytes that are remodelled without proliferation or the addition of new cells. One identity for each adult cardiac myocyte can therefore be inferred from its larval origin (Monier, 2005).

Transdifferentiation is the process by which a cell committed to one phenotypic fate acquires a different fate, implying that some characteristics are lost while novel ones are acquired. This study suggests that myocytes in segment A5 undergo a transdifferentiation process during cardiac tube remodelling: their shape and polarity are changed and they undergo transcriptional reprogramming. In addition, their physiological function is modified: they lose the rhythmic contractile activity they display during larval life, but gain specific innervations, implying that they may become involved in the regulation of cardiac activity. Indeed, adult A5 cardiac myocytes are directly innervated, whereas anterior innervations are targeted to ventral imaginal muscles but not to A1-A4 cardiac myocytes. Neurons innervating the terminal chamber have been described as being positive for Cardioacceleratory peptide and might be involved in the regulation of the anterograde heart beat (Monier, 2005).

Much attention has been given to transdifferentiation in recent years; however, the majority of studies have used artificial systems and the genetic control of the process is still largely unknown. This study has investigated the genetic and molecular control of a naturally occurring transdifferentiation process: the metamorphosis of the Drosophila larval cardiac tube. This situation offers the opportunity to analyse cell plasticity in an intact system, in which cells can be individually identified (Monier, 2005).

Like myocytes in segment A5, those in segments A1-A4 also gain a number of novel properties during remodelling. For example, Tin-expressing myocytes increase their myofibrillar content, activate I_h channel (Ih) transcription and acquire contractile activity; svp-expressing cells transiently express wg and differentiate into functional ostiae. However, this may not represent transdifferentiation per se, since these cells do not appear to lose any of the differentiated characteristics they would have acquired during embryonic or larval life. Rather, it is considered that larval A1-A4 myocytes are committed to differentiate into adult heart myocytes, but do not represent, on their own, a fully differentiated state. Interestingly, their final differentiation relies on an ecdysone-dependent modulation of Ubx expression: Ubx expression has to be downregulated in Tin-expressing myocytes, while its maintenance, and probably its overexpression, is required in svp-expressing cells. Ubx function, however, appears to be unchanged in these cells. Indeed, when Ubx is overexpressed in the embryonic cardiac tube, it is able to activate wg expression in A1-A4 svp-expressing myocytes, and to repress Ih transcription in Tin-expressing myocytes. This clearly indicates that, at least at the wg and Ih transcriptional level, Ubx activity is unmodified during remodelling (Monier, 2005).

By contrast, the reprogramming of A5 myocytes depends on the modification of AbdA activity. Although abdA is required, during embryogenesis, for Ih transcription and for the acquisition of rhythmic contractile activity by the A5 myocytes that constitute part of the beating larval heart, its cellular function is modified at metamorphosis. During cardiac tube remodelling, abdA is indeed required for Ih repression, and for changes in the shape and polarity of myocytes, as well as for the acquisition of specific innervations. In these myocytes, the functional outcome of AbdA activity is thus dramatically different during embryogenesis and metamorphosis, indicating that abdA is instrumental in myocyte reprogramming (Monier, 2005).

The developmental switch triggering cardiac tube remodelling is mediated by ecdysone, which impinges on Ubx expression and on AbdA activity. Interestingly, like retinoic acid (RA) in the developing vertebrate hindbrain, the outcome of ecdysone activity during the remodelling of segments A1-A4 is a 'posterior transformation': adult A1-A4 myocytes acquire properties characteristic of larval A5-A7 posterior myocytes. This is the first reported example of a steroid function in axial patterning in arthropods. In addition, it is shown that, like RA, which modulates Hox expression in vertebrates, the ecdysone signalling pathway modulates Ubx expression. These observations strongly suggest that steroid activity on axial patterning is conserved from flies to vertebrates (Monier, 2005).

In contrast to its effects on Ubx, ecdysone does not affect abdA expression but impinges on its activity. This is the first observation of such an activity for a steroid hormone and it certainly warrants further study in other animal models. Is this also a property of steroids (such as RA) in vertebrates? To date, the only known activity of RA on Hox gene function is a modulation of their transcriptional expression. The data presented in this study recommend a re-examination of RA/Hox functional interactions in vertebrates (Monier, 2005).

Cellular identities along the rostrocaudal axis mainly depend on the Hox code, i.e., on the combinatorial expression of Hox genes. The fact that, as is shown in this study, a steroid hormone can additionally control Hox activity greatly increases the number of cellular identities that can potentially be determined by the same Hox code. In addition, the situation described in this study concerning AbdA activity indicates the importance of the developmental context upon Hox specificity. An important unanticipated result from the present study is that the change of AbdA activity can occur in the same cells, leading to a reprogramming between two differentiated states that are both controlled by the same Hox gene (Monier, 2005).

A future challenge will be to understand the molecular basis of the steroid-dependent Hox activity switch. Ecdysone signalling might change AbdA transcriptional activity, by specifying new transcriptional targets or by switching its activity from an activator to a repressor (and vice versa) on the same transcriptional targets. What could be the molecular mechanism by which EcR activation impinges on AbdA activity? Given the pivotal role played by transcriptional cofactors in Hox function, one hypothesis is that EcR activates or represses such a Hox cofactor. Exd/Hth are obvious candidates but do not seem to be involved; they are not expressed in the cardiac myocytes, neither during embryogenesis nor during the remodelling process. Conversely, there is evidence that Hox transcriptional activity can be modified by phosphorylation. An alternate possibility would therefore be that EcR activates or represses the expression of some kinases or phosophatases, which would in turn modify AbdA activity. In any case, elucidation of the molecular mechanisms will rely on the identification of direct abdA targets in the cardiac tube. wg and Ih are potential candidates, and are currently under investigation (Monier, 2005).

In conclusion, this study may shed light on Hox gene function and regulation in other cell reprogramming processes, such as pathological remodelling of the human heart, in which steroids appear to play a key role. In addition, cellular therapies for inherited myopathies are based on the cellular plasticity of the donor cells (either satellite cells or muscle-derived stem cells). Cell transplantations for the treatment of muscular dystrophies appear promising, and understanding the molecular basis of one example of myocyte reprogramming should help to unravel the underlying mechanisms (Monier, 2005).

Decoding the regulatory logic of the Drosophila male stem cell system

The niche critically controls stem cell behavior, but its regulatory input at the whole-genome level is poorly understood. This study elucidated transcriptional programs of the somatic and germline lineages in the Drosophila testis and genome-wide binding profiles of Zfh-1 and Abd-A expressed in somatic support cells and crucial for fate acquisition of both cell lineages. Key roles were identified of nucleoporins and V-ATPase proton pumps, and their importance was demonstrated in controlling germline development from the support side. To make the dataset publicly available, an interactive analysis tool was generated, that uncovered conserved core genes of adult stem cells across species boundaries. The functional relevance of these genes was tested in the Drosophila testis and intestine, and a high frequency of stem cell defects was found. In summary, this dataset and interactive platform represent versatile tools for identifying gene networks active in diverse stem cell types (Tamirisa, 2018).

Using cell-type-specific transcriptome profiling and in vivo TF binding site mapping together with an interactive data analysis tool, this study comprehensively identified genes involved in controlling proliferation and differentiation within a stem cell support system. Importantly, many candidates that were functionally tested not only were required within the soma, but also had non cell-autonomous functions in the adjacent (germline) stem cell lineage (Tamirisa, 2018).

An interconnected network of TFs was identified that plays an important role in the maintenance and differentiation of both germline and somatic cell populations, signal processing V-ATPase proton pumps, and nuclear-transport-engaged Nups as regulators in the Drosophila male stem cell system. V-ATPases have been implicated in the regulation of various cellular processes in not only invertebrates but also vertebrates. For example, the V-ATPase subunit V1e1 was previously shown to be essential for the maintenance of NBs in the developing mouse cortex, as loss of this subunit caused a reduction of endogenous Notch signaling and a depletion of NBs by promoting their differentiation into neurons. Furthermore, two independent studies revealed that V-ATPase subunits and their isoforms are required for proper spermatogenesis in mice, in particular for acrosome acidification and sperm maturation. Thus, it is tempting to speculate that these proton pumps also have important functions in the stem cell pool of the mammalian testis and very likely many other stem cell systems, and some evidence is provided for their crucial role also in ISCs (Tamirisa, 2018).

This work also uncovered nuclear transport associated proteins, the Nups, as important control hubs in the somatic lineage of the Drosophila testis. This is of particular interest, since cell-type-specific functions of Nups have been identified only recently and may represent a critical feature of different stem cell systems. Examples include Nup153, one of the Nups expressed in all four stem cell systems, which interacts with Sox2 neural progenitors and controls their maintenance as well as neuronal differentiation; Nup358, which plays a role at kinetochores, and Nup98, which regulates the anaphase promoting complex (APC) and mitotic microtubule dynamics to promote spindle assembly. Interestingly, it has been shown just recently that Nups play a critical role in regulating cell fate during early Drosophila embryogenesis, thereby contributing to the commitment of pluripotent somatic nuclei into distinct lineages. Current results suggest that they may play a similar role in controlling the transition of continuously active adult stem cells toward differentiation. The next challenge will be to unravel how variations in the composition of an essential and basic protein complex like the NPC causes differential responses of cells, in particular in stem cells and their progenies (Tamirisa, 2018).

The datasets in conjunction with the versatile and easy-to-use analysis tool allowed identification of a substantial number of stem cell regulators for detailed mechanistic characterization. Importantly, this analyses have shed first light on processes and genes shared between diverse invertebrate and vertebrate stem cell systems and uncovered functionally relevant differences. Owing to its flexibility and the option to include datasets from any species, the online tool represents a valuable resource for the entire stem cell community. It not only provides an open platform for data analysis but also leverages the power of comparative analysis to enable researchers mining genomic datasets from diverse origins in a meaningful and intuitive fashion (Tamirisa, 2018).

Boundaries mediate long-distance interactions between enhancers and promoters in the Drosophila Bithorax complex

Drosophila bithorax complex (BX-C) is one of the best model systems for studying the role of boundaries (insulators) in gene regulation. Expression of three homeotic genes, Ubx, abd-A, and Abd-B, is orchestrated by nine parasegment-specific regulatory domains. These domains are flanked by boundary elements, which function to block crosstalk between adjacent domains, ensuring that they can act autonomously. Paradoxically, seven of the BX-C regulatory domains are separated from their gene target by at least one boundary, and must 'jump over' the intervening boundaries. To understand the jumping mechanism, the Mcp boundary was replaced with Fab-7 and Fab-8. Mcp is located between the iab-4 and iab-5 domains, and defines the border between the set of regulatory domains controlling abd-A and Abd-B. When Mcp is replaced by Fab-7 or Fab-8, they direct the iab-4 domain (which regulates abd-A) to inappropriately activate Abd-B in abdominal segment A4. For the Fab-8 replacement, ectopic induction was only observed when it was inserted in the same orientation as the endogenous Fab-8 boundary. A similar orientation dependence for bypass activity was observed when Fab-7 was replaced by Fab-8. Thus, boundaries perform two opposite functions in the context of BX-C-they block crosstalk between neighboring regulatory domains, but at the same time actively facilitate long distance communication between the regulatory domains and their respective target genes (Postaka, 2018).

The three homeotic (HOX) genes in the Drosophila Bithorax complex (BX-C), Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B), are responsible for specifying cell identity in parasegments (PS) 5-14, which form the posterior half of the thorax and all of the abdominal segments of the adult fly. Parasegment identity is determined by the precise expression pattern of the relevant HOX gene and this depends upon a large cis-regulatory region that spans 300 kb and is subdivided into nine PS domains that are aligned in the same order as the body segments in which they operate. Ubx expression in PS5 and PS6 is directed by abx/bx and bxd/pbx, while abd-A expression in PS7, PS8, and PS9 is controlled by iab-2, iab-3, and iab-4. Abd-B is regulated by four domains, iab-5, iab-6, iab-7 and iab-8, which control expression in PS10, PS11, PS12 and PS13 respectively (Postaka, 2018).

Each regulatory domain contains an initiator element, a set of tissue-specific enhancers and Polycomb Response Elements (PREs) and is flanked by boundary/insulator elements. BX-C regulation is divided into two phases, initiation and maintenance. During the initiation phase, a combination of gap and pair-rule proteins interact with initiation elements in each regulatory domain, setting the domain in the on or off state. In PS10, for example, the iab-5 domain, which regulates Abd-B, is activated by its initiator element, while the more distal Abd-B domains, iab-6 to iab-8 are set in the off state. In PS11, the iab-6 initiator activates the domain, while the adjacent iab-7 and iab-8 domains are set in the off state. Once the gap and pair-rule gene proteins disappear during gastrulation, the on and off states of the regulatory domains are maintained by Trithorax (Trx) and Polycomb (PcG) group proteins, respectively (Postaka, 2018).

In order to select and then maintain their activity states independent of outside influence, adjacent regulatory domains are separated from each other by boundary elements or insulators. Mutations that impair boundary function permit crosstalk between positive and negative regulatory elements in adjacent domains and this leads to the misspecification of parasegment identity. This has been observed for deletions that remove five of the BX-C boundaries (Front-ultraabdominal (Fub), Miscadestral pigmentation (Mcp), Frontadominal-6 (Fab-6), Frontadominal-7 (Fab-7), and Frontadominal-8 (Fab-8)) (Postaka, 2018).

While these findings indicate that boundaries are needed to ensure the functional autonomy of the regulatory domains, their presence also poses a paradox. Seven of the nine BX-C regulatory domains are separated from their target HOX gene by at least one intervening boundary element. For example, the iab-6 regulatory domain must 'jump over' or 'bypass' Fab-7 and Fab-8 in order to interact with the Abd-B promoter. That the blocking function of boundaries could pose a significant problem has been demonstrated by experiments in which Fab-7 is replaced by heterologous elements such as scs, gypsy or multimerized binding sites for the architectural proteins dCTCF, Pita or Su(Hw). In these replacements, the heterologous boundary blocked crosstalk between iab-6 and iab-7 just like the endogenous boundary, Fab-7. However, the boundaries were not permissive for bypass, preventing iab-6 from regulating Abd-B (Postaka, 2018).

A number of models have been proposed to account for this paradox. One is that BX-C boundaries must have unique properties that distinguish them from generic fly boundaries. Since they function to block crosstalk between enhancers and silencers in adjacent domains, an appealing idea is that they would be permissive for enhancer/silencer interactions with promoters. However, several findings argue against this notion. For one, BX-C boundaries resemble those elsewhere in the genome in that they contain binding sites for architectural proteins such as Pita, dCTCF, and Su(Hw). Consistent with their utilization of these generic architectural proteins, when placed between enhancers (or silencers) and a reporter gene, BX-C boundaries block regulatory interactions just like boundaries from elsewhere in the genome. Similarly, there is no indication in these transgene assays that the blocking activity of BX-C boundaries are subject to parasegmental regulation. Also arguing against the idea that BX-C boundaries have unique properties, the Mcp boundary, which is located between iab-4 and iab-5, is unable to replace Fab-7. Like the heterologous boundaries, it blocks crosstalk, but it is not permissive for bypass. A second model is that there are special sequences, called promoter targeting sequence (PTS), located in each regulatory domain that actively mediate bypass. While the PTS sequences that have been identified in iab-6 and iab-7 enable enhancers to 'jump over' an intervening boundary in transgene assays, they do not have a required function in the context of BX-C and are completely dispensable for Abd-B regulation (Postaka, 2018).

A third model is suggested by transgene 'insulator bypass' assays. In one version of this assay, two boundaries instead of one are placed in between an enhancer and the reporter. When the two boundaries pair with each other, the enhancer is brought in close proximity to the reporter, thereby activating rather than blocking expression. Consistent with a possible role in BX-C bypass, these pairing interactions can occur over large distances and even skip over many intervening boundaries. The transgene assays point to two important features of boundary pairing interactions that are likely to be relevant in BX-C. First, pairing interactions are specific. For this reason boundaries must be properly matched with their neighborhood in order to function appropriately. A requirement for matching is illustrated in transgene bypass experiments in which multimerized binding sites for specific architectural proteins are paired with themselves or with each other. Bypass was observed when multimerized dCTCF, Zw5 or Su(Hw) binding sites were paired with themselves; however, heterologous combinations (e.g., dCTCF sites with Su(Hw) sites) did not support bypass. A second feature is that pairing interactions between boundaries are typically orientation dependent For example, scs pairs with itself head-to-head, not head-to-tail (Postaka, 2018).

If both blocking and bypass activities are intrinsic properties of fly boundaries then the BX-C boundaries themselves may facilitate contacts between the regulatory domains and their target genes. Moreover, the fact that both blocking and bypass activity are non-autonomous (in that they depend on partner pairing) could potentially explain why heterologous Fab-7 replacements like gypsy and Mcp behave anomalously while Fab-8 functions appropriately. Several observations fit with this idea. There is an extensive region upstream of the Abd-B promoter that has been implicated in tethering the Abd-B regulatory domains to the promoter and this region could play an important role in mediating bypass by boundaries associated with the distal Abd-B regulatory domains (iab-5, iab-6, iab-7). Included in this region is a promoter tethering element (PTE) that facilitates interactions between iab enhancers and the Abd-B promoter in transgene assays. Just beyond the PTE is a boundary-like element, AB-I. In transgene assays AB-I mediates bypass when combined with either Fab-7 or Fab-8. In contrast, a combination between AB-I and Mcp fails to support bypass. The ability of both Fab-7 and Fab-8 to pair with AB-I is recapitulated in Fab-7 replacement experiments. Unlike Mcp, Fab-8 has both blocking and bypass activity when inserted in place of Fab-7. Moreover, its bypass but not blocking activity is orientation-dependent. When inserted in the same orientation as the endogenous Fab-8 boundary, it mediates blocking and bypass, while it does not support bypass when inserted in the opposite orientation (Postaka, 2018).

Boundaries flanking the Abd-B regulatory domains must block crosstalk between adjacent regulatory domains but at the same time allow more distal domains to jump over one or more intervening boundaries and activate Abd-B expression. While several models have been advanced to account for these two paradoxical activities, replacement experiments argued that both must be intrinsic properties of the Abd-B boundaries. Thus Fab-7 and Fab-8 have blocking and bypass activities in Fab-7 replacement experiments, while heterologous boundaries including multimerized dCTCF sites and Mcp from BX-C do not. One idea is that Fab-7 and Fab-8 are simply 'permissive' for bypass. They allow bypass to occur, while boundaries like multimerized dCTCF or Mcp are not permissive in the context of Fab-7. Another is that they actively facilitate bypass by directing the distal Abd-B regulatory domains to the Abd-B promoter. Potentially consistent with an 'active' mechanism that involves boundary pairing interactions, the bypass activity of Fab-8 and to a lesser extent Fab-7 is orientation dependent (Postaka, 2018).

This study has tested these two models further. For this purpose the Mcp boundary was used for in situ replacement experiments. Mcp defines the border between the regulatory domains that control expression of abd-A and Abd-B. In this location, it is required to block crosstalk between the flanking domains iab-4 and iab-5, but it does not need to mediate bypass. In this respect, it differs from the boundaries that are located within the set of regulatory domains that control either abd-A or Abd-B, as these boundaries must have both activities. If bypass were simply passive, insertion of a 'permissive' Fab-7 or Fab-8 boundary in either orientation in place of Mcp would be no different from insertion of a generic 'non-permissive' boundary such as multimerized dCTCF sites. Assuming that Fab-7 and Fab-8 can block crosstalk out of context, they should fully substitute for Mcp. In contrast, if bypass in the normal context involves an active mechanism in which more distal regulatory domains are brought to the Abd-B promoter, then Fab-7 and Fab-8 replacements might also be able to bring iab-4 to the Abd-B promoter in a configuration that activates transcription. If they do so, then this process would be expected to show the same orientation dependence as is observed for bypass of the Abd-B regulatory domains in Fab-7 replacements (Postaka, 2018).

Consistent with the idea that a boundary located at the border between the domains that regulate abd-A and Abd-B need not have bypass activity, this study found that multimerized binding sites for the dCTCF protein fully substitute for Mcp. Like the multimerized dCTCF sites, Fab-7 and Fab-8 are also able to block crosstalk between iab-4 and iab-5. Blocking activity of Fab-7 is incomplete and there are small clones of cells in which the mini-y reporter is activated in A4. In contrast, the blocking activity of Fab-8 is comparable to the multimerized dCTCF sites and the mini-y reporter is off throughout A4. One plausible reason for this difference is that Mcp and the boundaries flanking Mcp (Fab-4 and Fab-6) utilize dCTCF as does Fab-8, while this architectural protein does not bind to Fab-7(Postaka, 2018).

Importantly, in spite of their normal (or near normal) ability to block crosstalk, both boundaries still perturb Abd-B regulation. In the case of Fab-8, the misregulation of Abd-B is orientation dependent just like the bypass activity of this boundary when it is used to replace Fab-7. When inserted in the reverse orientation, Fab-8 behaves like multimerized dCTCF sites and it fully rescues the Mcp deletion. In contrast, when inserted in the forward orientation, Fab-8 induces the expression of Abd-B in A4 (PS9), and the misspecification of this parasegment. Unlike classical Mcp deletions or the Mcp^PRE replacement described here, expression of the Abd-B gene in PS9 is driven by iab-4, not iab-5. This conclusion is supported by two lines of evidence. First, the mini-y reporter inserted in iab-5 is off in PS9 cells indicating that iab-5 is silenced by PcG factors as it should be in this parasegment. Second, the ectopic expression of Abd-B is eliminated when the iab-4 regulatory domain is inactivated (Postaka, 2018).

These results, taken together with previous studies, support a model in which the chromatin loops formed by Fab-8 inserted at Mcp in the forward orientation brings the enhancers in the iab-4 regulatory domain in close proximity to the Abd-B promoter, leading to the activation of Abd-B in A4 (PS9). In contrast, when inserted in the opposite orientation, the topology of the chromatin loops formed by the ectopic Fab-8 boundary are not compatible with productive interactions between iab-4 and the Abd-B promoter. Moreover, it would appear that boundary bypass for the regulatory domains that control Abd-B expression is not a passive process in which the boundaries are simply permissive for interactions between the regulatory domains and the Abd-B promoter. Instead, it seems to be an active process in which the boundaries are responsible for bringing the regulatory domains into contact with the Abd-B gene. It also seems likely that bypass activity of Fab-8 (and also Fab-7) may have a predisposed preference, namely it is targeted for interactions with the Abd-B gene. This idea would fit with transgene bypass experiments, which showed that both Fab-7 and Fab-8 interacted with an insulator like element upstream of the Abd-B promoter, AB-I, while the Mcp boundary didn't (Postaka, 2018).

Similar conclusions can be drawn from the induction of Abd-B expression in A4 (PS9) when Fab-7 is inserted in place of Mcp. Like Fab-8, this boundary inappropriately targets the iab-4 regulatory domain to Abd-B. Unlike Fab-8, Abd-B is ectopically activated when Fab-7 is inserted in both the forward and reverse orientations. While the effects are milder in the reverse orientation, the lack of pronounced orientation dependence is consistent with experiments in which Fab-7 was inserted at its endogenous location in the reverse orientation. Unlike Fab-8 only very minor iab-6 bypass defects were observed. In addition to the activation of Abd-B in A4 (PS9) the Fab-7 Mcp replacements also alter the pattern of Abd-B regulation in more posterior segments. In the forward orientation, A4 and A5 are transformed towards an A6 identity, while A6 is also misspecified. Similar though somewhat less severe effects are observed in these segments when Fab-7 is inserted in the reverse orientation. At this point the mechanisms responsible for these novel phenotypic effects are uncertain. One possibility is that pairing interactions between the Fab-7 insert and the endogenous Fab-7 boundary disrupt the normal topological organization of the regulatory domains in a manner similar to that seen in boundary competition transgene assays. An alternative possibility is that Fab-7 targets iab-4 to the Abd-B promoter not only in A4 (PS9) but also in cells in A5 (PS10) and A6 (PS11). In this model, Abd-B would be regulated not only by the domain that normally specifies the identity of the parasegment (e.g., iab-5 in PS10), but also by interactions with iab-4. This dual regulation would increase the levels of Abd-B, giving the weak GOF phenotypes. Potentially consistent with this second model, inactivating iab-4 in the Mcp^F8 replacement not only rescues the A4 (PS9) GOF phenotypes but also suppresses the loss of anterior trichomes in the A6 tergite (Postaka, 2018).

The Hox proteins Ubx and AbdA collaborate with the transcription pausing factor M1BP to regulate gene transcription

In metazoans, the pausing of RNA polymerase II at the promoter (paused Pol II) has emerged as a widespread and conserved mechanism in the regulation of gene transcription. While critical in recruiting Pol II to the promoter, the role transcription factors play in transitioning paused Pol II into productive Pol II is, however, little known. By studying how Drosophila Hox transcription factors control transcription, this study uncovered a molecular mechanism that increases productive transcription. The Hox proteins AbdA and Ubx target gene promoters previously bound by the transcription pausing factor M1BP, containing paused Pol II and enriched with promoter-proximal Polycomb Group (PcG) proteins, yet lacking the classical H3K27me3 PcG signature. AbdA binding to M1BP-regulated genes results in reduction in PcG binding, the release of paused Pol II, increases in promoter H3K4me3 histone marks and increased gene transcription. Linking transcription factors, PcG proteins and paused Pol II states, these data identify a two-step mechanism of Hox-driven transcription, with M1BP binding leading to Pol II recruitment followed by AbdA targeting, which results in a change in the chromatin landscape and enhanced transcription (Zouaz, 2017).

Understanding Hox transcriptional networks is central to understanding their wide repertoire of functions, yet observing where they bind in the genome does not explain why they bind there. In using a homogenous cell-based system devoid of endogenous Hox expression to conditionally express the Hox protein Ubx or AbdA, this study has demonstrated that Drosophila Hox proteins target proximal promoters genome-wide, which is conserved (for Ubx at least) in developing embryos. While studies into Hox genomic binding have historically focussed on enhancer elements in spatially and temporarily controlling individual gene expression, genome-wide promoter enrichment of Hox proteins is known to occur for mouse HoxB4 in hematopoietic stem cells, mouse Hoxa2 in the second branchial arches and for zebrafish Hoxb1a in early embryogenesis. However, why Hox proteins target the promoter-proximal region has been little explored. A major advantage of the Drosophila S2 cell system is that the conditional Hox expression system allows studying in fine detail the sequence of events occurring upon promoter binding and the impact on gene expression (Zouaz, 2017).

The promoters targeted by both AbdA and Ubx in Drosophila are essentially promoters containing either GAF or M1BP. GAF controls mainly development and morphogenic genes, whereas M1BP controls genes mainly involved in basic cellular processes (Li, 2013), and this distinction in gene ontology is reflected in the genes whose promoters are targeted by AbdA. As AbdA and Ubx also target enhancer regions, it cannot be ruled out that the observed promoter binding is the result of enhancer~promoter interaction. However, given that the majority of genes controlled by M1BP do not have distal enhancers (Zabidi, 2015), it is unlikely that this is the case for M1BP-targeted promoters. Both GAF and M1BP are important and distinct Drosophila Pol II pausing factors, a role that proved important in understanding the nature of promoters targeted by AbdA and Ubx, since the majority of all promoters targeted by the Hox proteins contained poised Pol II. GAF binding sites have previously been shown enriched at Ubx targets, although a link between Hox and GAF in regulating gene transcription was not demonstrated. Similarly, in S2-AbdA cells, AbdA binding at GAF-regulated promoters has little clear-cut effect on poised Pol II status, although the amount of elongating Pol II and gene transcription appears reduced. It was at M1BP-bound promoters where AbdA was found to have an effect on Pol II pausing, whereby AbdA binding results in a reduction in poised Pol II giving rise to increased productive transcription. Taken together with the findings that both Ubx and AbdA target nearly identical promoters, that AbdA and M1BP synergise in reporter gene expression, that both Ubx and AbdA interact with M1BP in embryos and AbdA colocalises with M1BP on polytene chromosomes, these data suggest functional cooperation between M1BP and AbdA/Ubx. To this end, demonstrating that M1BP expression is essential in inhibiting autophagy in the larval fat body, an Exd-independent cellular function shared by all Drosophila Hox proteins where the loss of expression of all Hox genes is essential for autophagy induction, suggests that M1BP may function with Hox proteins in their generic function of autophagy inhibition (Zouaz, 2017).

Similar to the distinct mechanisms at play in pausing Pol II at M1BP- and GAF-controlled promoters, these data suggest that distinct mechanisms of release of paused Pol II exist at the two classes of poised Pol II promoters: additional factors that are not present in S2 cells are likely required to permit Hox-induced productive transcription at GAF-controlled promoters since little evidence is foundthat AbdA binding affects Pol II pausing, whereas at M1BP-controlled genes, AbdA binding is sufficient to increase gene transcription through the release of paused Pol II (Zouaz, 2017).

Testing the association of AbdA ChIP peaks in S2-AbdA cells with those of numerous publicly available histone-modifying proteins and histone marks in S2 cells, this study found that the M1BP- and GAF-poised Pol II promoters targeted by AbdA were enriched for PcG proteins and H3K4me3. The finding that AbdA-enhanced transcription at M1BP promoters was more consistently concomitant with a loss of promoter PcG protein binding than at GAF-controlled promoter, suggests that the emerging role for PcG proteins in maintaining a poised Pol II state can be perturbed by Hox binding. Indeed, it is noteworthy that of the PcG proteins tested here, it is promoter-bound dRing that is most affected upon AbdA binding, suggesting that, like in vertebrates where Ring1 plays a major role in restraining the poised Pol II at promoters, dRing may play a major role in tethering the poised Pol II state in Drosophila. Given that no clear effect on PcG binding occurs at GAF-controlled promoters, even when these genes are repressed upon AbdA binding, it reinforces the notion that contrary to M1BP targets, the control of gene expression by AbdA at GAF genes is unlikely to occur through the regulation of poised Pol II status (Zouaz, 2017).

Where PcG proteins are linked to maintaining gene repression, trithorax group proteins (trxG) are the PcG antagonists, responsible for maintaining gene expression. As a transcription factor, GAF has been traditionally classified as a trxG protein although it displays repressive activity and can recruit PcG complexes. As such, GAF can be classified as a member of the growing family of genes that display both PcG and trxG phenotypes, the so-called enhancers of trithorax and polycomb (ETP) family. This study shows that M1BP colocalises with PcG proteins at promoters in S2 cells and phenotypically enhances the PcG homeotic phenotype of extra male sex combs on the second and third pairs of legs. Indeed, M1BP-/Pc- transheterozygous males have an average of 5.3 legs displaying sex combs, which is more than most combinations of PcG mutant transheterozygotes, demonstrating the large increase in penetrance of the Pc phenotype upon M1BP mutation. As such, M1BP would be genetically classified as a PcG gene. However, PcG genes, by definition, display homeotic phenotypes due to the derepression of Hox genes when mutated and so since this study observed neither increased derepression of the upstream Hox gene responsible for sex comb development, Scr, upon M1BP mutation nor Hox expression in fat body cells following RNAi, M1BP cannot thus be classified as a PcG gene. Given that M1BP is a transcription factor involved in gene expression (Li, 2013), the hypothesis is therefore favoured that, like GAF, M1BP is likely to be a member of the ETP family. How ETP proteins can enhance the phenotypes of both repressors (PcG) and activators (trxG) has long remained a mystery. Demonstrating here that GAF and M1BP colocalise with PcG at poised Pol II promoters with the loss of PcG at those genes displaying increased expression upon AbdA binding, may go a long way to better understand how transcription factors and transcriptional repressors intricately cooperate to regulate gene transcription (Zouaz, 2017).

In summary, this work identifies a novel mechanism for Pol II pausing release mediated by AbdA: at genes bound by M1BP, targeting of AbdA results in the specific loss of PcG proteins, the release of poised Pol II and increases in H3K4me3 histone marks, which results in promoting productive transcription. Identified in S2 cells where Hox PBC-class cofactors are absent, this mechanism may more generally apply to Hox-generic functions that are independent of PBC-class cofactors, such as the repression of autophagy in the Drosophila fat body or sex comb development in Drosophila males. It may also apply to PBC-dependent Hox target gene regulatien by cooperating with Hox PBC-bound genomic regions located remote from the promoter. Further work aimed at studying Hox PBC-bound enhancers together with poised Pol II status, and promoter-proximal Hox and PcG binding, should provide further insight into how enhancer-bound protein complexes influence the basic mechanisms of transcription regulated through poised Pol II. Uncovering such a Hox-driven mechanism of gene regulation by sequence-specific transcription factors, PcG proteins and poised Pol II in the developing animal would have been fraught with difficulties, not least of which is the quagmire of PcG proteins being essential global repressors of all Hox genes (Zouaz, 2017).

GENE STRUCTURE

abdominal-A is one of three homeotic genes of the bithorax complex (BX-C) and is situated between Ultrabithorax and abdominal-B, and flanked by regulatory regions infra-abdominal 2 and 3.

PROTEIN STRUCTURE

Amino Acids - 330

Structural Domains

A comparison of the homeobox sequence of bithorax complex genes ANTP, UBX and ABD-A shows great intra-complex similarity, with only a few amino acid substitutions noted. A YPWM motif is shared by UBX, ANTP, DFD, SCR and LAB as well as an OPA repeat downstream of the homeobox sequence. Outside the homeodomain, the sequence of Abd-A has diverged from these other proteins (Karch, 1990 and Duboule, 1994).

date revised:  25 April 2024

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