trithorax encodes a positive regulatory factor required throughout development for normal expression of multiple homeotic genes of the bithorax and Antennapedia complexes (BX-C and ANTP-C). To determine how trx influences homeotic gene expression, the expression of the BX-C genes Ultrabithorax, abdominal-A, Abdominal-B and the ANTP-C genes Antennapedia, Sex combs reduced and Deformed were examined in trx embryos. Each of the genes examined exhibits different tissue-specific, parasegment-specific and promoter-specific reductions in their expression in response to trx. This implies that each of these genes have different requirements for trx in different spatial contexts in order to achieve normal expression levels, presumably depending on the promoters involved and the other regulatory factors bound at each of their multiple tissue- and parasegment-specific cis-regulatory sites in different regions of the embryo (Breen, 1993).

Embryos lacking both maternal and zygotic extra sex combs function display transient, general derepression of both the Ultrabithorax and Antp genes during germ band shortening. In addition, embryos that are maternally esc- but which receive two paternal copies of esc+ often are characterized by ectopic expression of the three homeotic genes, especially Ubx and Antp in the CNS (Glicksman, 1990).

ash1 and ash2 mutations were examined for the consequences on the expression of homeotic selector genes in imaginal discs. The results of these experiments demonstrates that both ash1 and ash2 are trans-regulatory elements of homeotic selector gene regulation. Hypomorphic ash1 mutations cause variegated expression of Antennapedia, Sex combs reduced, Ultrabithorax, and engrailed. (LaJeunesse, 1995).

The ash2 transcript detected in null mutant larvae is interpreted as a maternally derived product. This suggests that the zygotic requirement for ash2 begins during the third larval instar and is consistent with phenotypes of imaginal discs from mutant larvae. For example, in haltere discs from mid third instar mutant larvae there is no expression of an Antennapedia reporter gene and nearly uniform accumulation of Ultrabithorax protein as in normal haltere discs, whereas in haltere discs from late third instar mutant larvae there is ectopic expression of an Antennapedia reporter gene and patchy accumulation of Ultrabithorax protein. Finding the first detectable 53kDa Ash2 protein in late third instar larvae suggests that whatever activity is responsible for producing this smaller Ash2 product is not present before the end of the third larval instar. The fact that the final Ash2 protein is significantly different in larvae and pupae suggests that it has different functions in larvae and pupae. This may represent a novel mechanism for a single gene product to have multiple functions (Adamson, 1996).

One of the homeotic transformations caused by ash2 mutations is antenna to leg. This transformation is also caused by gain of function mutations in the Antennapedia gene and is a consequence of ectopic accumulation of Antp. The ectopic accumulation of Antp is caused by juxtaposition of an antennal promoter and the Antp gene. Since no ash2 alleles affect the penetrance or expressivity of antennal transformations caused by heterozygosity for Antp73B, this suggests that ash2 does not affect transcription from this antennal promoter nor does it have any affect on translation of the Antp protein. The ectopic accumulation of Antp is caused by alteration of the Antp promoter itself. Since heterozygotes, amorphic alleles of ash2 increase the penetrance and expressivity of antennal transformations caused by heterozygosity for AntpNs. Other ash2 alleles that cause a similar enhancement are probably also amorphic. In contrast, two alleles suppress the penetrance and expressivity of antennal transformations caused by heterozygosity of AntpNs. These two alleles appear to be gain-of-function mutations. The levels of ectopic Antp in the eye-antenna discs of AntpNs+/+ ash2 double heterozygotes correlate well with the transformation frequencies observed. Enhancers of AntpNs, such as ash21 and ash218, increase the accumulation of ectopic Antp protein in antennal discs from doubly heterozygous larvae, while suppressors of AntpNs decrease the accumulation of ectopic Antp protein in antennal discs from doubly heterozygous larvae. The ectopic accumulation of Antp caused by AntpNs is primarily due to expression from the P2 promoter of Antennapedia. If the action of Ash2 on the Antennapedia gene were direct, the enhancement of AntpNs caused by amorphic alleles would imply that the normal function of Ash2 is to repress the P2 promoter of Antennapedia. However, it is more likely that action of Ash2 on the Antennapedia gene is not direct. In haltere discs from ash2 mutant larvae there is ectopic accumulation of Antp in cells that have lost Ubx accumulation. This ectopic Antp was interpreted as a consequence of derepression of the P1 promoter of Antennapedia due to the absence of Ubx. By analogy, it is possible that amorphic mutations of ash2 cause reduced accumulation in eye-antenna discs of a protein that represses transcription from the P2 promoter of Antennapedia. According to this hypothesis it is reduced accumulation of this other protein that leads to ectopic accumulation of Antp in the antenna (Adamson, 1996).

The cis-acting control sequences of the homeotic Antennapedia gene regulated by Polycomb has been mapped. Using Antp P1 and P2 promoter fragments linked to the E. coli lacZ reporter gene, different expression patterns of beta-galactosidase (beta-gal) can be observed in transformed Pc+ and Pc- embryos. The direct binding of the PC protein to the transposed cis-regulatory promoter fragments can be visualized on polytene chromosomes. However, short Antp P1 promoter constructs which, due to position effects are ectopically activated in salivary glands, do not reveal a PC binding signal (Zink, 1991).

The distribution of Polycomb protein has been mapped at high resolution on the bithorax complex of Drosophila tissue culture cells, using an improved formaldehyde cross-linking and immunoprecipitation technique. Sheared chromatin was immunoprecipitated and amplified by linker-modified PCR, before using as a probe on a Southern of the entire PX-C walk. Polycomb protein is not distributed homogeneously on the regulatory regions of the repressed Ultrabithorax and abdominal-A genes, but is highly enriched at discrete sequence elements, many of which coincide with previously mapped Polycomb group response elements (PREs). Among the identified sites are peak F (the bxd PRE) and peak G (the bx PRE), both of which contain GAGA consensus sequences. Three other sites, E, D and C correspond to iab2, iab3 and iab4. No PC binding is seen in the regulatory domains iab6, iab7 or iab8, indicating that these domains positively regulate Abd-B expression. These results suggest that Polycomb protein spreads locally over a few kilobases of DNA surrounding PREs, perhaps to stabilize silencing complexes. GAGA factor/Trithorax-like, a member of the trithorax group, is also bound at those PREs which contain GAGA consensus-binding sites. Two modes of binding can be distinguished: a high level binding to elements in the regulatory domain of the expressed Abdominal-B gene, and a low level of binding to Polycomb-bound PREs in the inactive domains of the bithorax complex. The Abd-B sites include the iab7/iab8 regulatory region, and the Fab-7 PRE. The Fab-7 PRE does not bind Polycomb. It is proposed that GAGA factor binds constitutively to regulatory elements in the bithorax complex, which function both as PREs (silencing elements) and as trithorax group response elements. It is suggested that a GAGA site in the Antennapedia promoter is both a PRE (binding PC protein) and a TRE (binding GAGA) factor (Strutt, 1997).

moira (mor) is a member of the trithorax group of homeotic gene regulators in Drosophila. moira is required for the function of multiple homeotic genes of the Antennapedia and bithorax complexes (HOM genes) in most imaginal tissues. Heterozygous mor mutations suppress the following Polycomb-induced phenotypes:

Derepression of the Antp gene in the eye-antennal disc causes replacement of adult antennal structures with leg structures. Derepression of the Scr gene in the second and third leg discs causes the appearance of first leg structures in the second and third legs of the adults. Derepression of the Ubx gene in the wing discs causes the appearance of haltere tissue in the adult wing. Derepression of the genes in the BXC (abd-A and Abd-B) causes cells of the fourth abdominal segment of the adult to differentiate structures of a more posterior identity.

The requirement for moira function is at the level of transcription. The ability of moira mutations to supppress Antp homeotic phenotypes is dependent on the promoter. moira is also required for transcription of the engrailed segmentation gene in the imaginal wing disc. Because homozygous mor clones have phenotypes similar to those seen in clones of cells that have lost en function, en transcription was examined in clones of cells in the posterior wing. In the absence of transcriptional activation by mor, the pattern of en is altered. Greatly reduced en expression is found in wing clones. The abnormalities caused by the loss of moira function in germ cells suggest that at least one other target gene requires moira for normal oogenesis (Brizuela, 1997).

If mod(mdg4) functions as a trithorax-group gene, it should play a positive role in controlling the expression of homeotic genes, both during embryonic and later stages of development. To determine whether mod(mdg4) mutations affect homeotic gene expression, their effects were analyzed on the expression of homeotic genes during larval development. The effect of mod(mdg4) mutations were examined on the expression of the Antennapedia (Ant) gene. To this end, a combination of two mod(mdg4) alleles were used, mod(mdg4)16/mod(mdg4)E(var)3-93D, resulting in lethality at the early pupa stages. Tissues were taken from live individuals during late larval stages of development. At this time, the Antp protein is expressed in the ventral ganglion in three bands of cells that correspond to the three thoracic segments. In the mod(mdg4)16/mod(mdg4)E(var)3-93D mutant individuals examined, the brain lobes are small, the ventral ganglion is malformed, and expression of the Antp protein is undetectable. A second homeotic member of the Antp complex, Sex combs reduced (Scr), is expressed in a stripe of cells located in the most anterior region of the ventral ganglion in wild-type third-instar larvae. This band is not observed in mod(mdg4)16/mod(mdg4)E(var)3-93D mutants. Mod(mdg4) also regulates homeotic genes involved in the development of posterior body segments. Ubx is expressed in a band of cells in the ventral ganglion located posterior to the domain of Antp expression. This stripe of Ubx expression is not detectable in the ventral ganglion of mod(mdg4)16/mod(mdg4)E(var)3-93D larvae, suggesting that the Mod(mdg4) protein positively regulates Ubx expression. Mutations in mod(mdg4) also affect the expression of the Abdominal B (Abd B) gene, which is expressed in the most posterior region of the ventral ganglion during larval development but is lacking in mod(mdg4)16/mod(mdg4)E(var)3-93D mutants. A similar effect is observed for the expression of homeotic proteins in the wing and leg imaginal discs; in mod(mdg4) mutants, these structures often appear malformed, and there is no detectable accumulation of Antp, Scr, Ubx, or Abd-B proteins. These results indicate that several homeotic genes of the Antennapedia and bithorax complexes are not properly expressed in mod(mdg4) mutants, suggesting that the Mod(mdg4) product plays a positive role in regulating their expression, in agreement with its putative role as a trxG gene (Gerasimova, 1998).

The trithorax group gene brahma (brm) encodes the ATPase subunit of a chromatin-remodeling complex involved in homeotic gene regulation. brm interacts with another trithorax group gene, osa, to regulate the expression of the Antennapedia P2 promoter. The osa gene was first identified as a trxG gene in the same genetic screens that identified brm (Kennison, 1988). osa turns out to code for the same transcript as eyelid. Regulation of Antennapedia by Brm and Osa proteins requires sequences 5' to the P2 promoter. Loss of maternal osa function causes severe segmentation defects, indicating that the function of osa is not limited to homeotic gene regulation. The Osa protein contains an ARID domain, a DNA-binding domain also present in the yeast SWI1 and Drosophila Dead ringer proteins. It is proposed that the Osa protein may target the BRM complex to Antennapedia and other regulated genes (Vázquez, 1999).

osa and brm were first identified as suppressors of both the antenna to leg transformation caused by the Nasobemia (Ns) allele of Antp and the extra sex combs phenotype caused by derepression of Sex combs reduced (Scr) in Polycomb (Pc) mutants (Kennison, 1988). While examining genetic interactions among trxG mutations, it was noted that flies heterozygous for both brm and osa mutations have a held-out phenotype rarely seen in flies heterozygous for either mutation alone. The expressivity of the held-out wings phenotype is more severe in combinations of brm with some point mutations in osa than it is with the osa deficiency, suggesting that the osa point mutations make altered proteins that still bind to something in competition with wild-type Osa proteins, but then fail to function. Increasing the dosage of wild-type brm reduces the held-out wings phenotype, as expected (Vázquez, 1999).

The held-out wings phenotype is not rare in Drosophila. It is caused by mutations in many other genes, including dpp. This phenotype was also observed in flies trans-heterozygous for partially complementing brm alleles. Nevertheless, the interaction between brm and osa alleles is unusual because it results from the failure of complementation between mutations in two different genes (non-allelic non-complementation). Although a few other trxG mutations have been shown to interact in double heterozygotes, the penetrance in every other case is far less than that observed for the brm/osa interactions. In fact, the majority of trxG mutations show little if any interaction in double heterozygotes. brm interacts with the trxG genes trx and ash1 to cause partial transformation of the fifth abdominal segment to fourth, and metathorax to mesothorax, but these flies do not hold their wings out at any significantly higher frequency. The basis for the held-out wings phenotype in the brm/osa transheterozygotes was investigated. The Antp gene has two alternative promoters, P1 and P2. Genetic studies have shown that the functions of both promoters are essential. Two mutations that inactivate only the P2 promoter have been described. Flies heterozygous for the P2-specific mutations and the chromosome aberrations that remove P1 function were examined. All combinations appear as wild type, except flies carrying either one of two very specific Antp mutations, which produce chromosome aberrations that remove P1 function in combination with the P2- specific mutations. Many of these flies have held-out wings phenotype indistinguishable from the held-out wings phenotype of the brm/osa transheterozygotes. It is suggested that disruption of P2 promoter activity can result in a held-out wings phenotype. Moreover, when a brm mutation is introduced, there is a significant increase in the penetrance of the held-out wings phenotype. These results strongly suggest that brm is one of the factors required for normal expression of the P2 promoter to prevent the held-out wings phenotype (Vázquez, 1999).

That both brm and osa are required for activation of the Antp P2 promoter is also suggested by their interaction with the Antp Ns mutation. The Antp Ns mutant chromosome has a large insertion (including a second copy of part of the P2 promoter) upstream of the P2 promoter. This insertion derepresses the P2 promoter and causes the antennae to differentiate leg structures. The first alleles of both brm and osa were isolated because they fail to derepress the P2 promoter in the Antp Ns mutant. As noted by Kennison and Tamkun (1988) the trxG genes identified in their screen, including the osa gene, might regulate HOM gene function at a variety of different levels. They might regulate transcription or translation of the HOM genes, or encode cofactors that interact with the HOM proteins in regulating target genes. Since brm has been shown to affect HOM gene transcription, the genetic interaction with brm suggests that osa may also act at the level of HOM gene transcription. Antp proteins are normally not expressed in the cells that form the adult antenna. Misexpression of Antp proteins during the larval stage in these cells causes them to differentiate leg structures instead of antennal structures. The Antp Ns allele derepresses the Antp P2 promoter in the eye-antennal disc, expressing wild-type Antp transcripts from the Antp promoter. The penetrance of the antenna-to-leg transformation of Antp Ns mutants is greatly reduced in osa heterozygotes. High levels of osa expression are required only for the Antp P2 promoter, and not for the function of Antp proteins expressed from other promoters (Vázquez, 1999).

osa is also required maternally for proper embryonic segmentation. Although osa function appears to be important for expression of some HOM and segmentation genes in imaginal tissues, homozygous osa mutants die late in embryogenesis with no clear defects in either segmentation or segment identity. To determine whether wild-type maternal osa gene products deposited in the egg might be sufficient for segmentation and segment identity, homozygous germ cells were generated for the osa alleles that are strong Antp Ns suppressors. Loss of maternal osa functions has dramatic effects on the segmentation of the embryo. When rescued by a wild-type allele inherited from the father, the embryos secrete cuticle but have severe defects in segmentation, resembling mutants for the early-acting gap segmentation genes. When both maternal and zygotic osa functions are lacking, the embryos fail to differentiate any cuticle at all. The failure to detect obvious changes in the homozygous osa mutants from heterozygous mothers is clearly a consequence of the maternally encoded osa gene products, which function early in embryogenesis to activate transcription of target genes. Because of the severe defects in embryos lacking maternal osa function and the cascade of regulatory interactions between the segmentation and HOM genes early in embryogenesis, no attempt was made to identify the earliest-acting genes affected by loss of osa function (Vázquez, 1999).

Is OSA essential for the function of the BRM complex? If so, one might expect brm and osa mutants to have identical phenotypes, and the mutation with the strongest effects in one assay should be the mutation with the stronger effects in all other assays. This is not observed. For example, there are much greater effects on Scr, Ubx, and Abd-B in brm heterozygotes than in osa heterozygotes, but the reverse is observed for Antp. Another important difference is the germ line requirements for brm and osa, i. e., brm clones do not make eggs while osa clones make normal appearing eggs that are fertilized but fail during embryogenesis. Thus, brm is required under conditions that do not appear to require osa. If Osa is a subunit of the BRM complex, it is not essential for all of the complexs functions. Consistent with this possibility, the Osa protein was not identified as one of the major subunits of the BRM complex in the Drosophila embryo. However, it remains possible that Osa is a substoichiometric subunit of the BRM complex, or that it is associated with Brm at other stages of development. Another possibility is that the Osa protein targets the BRM complex to specific promoters (e.g., Antp P2). To date, no protein from the SWI/SNF complex (including SWI3 or the ARID-domain protein SWI1), has been shown to bind DNA in a sequence-specific manner (Vázquez, 1999 and references).

It is proposed that the Osa protein may be involved in the targeting of the BRM complex in Drosophila. Whether an intrinsic member of the BRM complex or merely an associated partner, the OSA protein may interact with specific target sequences in cis-regulatory elements to anchor or recruit the BRM complex. Given the patterns of expression driven by Antp cis-regulatory sequences in a reporter gene transposon, it is likely that there are En DNA-binding sites in the 10 kb region 5' to the Antp P2 promoter. Since the ARID domain found in the Osa protein may bind to En target sites, it is possible that Osa proteins will bind directly to these sequences. It is also possible that Osa may bind AT-rich regions of DNA with little specificity. The delineation of brm and osa response elements should allow a clarification of whether they act in concert or independently. It is also possible that the BRM complex alters chromatin structure in order to facilitate the binding of Osa to its target sites. Subsequent to this, Osa would act independently of the BRM complex to activate transcription (Vázquez, 1999 and references).

Mutations in the lawc gene result in a pleiotropic phenotype that includes homeotic transformation of arista into leg. lawc mutations enhance the phenotype of trx-G mutations and suppress the phenotype of Pc mutations. Mutations in lawc affect homeotic gene transcription, causing ectopic expression of Antennapedia in the eye-antenna imaginal disc. These results suggest that lawc is a new member of the trithorax family. The lawc gene behaves as an enhancer of position-effect variegation and interacts genetically with mod(mdg4), which is a component of the gypsy insulator. In addition, mutations in the lawc gene cause alterations in the punctated distribution of Mod(mdg4) protein within the nucleus. These results suggest that the Lawc protein is involved in regulating the higher-order organization of chromatin (Zorin, 1999).

As expected from its properties as a trx-G gene, mutations in lawc affect the expression of homeotic genes. The observation that the lawc/Df background leads to the reduction of some homeotic gene products (Ubx, Lab, Dfd) and not others (Scr, Antp) is not exceptional. In the case of ash-2, the level of Antp is not reduced in the first leg disc, and there is no change in the level of Ubx expression in the CNS, although there is patterned loss in the haltere and third leg disc. ash-2 also causes a reduction of Scr in the first leg disc. ash-1 mutations lead to a reduction of Antp in the first leg disc and lower levels of Ubx in the CNS, but only variable loss of Scr in the first leg disc. Because the trx-G gene products appear to form a complex, it is possible that different trx-G gene proteins interact with different homeotic genes. In forming this complex, some trx-G products such as Trl might bind to DNA, whereas others bind to each other. trx-G members are diverse and range from transcription factors, such as the Trl GAGA factor, to putative nucleosome displacement factors, such as Brahma. trx-G proteins could exert their effects on gene expression at various levels in the process of regulating transcription. Some trx-G products must have a general role in transcription because they bind to many sites on polytene chromosomes, other than the sites of homeotic genes (Zorin, 1999 and references).

The trithorax group genes are required for positive regulation of homeotic gene function. The trithorax group gene brahma encodes a SWI2/SNF2 family ATPase that is a catalytic subunit of the Brm chromatin-remodeling complex. The Drosophila tonalli (tna) gene was identified by genetic interactions with brahma. tna mutations suppress Polycomb phenotypes and tna is required for the proper expressions of the Antennapedia, Ultrabithorax and Sex combs reduced homeotic genes. The tna gene encodes at least two proteins, a large isoform (TnaA) and a short isoform (TnaB). The TnaA protein has an SP-RING Zn finger, conserved in proteins from organisms ranging from yeast to human and thought to be involved in the sumoylation of protein substrates. Besides the SP-RING finger, the TnaA protein also has extended homology with other eukaryotic proteins, including human proteins. tna mutations also interact with mutations in additional subunits of the Brm complex, with mutations in subunits of the Mediator complex, and with mutations of the SWI2/SNF2 family ATPase gene kismet. It is proposed that Tna is involved in postranslational modification of transcription complexes (Gutiérrez, 2003).

The Antp gene has two alternative promoters, P1 and P2. The AntpNs allele derepresses the Antp P2 promoter in the eye-antennal disc and expresses wild-type Antp transcripts from the Antp promoter. Derepression of the Scr gene causes the appearance of extra sex combs on the second and third legs of males. This derepression can be caused by gain-of-function alleles of Scr, such as ScrMsc, or by loss-of-function mutations in Polycomb group genes, such as Pc3 or Pc4. Several trithorax group genes (including brm, mor, osa, kis, skd and kto) were first identified as suppressors of the extra sex combs phenotype caused by derepression of Scr or as suppressors of the antenna to leg transformation caused by derepression of Antp in the Nasobemia (Ns) allele of Antp. Since the tna gene was identified on the basis of genetic interactions with brm, tests were performed to see whether tna mutations could also suppress these two homeotic derepression phenotypes. It was found that all tna mutations strongly suppress the extra sex combs phenotype caused by Pc3, Pc4 or ScrMsc, but only weakly suppress the antenna to leg transformation caused by the AntpNs mutation (Gutiérrez, 2003).

Interactions among Polycomb domains are guided by chromosome architecture

Polycomb group (PcG) proteins bind and regulate hundreds of genes. Previous evidence has suggested that long-range chromatin interactions may contribute to the regulation of PcG target genes. This study adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue. The results demonstrate that PcG target genes interact extensively with each other in nuclear space. These interactions are highly specific for PcG target genes, because non-target genes with either low or high expression show distinct interactions. Notably, interactions are mostly limited to genes on the same chromosome arm, and it was demonstrated that a topological rather than a sequence-based mechanism is responsible for this constraint. These results demonstrate that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture (Tolhuis, 2011).

This study successfully adapted the 4C method to systematically map long-range chromatin contacts with limited material from a single fly tissue. With this method, interactions were detect between the ANT-C and BX-C in central brain. This observation is in good agreement with earlier microscopic reports, underscoring the strength of the method. Importantly, it was further demonstrated that not only the two Homeotic gene clusters, but also many other PcG target genes interact, suggesting that long-range chromatin contacts between PcDs are common in central brain tissue. The control fragments (wntD, CG5107, Crc, and RpII140), which do not reside in PcDs, have interactions that are distinct from PcDs, emphasizing the specificity of the findings (Tolhuis, 2011).

PcG targets show a strong preference for interaction with other PcG targets, suggesting that PcG proteins help to establish these interactions. This is in line with earlier in vivo and in vitro results that indicated that PcG proteins can keep certain DNA sequences together. However, interactions among PcG target genes are constrained by overall chromosome architecture, because the data demonstrate that loci need to be on the same chromosome arm for efficient interaction (Tolhuis, 2011).

Discrete interaction domains (DIDs) range in size from 6 to 600 kb, with an average of ~170 kb. Thus, highly local strong enrichments are found as well as moderate enrichments over larger regions, which may reflect different types of long-range interactions. It is emphasized that interactions as detected by 4C technically represent events of molecular proximity of DNA sequences, and not necessarily physical binding. Based on the current data it is therefore not possible to identify within the DIDs sequence elements that may mediate direct contact with other DIDs. It is conceivable that contacts between PcDs may occur at any position within the PcDs; if PcG protein complexes have an intrinsic propensity to aggregate, as has been observed in vitro, then large PcDs may have a higher chance of interacting with each other due to their larger sticky surface area (Tolhuis, 2011).

The 4C method is a cell population based assay that only detects the most frequent interactions in the population. Previous 4C studies in mammalian cells suggested an extensive network of long-range interactions. The current data also suggests an extensive network among interacting PcDs. However, microscopic studies in mammalian cells revealed that specific long-range interactions occur only in a small proportion of the cells. Likewise, only a proportion of D. melanogaster cells show contacts between the two Homeotic clusters. Therefore, 4C data have to be carefully interpreted, because the identified interactions are in part stochastic and do not all occur simultaneously. As a consequence, it is not known how many PcDs interact in a single cell, but the most common interactions are known in the population of larval brain cells (Tolhuis, 2011).

Interphase chromosomes in most eukaryotes occupy distinct 'territories' inside the nucleus, with only a limited degree of intermingling. Although some studies have reported interactions between some loci that are on two different chromosomes (interchromosomal), unbiased 4C mapping in mouse tissues has indicated that interactions within the same chromosome (intrachromosomal) occur much more frequently than between different chromosomes. In addition, a recent genome-wide map of chromatin interactions in human cells showed that intrachromosomal interactions occur with higher frequency than interchromosomal contacts. The current data are in agreement with these observations, and show that most interactions are even limited to single chromosome arms, at least in D. melanogaster larval brain. Since the experiments indicate that a topological mechanism prevents interactions between the two arms of a chromosome, it is proposed that each arm (rather than the chromosome as a whole) forms a distinct territory. This is consistent with early microscopy studies of chromosome architecture in D. melanogaster, which suggested that chromosome arms are units of spatial organization (Tolhuis, 2011).

What topological mechanism may limit contacts between the two chromosome arms? About ~16 Mbp of pericentric heterochromatin are located in between the two euchromatic arms of chromosome 3. This heterochromatin region could act as a long spacer and prevent efficient interactions between DNA fragments that are located on either chromosome arm. However, the data show that interactions within one arm can span even longer distances, such as between Ptx1 and grn (~22.7 Mbp). Another explanation may be that the pericentric regions of all chromosomes assemble into a nuclear compartment, called chromocenter. This large structure could physically obstruct interactions between chromosome arms (Tolhuis, 2011).

Previous reports have demonstrated that certain PcG-bound PREs can pair in trans (i.e. when they are located on different chromosomes). First, a Fab7 PRE-element integrated on the X chromosome (Fab-X) was often found in close spatial proximity to the endogenous Fab-7 in the BX-C (chromosome 3R), although this phenomenon appears to be tissue-specific and dependent on the transgene integration site. Second, a microscopy assay based on Lac repressor/operator recognition showed that the Mcp PRE-element is able to pair with copies of that same element inserted at remote sites in the genome either in cis (i.e. when they are located on the same chromosome) or in trans. The 4C experiments also identified cases of trans interactions between endogenous loci, although they occur with low frequency (approximately 5% occurs in trans) (Tolhuis, 2011).

Although rare, such interactions between loci on different chromosome arms are of interest, because they indicate that the topological constraints imposed by chromosome architecture can in principle be overcome. In mammalian cells, there is evidence that the relative position of a gene locus within its chromosome territory (CT) influences its ability to form either cis- or trans-interactions. Peripheral regions of mammalian CTs intermingle their chromatin, which may allow for interactions between chromosomes. Indeed, more trans contacts are identified by 4C using a bait that often resides in the CT periphery compared to a bait located in the interior of a CT. Likewise, activation of the HoxB gene cluster during differentiation coincides with relocation away from its CT interior, and the active HoxB1 gene more frequently contacts sequences on other chromosomes compared to the inactive gene. In line with this, a varying degree of trans interactions are observed among eight bait sequences, suggesting distinct capacities to contact chromatin on other chromosomes. The trh gene has the strongest capacity to contact other chromosome arms. Interestingly, trh is located within 500 Kbp of the telomere of chromosome 3L, and 5 out of 6 contacts in trans occur within 500 Kbp of other telomeres. Thus, interactions between chromosome arms may be possible if loci are favorably positioned on the edge of chromosome (arm) territories, which could be the case for telomeric sequences in larval brain cells (Tolhuis, 2011).

The experiments with strain In(3LR)sep showed dramatic changes in interactions, such as loss of contacts between the Homeotic gene clusters and gain of contacts with other PcDs. Despite these changed interactions, no convincing evidence was found for global gene expression alterations on the In(3LR)sep chromosome. This raises the question: how relevant are long-range chromatin contacts between PcG-target genes for regulation of expression?

The lack of detectable expression changes may indicate that long-range interactions have only quantitatively subtle effect on the regulation of gene expression. Nevertheless, such subtle effects on gene expression could be very important for long-term viability and species survival. In(3LR)sep animals do suffer from an overall reduced viability during several stages of development, which may indicate a generally reduced fitness, possibly due to a dimly altered regulation of gene expression (Tolhuis, 2011).

Alternatively, PcG gene regulation may not be affected in strain In(3LR)sep, because Abd-B and Antp, although they no longer interact with each other, still prefer to interact with other PcDs, suggesting that it is not relevant which PcDs interact. In such a model, the complement of all interactions contributes to PcG-mediated gene silencing in a population of cells (Tolhuis, 2011).

Finally, it is interesting to note that over ~100 million years of evolution of the Drosophila genus, exchange of genes between chromosome arms has been rare despite extensive rearrangements within each arm. Chromosome arm territories ensure that genes within a single arm are relatively close compared to genes on other arms, which may have resulted in an increased chance of rearrangements within one arm. Alternatively, the importance of long-range interactions among sets of genes, which are topologically limited to the same arm, may have contributed to the selective pressure that has led to this remarkable conservation of the gene complement of each chromosome arm (Tolhuis, 2011). 7600982

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