BIOLOGICAL OVERVIEW

What are the targets of Polycomb-group (Pc-G) proteins and how do they regulate gene silencing? Pc-G proteins are usually considered to be inhibitors of homeotic genes, since Pc-G mutants were originally identified on the basis of their causing expression of homeotic genes in unusual locations, areas in which they normally would not be expressed. This so-called ectopic expression is attributed to the failure of proper gene silencing in Pc-G mutants. In fact bithorax complex contains Pc-G response elements (PREs) that are involved in initiating and maintaining silencing of the whole bithorax complex (Busturia, 1993).

Pc-G genes also regulate gap genes giant and knirps, restructuring their transcription to the posterior half of the embryo. This silencing is inititated by high levels of Hunchback protein in the anterior portion of the embryo, and maintained by Pc-G action (Pelegri, 1994).

The action of Pc-G proteins is thought to be mediated through chromatin. Chromatin is a combination of DNA and protein that derives its name from a staining reaction with dyes. About a third of the DNA in chromosomes is held in heterochromatin, a type of chromatin with which few genes are associated. Placing active genes next to heterochromatin results in their inactivation or variation in expression, an effect termed position effect variation. It is thought that heterochromatin is capable of spreading to active genes, resulting in their silencing (Spofford, 1976).

These classic biological observations have taken on a new and more literal meaning recently with the discovery of common sequence domains in Pc-G genes. Polycomb itself has a domain called the chromodomain, which is shared with HP1, a heterochromatin-associated protein of Drosophila (Paro, 1991). Many of the Pc-G proteins, including Polycomb itself, are unable to bind DNA.

Considering that they lack the ability to bind DNA, how do Pc-G proteins function to establish gene silencing? It is thought that the initial repression of a gene is carried out by transcription factors that possess the ability to recognize DNA. In the cases of giant and knirps, for example, the maternal protein Hunchback represses their expression in the anterior part of the embryo (Pelegri, 1994). Pc-G proteins provide a mechanism whereby initial repression becomes permanent. They carry out this role by assembling at the site of initial repression and forming a multiprotein complex involved in modifying chromatin to promote gene silencing. Polycomb itself has this ability to self associate (Franke, 1992).

Thus many of the roles of Pc-G proteins are played out not in association with DNA but in association with each other and with other chromosomal proteins, especially histones, the principle scaffolding protein of chromosomes. Interest in the silencing of gene expression has shifted from a concern about DNA-protein interaction to an emphasis on protein-protein interaction, and the maintenance and modification of chromatin structure (Reviewed by Orlando, 1995, Pirrotta, 1995 and Simon, 1995).

Trithorax group (trx-G) genes are able to reverse the inactivating effects of chromatin. These proteins are thought to function as transcriptional activators, removing the block on gene expression put in place by arrays of inactivating proteins.

Cooperativity, specificity, and evolutionary stability of polycomb targeting in Drosophila

Metazoan genomes are partitioned into modular chromosomal domains containing active or repressive chromatin. In flies, Polycomb group (PcG) response elements (PREs) recruit Pho and other DNA-binding factors and act as nucleation sites for the formation of Polycomb repressive domains. The sequence specificity of PREs is not well understood. This study used comparative epigenomics and transgenic assays to show that Drosophila domain organization and PRE specification are evolutionarily conserved despite significant cis-element divergence within Polycomb domains, whereas cis-element evolution is strongly correlated with transcription factor binding divergence outside of Polycomb domains. Cooperative interactions of PcG complexes and their recruiting factor Pho stabilize Pho recruitment to low-specificity sequences. Consistently, Pho recruitment to sites within Polycomb domains is stabilized by PRC1. These data suggest that cooperative rather than hierarchical interactions among low-affinity sequences, DNA-binding factors, and the Polycomb machinery are giving rise to specific and strongly conserved 3D structures in Drosophila (Schuettengruber, 2014).

Comparative epigenomics was used to demonstrate that Polycomb domains are an extremely well conserved feature of the genome during fly evolution. In fact, the evolutionary profile of epigenomic domain organization in embryos of five Drosophila species indicates a complete lack of divergence of H3K27me3- marked Polycomb domains in syntenic regions. A similar high conservation of the H3K27me3 pattern across Drosophila species was recently described. Polycomb domains typically harbor several PH-marked PREs, and a comparative analysis showed that these are also highly conserved and the few loci that show a divergence of PRC1 occupancy patterns are not correlated with overall domain divergence. Likewise, the binding of PHO and DSP1 is highly conserved (to a degree at least as strongly, and possibly more strongly, than binding of individual factors), but even cases of diverged factor occupancies are usually not correlated with overall PRE divergence. In marked contrast, the sequences underlying PREs and Polycomb domains are diverging extensively, and sequence-based prediction of PREs across Drosophila species suggested that divergence of PREs could occur frequently. However, neither ChIP-seq experiments nortransgenic reporter assays support this dynamic behavior. Instead, such sequence divergence is buffered by the epigenetic targeting mechanisms to maintain Polycomb domains. It is suggested that the multilayered organization uses redundancy and cooperativity to facilitate the remarkable Polycomb domain conservation. This is occurring both in cis, where several TFs collaborate to define a regulatory element even when the underlying sequence is imperfect, and at the domain level, where several PREs participate to define the PcG domain structure and possibly stabilize each other (Schuettengruber, 2014).

Although PREs are associated with several known sequence features (such as GAGA- and PHO-binding motifs) in a statistically significant way, these features are not sufficient to distinguish many PREs from the genomic background and from other PHO- or DSP1-bound active chromatin elements. There are many possible explanations for this lack of specificity, including the existence of additional, yet-to-be-characterized sequence-specific recruiting factors; the involvement of nucleosome positioning; transcription of non-coding RNAs; or imperfect modeling of the sequence specificity of the known factors. The data presented in this study, however, introduce a new perspective that can help resolve this conundrum. In contrast to previous hypotheses, the data show that even when strong binding sites are lacking, PHO and DSP1 may bind PREs directly through weak (but highly nonrandom) motifs. Remarkably, sequence affinities that are completely nonspecific on a genomic scale (possibly defining millions of spurious sites) are still highly informative for predicting the binding intensity within the context of a PRE. The strong correlation of PHO binding with weak but nonrandom motifs makes it unlikely that binding to these sites represents indirect binding via interaction/looping with strong binding sites. The data show that in order to understand PRE sequence specificity, multiple potential binding sites with variable affinities and fidelities must be taken into account, and their cooperative interaction must be considered in the context of the PRE chromosomal landscape. This idea is compatible with the evolutionary constraints on PRE sequences, which has been demonstrated in this study to affect a spectrum of binding affinities rather than to conserve classical binding sites alone (Schuettengruber, 2014).

What might be the molecular mechanism that allows the specific binding of weak sites in the context of Polycomb domains? One possibility is that cooperative binding of TFs at PREs supports their occupancy of weak motifs. Indeed, this study found that PHO and DSP1 are bound jointly at PREs (with weak underlying sequence motifs), whereas at other regions of the genome where the factors bind alone, they are usually associated with strong sequence motifs. This observation is in agreement with the recently proposed 'TF collective model,' according to which combinatorial TF binding occurs with little or no apparent sequence motifs for at least a subset of the bound factors (Schuettengruber, 2014).

In addition, it was shown that transient interactions of DNA-binding proteins with weak affinity sites are stabilized by the presence of the PcG proteins themselves. A similar observation of a positive feedback of PRC1 on PHO binding was recently reported (Kahn, 2014) and is further supported by the fact that cooperative binding of PHO and Polycomb to PREs can occur even in vitro. In vivo, long-range contacts involving remote PREs within the same (or even a different) Polycomb domain may contribute to this process. Clustering of multiple flanking PREs in the 3D space of the nucleus might generate Polycomb compartments characterized by high concentrations of PcG proteins as well as their recruiting DNA-binding proteins. In this scenario, loss of occupancy following the dissociation of any of these factors from DNA may be more easily replenished by the concentrated stock of factor within a Polycomb compartment compared with individual binding sites present elsewhere in the genome. This may push the equilibrium toward increased PHO and DSP1 binding to low-affinity sites and partially reduce the evolutionary pressure to maintain the nucleotidic sequence of recruiter motifs at PREs. Structural long-range effects may also inhibit PcG recruitment in cases where active enhancers and TSSs are in proximity to a candidate PRE sequence. This analysis suggests that H3K4me3-marked loci are also highly conserved, but the low-affinity PHO- or DSP1-binding sites in them are completely uncorrelated with occupancy of these factors, further supporting a model of highly organized and cooperative epigenomic organization (Schuettengruber, 2014).

In conclusion, the data indicate that sequence conservation collaborates with 3D chromatin architecture to maintain an exceptional evolutionary stability of Polycomb-regulated loci in fly genomes. This phenomenon highlights the contribution of chromosome domains and their particular looping structures to epigenomic specificity and genome evolution. Hi-C analysis in mammals has revealed that topological domains are a strikingly conserved feature between the mouse and human genomes. The current data raise the possibility that, beyond combinatorial contributions by TF-binding sites in close proximity, the confinement of regulatory elements within TADs and their frequent DNA contacts constitute significant driving forces that also affect DNA sequence evolution in these and possibly many other specie (Schuettengruber, 2014).

Propagation of Polycomb-repressed chromatin requires sequence-specific recruitment to DNA

Epigenetic inheritance models posit that during Polycomb repression, Polycomb Repressive Complex 2 (PRC2) propagates histone H3K27 tri-methylation (H3K27me3) independently of DNA sequence. This study shows that insertion of Polycomb Response Element (PRE) DNA into the Drosophila genome creates extended domains of H3K27me3-modified nucleosomes in the flanking chromatin and causes repression of a linked reporter gene. After excision of PRE DNA, H3K27me3 nucleosomes become diluted with each round of DNA replication and reporter gene repression is lost, whereas in replication-stalled cells, H3K27me3 levels stay high and repression persists. Hence, H3K27me3-marked nucleosomes provide a memory of repression that is transmitted in a sequence-independent manner to daughter strand DNA during replication. In contrast, propagation of H3K27 tri-methylation to newly incorporated nucleosomes requires sequence-specific targeting of PRC2 to PRE DNA (Laprell, 2017).

The ability of certain histone-modifying enzymes to bind to the modification they generated has led to models where such enzymes might propagate modified chromatin domains by a positive feedback loop, independently of the underlying DNA sequence. Two paradigms of chromatin states have been proposed to be maintained by such an epigenetic inheritance mechanism: constitutive heterochromatin with histone H3 lysine 9 di- and tri-methylation (H3K9me2/3) generated by Suv39/Clr4 enzymes, and Polycomb-repressed chromatin marked with H3K27me3 by PRC2. In both chromatin states, these histone modifications are essential for repressing gene transcription. To date, there is compelling evidence that H3K9me2/3- and H3K27me3-modified nucleosomes are transmitted to daughter strand DNA during replication. However, the steps required to propagate these modifications are much less understood. Fission yeast Clr4 has the capacity to propagate ectopically induced H3K9me2/3 domains over many cell divisions by an H3K9me2/3-based positive feedback loop but only in cells mutated for H3K9me2/3 demethylase activity. In the case of PRC2, allosteric activation of the enzyme induced by binding to H3K27me3 has been proposed to be the foundation for propagating H3K27me3 chromatin. In mammalian cells, transient DNA-tethering of PRC2 generates short ectopic H3K27me3 domains that were at least partially maintained for several cell divisions after release of DNA-tethered PRC2. However, in Drosophila, where PRC2 and other Polycomb group (PcG) protein complexes are targeted to PREs, repression imposed by insertion of PRE DNA next to a reporter gene was lost upon excision of PRE DNA. This study investigated how insertion and excision of PRE DNA at ectopic sites in Drosophila affects binding of PcG proteins and H3K27me3 at the molecular level (Laprell, 2017).

Two previously described strains were analyzed that each carried a single copy of the >PRE>dpp^WE-TZ reporter gene, integrated at different chromosomal locations. >PRE>dpp^WE-TZ contains a 1.6 kilobase (kb) DNA fragment of the bxd PRE from the HOX gene Ultrabithorax (Ubx), flanked by FRT recombination sites (>PRE>) to permit excision of PRE DNA by Flp-mediated recombination. Adjacent to the >PRE> cassette, the construct contains a reporter gene comprising the wing imaginal disc enhancer from decapentaplegic (dpp) (E), linked to the hsp70 TATA box minimal promoter (T) and LacZ sequences encoding β-galactosidase (Z) . In the presence of the >PRE> cassette, the transgene was silenced and no β-galactosidase activity could be detected in wing imaginal discs of >PRE>dpp^WE-TZ transgenic animals. In contrast, >dpp^WE-TZ transgenic animals, generated by excision of the >PRE> cassette in the germ line, showed strong β-galactosidase expression in the characteristic pattern driven by the dpp enhancer. The observation that silencing of the intact >PRE>dpp^WE-TZ reporter gene is lost in mutants lacking PRC2 function, prompted determination of the H3K27 methylation profile and binding of PcG proteins across the transgene. In both lines, the transgene had inserted into a genomic location normally devoid of H3K27me3 and PcG protein binding. Chromatin immunoprecipitation (ChIP) assays were performed on batches of wing imaginal discs from >PRE>dpp^WE-TZ and the corresponding >dpp^WE-TZ transgenic animals, and the immunoprecipitates were analyzed by quantitative real-time PCR (qPCR). For qPCR, primer pairs were used that selectively amplified transgene sequences and sequences in the genomic regions flanking the transgene insert. As controls, primer pairs were used amplifying sequences at the endogenous bx PRE in Ubx that are known to be bound by PcG proteins (C2) or enriched for H3K27me3 (C1 and C3) and at two regions elsewhere in the genome (C4 and C5) without PcG protein binding or H3K27me3 (Laprell, 2017).

The PRC1 subunits Polycomb (Pc), Polyhomeotic (Ph) and the PRC2 subunit E (z) were specifically enriched at the transgene PRE in animals carrying >PRE>dpp^WE-TZ and, as expected, no binding was detected in >dpp^WE-TZ animals. In both >PRE>dpp^WE-TZ transgenic lines, H3K27me3 was present at high levels across a domain that extended about 4-5 kb to either side of the >PRE> cassette, spanning almost the entire construct. No enrichment of H3K27me3 was detectable at the >dpp^WE-TZ transgene. At >PRE>dpp^WE-TZ, PRC2 thus tri-methylates H3K27 across a chromatin interval that spans about 8-10 kb (Laprell, 2017).

To estimate to what extent nucleosomes at the >PRE>dpp^WE-TZ transgene are tri-methylated at H3K27, the H3K27me2 profile was determined. H3K27me2 levels across the >PRE>dpp^WE-TZ transgene were much lower than at C4 and C5 and comparable to the levels at Ubx (regions C1-C3) that is repressed and predominantly tri-methylated at H3K27 in wing imaginal discs. Conversely, across >dpp^WE-TZ, H3K27me2 levels were much higher and comparable to those seen at C4 and C5. This suggest that the nucleosomes across the >PRE>dpp^WE-TZ transgene are predominantly tri-methylated at H3K27 (Laprell, 2017).

Excision of the >PRE> cassette from >PRE>dpp^WE-TZ transgenic animals by heat-shock induced expression of Flp during larval development results in appearance of β-galactosidase expression in the dpp pattern 12 hours after the heat shock. Efficiency of PRE excision was measured and it was found that 8 hours after a single 1-hour heat shock, excision had occurred in about 95% of wing imaginal disc cells. The delayed increase of β-galactosidase expression over time suggests a gradual rather than abrupt loss of repression. ChIP analyses were performed on chromatin prepared from batches of entire wing imaginal discs dissected from >PRE>dpp^WE-TZ transgenic animals 12, 32 or 56 hours after Flp-induction. This allowed monitoring the consequences of PRE excision in cells that had undergone at least one (+12 hours), at least two (+32 hours), or more than four (+56 hours) cell divisions. 12 hours after Flp-induction, H3K27me3 levels were at least two-fold reduced across the entire transgene and further reduced by at least two-fold at the 32 hours time point. 56 hours after Flp-induction, H3K27me3 levels across the transgene were nearly as low as in >dpp^WE-TZ animals derived from >dpp^WE-TZ germ cells. The histone H3 profile was unaltered at all time points, suggesting that PRE excision does not cause global disruption of nucleosome occupancy across the transgene. The loss of H3K27me3 after PRE excision suggests that PRC2 is unable to propagate H3K27me3 across the >dpp^WE-TZ transgene in the absence of PRE DNA (Laprell, 2017).

In parallel, Pc protein binding was monitored after PRE excision. Pc, unlike Ph or other PRC1 subunits, is not only bound at PREs but also associates with the chromatin flanking PREs likely reflecting its interaction with H3K27me3-modified nucleosomes. 12 hours after PRE excision, Pc binding at the transgene was already almost reduced to background levels (Laprell, 2017).

The H3K27me3 profile at the >PRE>dpp^WE-UZ transgene that contains a 4.1 kb fragment of the Ubx promoter instead of the hsp70 minimal promoter was then analyzed. At >PRE>dpp^WE-UZ, the H3K27me3 domain spans about 12 kb and is thus about 4 kb longer than at >PRE>dpp^WE-TZ. Nevertheless, after PRE excision, H3K27me3 at dpp^WE-UZ was lost at a rate comparable to that seen at dpp^WE-TZ. Ubx promoter DNA thus does not enable H3K27me3 propagation. It is concluded that even at a domain that spans 12 kb and therefore comprises about 60 nucleosomes, PRC2 is unable to propagate H3K27me3 in the absence of PRE DNA (Laprell, 2017).

The H3K27me3 profile and reporter gene repression was then analyzed after PRE excision in animals in which DNA replication had been blocked. Larvae were reared in liquid medium containing Aphidicolin, an inhibitor of DNA polymerases A and D, which resulted in a complete block of DNA replication in imaginal discs. In larvae reared in Aphidicolin-containing medium, Flp-induced PRE excision from >PRE>dpp^WE-TZ was as efficient as under normal growth conditions but 12 hours after excision, H3K27me3 levels at the transgene were undiminished compared to +PRE control larvae. In larvae reared in liquid medium without Aphidicolin, PRE excision resulted in the expected two-fold reduction of H3K27me3 levels after 12 hours. Together, this suggests that the loss of H3K27me3 nucleosomes after PRE excision in proliferating cells reflects their dilution as they become transmitted to DNA daughter strands during replication. Unlike under normal growth conditions, Aphidicolin-treated larvae lacked detectable β-galactosidase expression 12 hours after PRE excision. When these animals were permitted to recover in medium lacking Aphidocolin, they resumed DNA replication and began expressing β-galactosidase. If DNA replication is blocked and H3K27me3 levels stay high, repression is thus also sustained in the absence of PRE DNA, possibly by PRC1 (Laprell, 2017).

Finally, PRE excision was induced from >PRE>dpp^WE-TZ in larvae that were hemizygous for Utx^Δ, a null mutation in the single H3K27me3 demethylase in Drosophila. 12 hours after Flp-induction, H3K27me3 levels at the transgene were reduced about two-fold, like in wild-type animals. This suggest that demethylation of H3K27me3 by Utx does not contribute to the disappearance of H3K27me3 from transgene chromatin after PRE excision (Laprell, 2017).

These results lead to the following conclusions. First, PRE cis-regulatory DNA provides the genetic basis not only for generating but also for propagating H3K27me3-modified chromatin. This argues against a simple epigenetic model where PRC2 binding to parental H3K27me3 nucleosomes after replication would suffice to propagate H3K27 tri-methylation in daughter strand chromatin. PRC2 needs to be recruited to PRE DNA first, before allosteric activation through interaction with H3K27me3 nucleosomes in flanking chromatin may then facilitate methylation of newly incorporated nucleosomes. Secondly, following PRE excision and replication, parental H3K27me3 nucleosomes remain associated with the same underlying DNA in daughter cells and thus provide epigenetic memory. However, while in replication-stalled cells high H3K27me3 levels permit to sustain repression also in the absence of PRE DNA, their dilution in proliferating cells is accompanied with loss of repression after one cell division. H3K27me3 nucleosomes therefore only appear to provide short-term epigenetic memory of the repressed state. Hence, DNA targeting of PRC2 after replication to replenish H3K27me3 is critical to preserve repression (Laprell, 2017).

Drosophila HOX and other large-size PcG target genes often contain multiple PREs and H3K27me3 domains that span dozens of kilobases. Deletion of single PREs from these genes typically results in only minor diminution of the H3K27me3 profile and misexpression is less severe than misexpression of the native genes in PcG mutants. Furthermore, when the same >PRE> cassette that was used in this study was excised from a Ubx-LacZ reporter gene with more extended Ubx upstream regulatory sequences, repression was lost with a longer delay\, suggesting that additional elements with PRE properties in those Ubx sequences permitted to sustain repression through more cell divisions. The evolution of PRE DNA sequences and of their frequency and arrangement within target genes may thus ultimately determine stability and heritability of H3K27me3 chromatin and PcG repression (Laprell, 2017).

Polycomb-mediated chromatin loops revealed by a subkilobase-resolution chromatin interaction map

The locations of chromatin loops in Drosophila were determined by Hi-C (chemical cross-linking, restriction digestion, ligation, and high-throughput DNA sequencing). Whereas most loop boundaries or "anchors" are associated with CTCF protein in mammals, loop anchors in Drosophila were found most often in association with the polycomb group (PcG) protein Polycomb (Pc), a subunit of polycomb repressive complex 1 (PRC1). Loops were frequently located within domains of PcG-repressed chromatin. Promoters located at PRC1 loop anchors regulate some of the most important developmental genes and are less likely to be expressed than those not at PRC1 loop anchors. Although DNA looping has most commonly been associated with enhancer-promoter communication, the results indicate that loops are also associated with gene repression (Eagen, 2017).

The locations of loop anchors in Drosophila determined in this study are notable both for correlations with ChIP-seq data and for the lack thereof. The lack of correlation with locations of CTCF protein was unexpected, inasmuch as most loop anchors in mammals are associated with CTCF protein, apparently bound to CTCF sequence motifs in a convergent orientation. There are evidently multiple patterns of protein association with loop anchors in metazoans. The association of loop anchors in Drosophila with Pc protein is noteworthy because it points to a role of looping not only in gene activation, as widely observed in the past, but in gene repression as well (Eagen, 2017).

Regions of PcG-repressed chromatin ('PcG domains') that are separated by hundreds of kilobases to megabases are known to be in enhanced spatial proximity, but details of their internal organization have only been investigated by averaging over many PcG domains. The high resolution of Hi-C contact maps in this study revealed chromatin loops within individual PcG domains, giving insight into their internal organization. PRC1 is known to compact nucleosome arrays in vitro. Knockdown of the PRC1 subunit, Polyhomeotic (Ph), in vivo decompacts PcG-repressed chromatin, and Ph that is unable to polymerize impairs the ability of PRC1 to form clusters. Together with these findings, the current results suggest that PRC1-bound chromatin loops within PcG-repressed domains either establish or maintain a condensed state (Eagen, 2017).

Previous analyses by 3C have pointed to associations of PcG proteins with chromatin loops for the Bithorax complex (BX-C) in S2 cells; for inv and en in BG3 and Sg4 cells; and for an embryonic, pupae, and adult transgenic reporter system. The current Hi-C data are, however, at higher resolution and genome-wide. Higher resolution allowed more comprehensive analysis, such as the unambiguous identification of loops and the segmentation of ANT-C into a series of TADs with one or two homeotic gene promoters per TAD. Genome-wide analysis revealed both the pervasive nature of Pc protein association and the absence of significant CTCF protein association, despite conservation of CTCF from Drosophila to humans (Eagen, 2017).

A report by Cubeñas-Potts (2017) on Drosophila chromatin loops in Kc cells appeared while this manuscript was in preparation. Cubeñas-Potts noted an enrichment of cohesin but a lack of Drosophila CTCF at loop anchors, consistent with the current observations. Cubeñas-Potts did not mention Pc, but a current analysis of their data revealed an enrichment of loop anchors at Pc ChIP peaks and an enrichment of Pc ChIP peaks at loop anchors. This study found a likelihood of repression of promoters at Pc-bound loop anchors, especially for developmental genes; Cubeñas-Potts observed an enrichment of active developmental enhancers at loop anchors, possibly because these are among the many loop anchors not bound by Pc, or because the chromatin at these loop anchors is bivalent, bound by nonhistone proteins and histone posttranslational modifications associated with both gene activation and repression (Eagen, 2017).

The occurrence of PRC1 at loop anchors could reflect a role in loop formation similar to that proposed for CTCF in mammals, wherein cohesion complexes extrude loops, in a process halted upon reaching bound CTCF. Consistent with this model, a large majority (72.8%) of Drosophila loop anchors are bound by the Rad21 subunit of cohesin. Regardless of whether PRC1 performs such a role, additional proteins must be involved, because PRC1 is present at only 26% of Drosophila loop anchors (Eagen, 2017).

Interactions between the genome and the nuclear pore complex (NPC) have been implicated in multiple gene regulatory processes, but the underlying logic of these interactions remains poorly defined. This study reports high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. This investigation uncovered differential binding of these NPC subunits, where Nup107 preferentially targets active genes while Nup93 associates primarily with Polycomb-silenced regions. Comparison to Lamin-associated domains (LADs) revealed that NPC binding sites can be found within LADs, demonstrating a linear binding of the genome along the nuclear envelope. Importantly, this study identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. These findings lend to a model where different nuclear pores bind different types of chromatin via interactions with specific NPC sub-complexes, and a subset of Polycomb domains is stabilized by interactions with Nup93 (Gozalo, 2019).

Spatial architecture in the nucleus is set up by interactions between the genome and protein components of nuclear macro-complexes and scaffolds. The most prominent nuclear scaffold is the nuclear envelope (NE), which consists of a double membrane interspersed by a variety of trans-membrane and closely associated proteins. Chromatin re-organization and gene re-positioning during cellular differentiation involves losing or gaining interactions between the genome and the NE, and such rearrangements can influence gene expression programs. For instance, the nuclear lamina, which is a filamentous protein network underlying the NE, has been extensively implicated in setting up tissue-specific genome organization by sequestering genes destined for silencing. Genome-wide mapping of Lamin-associated domains (LADs), as well as related functional studies, have led to the current view of the nuclear lamina as a compartment for stable gene repression (reviewed in van Steensel, 2017). Another major component of the NE is the nuclear pore complex (NPC), which consists of multiple copies of approximately 30 different proteins termed nucleoporins (Nups) and is responsible for selective nucleo-cytoplasmic transport. In addition to transport-related functions, NPCs and individual Nups are also involved in genome organization and gene regulation through physical interactions with the genome. Yet unlike the nuclear lamina, the functional relationship between NPCs and genome regulation appears to be considerably more varied and remains less understood (Gozalo, 2019).

Given the close proximity of nuclear pores to the underlying chromatin, it is not surprising that multiple studies have now identified binding of Nups to subsets of genes and regulatory elements in a number of species. Many of these studies have reported preferential association of particular Nups with actively transcribing genes or re-localization of genes to the NPCs during activation. These findings have led to the predominant view of the NPC as a nuclear compartment for active processes, functionally opposed to those of the nuclear lamina. However, at least in metazoan systems, this view is confounded by the reported intranuclear presence of Nups that have been classified as dynamic. The ~30 conserved Nups that comprise the NPC can be either dynamic, meaning they are able to come on and off the NPC during interphase, or stable, meaning they are core components of the NE-embedded NPC for the majority of the cell cycle. Currently, many of the reported contacts between active genes and Nups have been described for dynamic Nups, such as Nup98, Nup153, and Nup62, and can frequently occur in the nucleoplasm. Consequently, it is unclear whether genomic binding to actual NPCs is functionally distinct from intranuclear Nup binding (Gozalo, 2019).

Genomic binding to actual NPCs can be determined by mapping chromatin-binding patterns of stable Nups, which are components of the outer-ring Nup107-Nup160 and the inner-ring Nup93-Nup205 sub-complexes. Interestingly, previous studies that profiled chromatin binding of stable Nups did not identify enrichment for transcribing loci and reported prevalence of repressive chromatin. Similarly, DamID profiling of Nup98, artificially tethered to the NPC and thus used as a marker for actual NPC binding, showed no enrichment for active genes and instead exhibited high incidence of motifs for the architectural protein Su(Hw) in Drosophila cells. These studies conflict with the simplified view of the NPC as a scaffold for gene activation and highlight the complexity of NPC-genome interactions. It should be noted that the majority of the NPC genome-binding datasets, mentioned above, were produced using either the DamID technique, which tends to generate wide binding peaks, or the lower-resolution ChIP-chip approach, and thus may have given an incomplete picture of the locations and functions of NPC-genome contacts (Gozalo, 2019).

One hypothesis, which can explain this dichotomy of both active and silent regions at the NPC, is that individual stable Nups bind distinct regions of the genome and regulate distinct chromatin-associated processes. This study set out to explore this hypothesis by generating precise binding maps of stable Nups, using an optimized ChIP-seq approach. ChIP-seq maps revealed that Nup107, a core component of the outer ring sub-complex, and Nup93, a core component of the inner ring sub-complex, bind highly non-overlapping regions of the genome. Specifically, while Nup107 preferentially targets active promoters, as has been reported for other Nups, Nup93 associates primarily with silenced regions bound by Polycomb group (PcG) proteins. PcG proteins are conserved regulators of epigenetically maintained gene repression, which often bind the genome in long Polycomb (Pc) domains. In agreement with its binding pattern, this study found that Nup93 plays a functional role in the silencing and long-range interactions of Pc targets. Together, the results emphasize the concept that different sub-complexes of the nuclear pore interact with and influence distinct chromatin states, revealing a complex landscape of NE-genome interactions (Gozalo, 2019).

The results provide high-resolution chromatin binding maps of stable NPC components and offer a resource for future comparisons to a variety of genomic features. These maps and analysis contribute several insights into the nuclear organization field. First, it was found that representative members of the two core sub-complexes of the NPC, Nup107 and Nup93, bind to active and silenced regions, respectively. This differential binding helps explain the variability in previous conclusions on NPC-genome contacts and extends understanding of how NPC-genome contacts shape three-dimensional genome architecture. ChIP-seq maps of Nup107 are consistent with the predominant view of the NPC as a place for targeting active genes and suggest that this function is carried out primarily through associations of the genome with the outer-ring NPC sub-complex. Although currently it is not possible to definitively prove that all identified binding peaks of Nup107 and Nup93 represent NPC binding, comparison to LADs and immunofluorescence localization analysis, as well as DNA FISH analysis of select loci, suggest that a large proportion of these binding peaks represent regions present at the nuclear periphery, at actual NPCs (Gozalo, 2019).

Second, the findings describe a functional connection between Nup93, a conserved subunit of the inner-ring NPC sub-complex, and Polycomb complexes, which are key regulators of developmental gene silencing. ChIP-seq map of Nup93 demonstrates preferential targeting of Nup93 to a large subset of PcG domains, particularly those that exhibit the highest level of Pc binding. Importantly, it was found that lowering levels of Nup93 leads to de-repression of Pc targets in both cultured cells and fly tissues. These findings suggest that (1) a core NPC subunit is involved in the epigenetic maintenance of silencing via its chromatin binding role; and (2) a subclass of particularly stable PcG chromatin domains are targeted to the nuclear periphery, where they require Nup93 for optimal silencing. The function of the Nup93 sub-complex in gene repression appears to be highly conserved, as strikingly, the S. pombe homolog of Nup93 was found by Moazed and colleagues to be required for silencing and nuclear clustering of heterochromatin (Iglesias, 2019). Nup93 has also been previously shown to be required for the repression of the HoxA gene cluster in mammalian cells (Labade, 2016). This is in line with previous findings that another subunit of this sub-complex, Nup155, associates with histone deacetylases in mammalian cells and targets repressed heterochromatin in yeast. The role of Nup93 in PcG silencing is also potentially related to the previously reported link of Nup153 to PcG-mediated repression in mouse ES cells. In further support of this notion, several Nups have been previously identified in a genome-wide imaging screen for factors that affect nuclear distribution of PcG proteins (Gozalo, 2019).

Thus, a proposed model envisions that certain nuclear pores may interact with active chromatin via the Nup107 sub-complex, while other nuclear pores may associate with silent chromatin via the Nup93 sub-complex. This model is based on the ability to ChIP-seq distinct regions of the genome with different structural NPC components, and it is further supported by biochemical interaction data and the specificity of the functional effect of Nup93. The proposed model is consistent with previous findings in yeast, which reported the binding of a component of the Nup93 sub-complex to silent chromatin and the possible existence of this sub-complex as a type of an independent nuclear pore-related complex, present at the nuclear periphery. In this context, it remains to be determined whether some of the Nup93-PcG interactions similarly occur as an independent complex or if they are normally part of actual NPCs and whether the observed functional effect of Nup93 on PcG silencing always takes place at the NPC (Gozalo, 2019).

Interestingly, it was found that the Nup93-targeted PcG domains tend to preferentially interact with each other in nuclear space. It is intriguing that other Nups, such as Nup98 and Mlp1/2, have been previously shown to facilitate long-range contacts of transcribing genes, such as enhancer-promoter and 5'-3' loops. It appears that stabilization of long-range contacts, either at active or silent genes, may be generally promoted by NPC binding, but the nature of contacts depends on the particular Nup involved. Based on the combined results, it is hypothesized that Nup93 binding may promote stabilization of PcG domains that are destined to be highly repressed. This stabilization may involve promoting long-range interactions between Pc sites, as well as possibly helping sequester PcG domains into specific nuclear compartments, away from gene activity. The findings also suggest that in the case of PcG silencing, long-range interactions are more functionally involved in gene repression than localization to the nuclear periphery is, since de-repression of PcG targets is consistently associated with loss of long-range interactions (Gozalo, 2019).

Furthermore, the results suggest that some of the previously defined LADs are in fact interrupted or flanked with NPC-associated chromatin. In this manner, it appears that at least a fraction of mapped LADs may be complex, containing Nup-targeted sub-environments. These conclusions are also supported by the recent refined analysis of mammalian LADs, which revealed LAD interruptions that contain marks of active chromatin, termed 'Disruption in Peripheral signal' (DiPs). If such DiPs are biologically meaningful, the data would suggest that some such DiPs may be NPC-bound areas of the genome, characterized by functions distinct from the surrounding LADs. An intriguing conjecture is that positioning genes at NPCs within LADs may facilitate ready switching of transcriptional states, such that genes can shift between adjacent active and silent states, depending on incoming signals (Gozalo, 2019).

Finally, this analysis demonstrated widespread genomic binding by a non-stable Nup Elys, which is currently the only Nup with a known direct chromatin binding activity (Zierhut, 2014). Interestingly, Nup107 is almost exclusively found at Elys binding sites. It is tempting to speculate that Elys serves as a chromatin tethering Nup for the Nup107 sub-complex components in the interphase genome, much like it has been demonstrated to do post-mitotically, during NPC assembly. The reproducibility of Elys-Nup107 binding patterns further invokes the possibility that post-mitotic targeting of the Elys/Nup107 sub-complex to chromatin occurs at specific sites in the genome and as such, may participate in the correct re-establishment of chromatin states and nuclear architecture after mitosis. On the other hand, Nup93 similarly shares a large fraction of its binding sites with Elys, suggesting that Elys may carry a similar function in targeting the inner ring sub-complex to chromatin. Presently it remains unclear how this specificity of Nup93 versus Nup107 genome targeting may be established. But together, the findings support the model where different subunits of the NPC have evolved unique functions in chromatin regulation. Individual Nups appear to be able to facilitate either activating or repressive processes and to assist nuclear organization of chromatin domains and key proteins complexes (Gozalo, 2019).

Stuxnet facilitates the degradation of polycomb protein during development

Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. This study reports a Drosophila gene, stuxnet (stx), that controls Pc protein stability. Heightened stx activity leads to homeotic transformation, reduced Pc activity, and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyperactivation of Pc. Biochemical analyses provide a mechanistic basis for the interaction between stx and Pc; stx facilitates Pc degradation in the proteasome, independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells and induces homeotic transformation in Drosophila. These results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity (Du, 2016).

Polycomb-group (PcG) genes were first identified in Drosophila for their roles in maintaining correct expression patterns of homeotic genes. PcG-mediated transcription silencing was later proved to be a well-conserved regulatory mechanism throughout metazoans. Classical PcG targets, such as Hox genes, play important roles in biological processes ranging from stem cell maintenance to genomic imprinting. Recent genome-wide studies unveiled additional PcG targets, many of which encode transcription factors and cell-signaling proteins that regulate a diverse array of downstream effectors. Thus, PcG may act in a much broader spectrum of cellular processes than previously anticipated (Du, 2016).

PcG silencing depends primarily on the activities of two Polycomb repressive complexes (PRC). In Drosophila, PRC1 is composed of Pc (Polycomb), Ph (Polyhomeotic), Psc (Posterior sex combs), and Sce (Sex combs extra). The main subunits of the PRC2 include Esc (Extra sex combs), E(z) (Enhancer of zeste), Su(z)12 (Suppressor of zeste 12) and Caf1 (Chromatin assembly factor 1). Relying on the presence of a conserved enzymatic SET domain in E(z), PRC2 catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3). Pc then employs its chromo domain to recognize H3K27me3 mark, resulting in recruitment of PRC1 to PcG targets. Mechanisms utilized by PRC1 to silence target genes include histone H2A mono-ubiquitination, chromatin compaction, and direct interaction with the general transcription machinery (Du, 2016).

While intensive studies have been focused on uncovering mechanisms by which PcG proteins epigenetically repress target gene expression, few are devoted to define how the PcG activities are regulated. Nevertheless, several transcription factors and microRNAs are known to directly modulate PcG expression. Feedback regulatory loops may also be important to maintain proper expression of PcG, which themselves are subject to epigenetic repression. Furthermore, post-translational modifications on several PcG proteins have been reported, and the importance of such modifications has only been revealed recently. For example, SUMOylation is shown to modulate PcG activity by affecting chromatin targeting of the Pc protein, and O-GlcNAcylation has been demonstrated to prevent aggregation of PRC1 subunit Ph in Drosophila (Du, 2016).

This report describe that a Drosophila gene CG32676, which was named stuxnet (stx), functions through ubiquitin-independent degradation (UID) to control Pc protein stability and thereby PcG-mediated epigenetic repression. This study shows further that vertebrate Stx regulates orthologous Pc protein in the same fashion. Together, these results highlight a conserved regulatory mechanism for Pc, the founding member of the PcG family of proteins (Du, 2016).

Taking advantage of genetic tools available in Drosophila, the function of a UBL-domain-containing protein, Stx, was examined, and its unexpected role of regulated Pc protein degradation in epigenetic repression. These analyses on classical PcG targets demonstrate that Stx functions as a Pc-specific regulator that negatively modulates the PcG activity. Importantly, this mode of regulation was found to be conserved from flies to vertebrates (Du, 2016).

stx activity is essential for Drosophila development. The fact that pupal lethal phenotype associated with loss-of-function stx mutations can be rescued by removing 50% of Pc activity strongly supports that modulating the Pc expression is the major developmental process regulated by stx. Stx might not be a constitutive component of the canonical PRC1. However, the ability of Stx to reduce Pc recruitment to target gene loci argues that Stx may act as a gatekeeper for control of Pc availability to form highly dynamic PRC complexes on target chromatin. As stx activity is necessary for PcG target expression, Stx could function in an intrinsic machinery to regulate Pc protein homeostasis. Stx directly binds Pc through a serine-rich PcB domain and interacts with the proteasome through the UBL domain. As Pc protein degradation does not rely on ubiquitination, the UBL domain in Stx, upon interaction with Pc, could serve as a recognition signal that marks Pc protein for degradation in the proteasome. Thus, a model is proposed in which Stx acts first as an adapter and then a chaperone-like protein to facilitate proteasomal degradation of Pc, resulting in altered PcG activity in animal development. Intriguingly, upon inspection of modENCODE database, multiple binding sites were found for PcG components, including Pc, Psc, Sce, and Pho, and Ubx, which is itself a PcG target, thus pointing to the existence of a potential feedback loop between Stx and PcG activity (Du, 2016).

Altered Pc protein abundance has been noted in several biological processes. In the Sce mutant fly embryos, the bulk level of Pc protein is significantly reduced, but Ph and Psc are not affected. Similar results have been reported in mouse ES cells for RING1B and Cbx4, mammalian orthologs of Sce and Pc. However, the significance of such regulation was not understood. It is suspected that binding with Sce might stabilize Pc, which is crucial for PRC1 assembly. It is interesting to note that the level of Pc changes rapidly in the cell cycle. The oscillation of Pc protein during the cell cycle is thought to be important for establishment and maintenance of cellular epigenetic memory. The observation of the reciprocal expression pattern of Pc and Stx as well as the ability of Stx to control Pc abundance in cell cycle are in favor of a notion that regulated Pc protein stability may be one way to dynamically control Pc activity in physiological contexts. How Stx participates in such regulation is an interesting question that awaits further exploration (Du, 2016).

The PRC1 is composed of four core subunits, each of which has unique molecular activities non-exchangeable among each other. However, the loss-of-function phenotypes of individual PRC1 subunits in Drosophila only partially overlap, revealing the complexity of PRC1 regulation in various cellular processes. The differential requirement of PRC1 subunits in development might be due to the presence of distinct PRC complexes in a temporal and tissue-specific manner. This view is further complicated in vertebrates by partially redundant orthologous PRC1 proteins and the formation of multiple non-canonical complexes. Thus, it will be necessary to explore the regulatory machineries utilized by individual PRC1 components to better understand how PRC complexes exert versatile functions in vivo. This study has shown that Stx targets Pc for proteasomal degradation, but whether parallel regulators exist for other PRC1 components is still unknown (Du, 2016).

This study of Stx regulation on Pc stability reveals that the activity of Pc protein, the founding member of the PRC complexes, can be controlled through regulated protein degradation. Surprisingly, it was found that fly Pc protein is largely regulated by UID. The list of substrates that undergo UID has expanded rapidly in recent years. Intriguingly, many UID substrates are localized to the nucleus, including transcription factors and chromatin remodeling factors. The addition of Pc, a key epigenetic regulator, to this list leads to the belief that UID in the nucleus may participate in the control of gene expression (Du, 2016).

Consistent with a role of Stx on Pc stability in Drosophila development, proteasomal degradation has been reported to affect the stability of several PcG components in cultured vertebrate cells, including three PRC1 proteins BMI1, RING1B, and PHC, and one PRC2 protein EZH2. It is thus highly likely that protein degradation may play a general role in regulating PcG activity (Du, 2016).

Appropriate PcG activity is essential for stem cell maintenance and lineage specification in vertebrates. Altered PcG activity is associated with malignant human diseases, including cancer. Furthermore, dysregulated stx expression and Stx mutations are reported in several forms of cancer in the COSMIC database. Consistently, genes co-expressed with stx shown in COXPRESdb are clustered into pathways in cancer as well as Notch and MAPK signaling pathways. Very recently, Stx mutations were found in patients with autism spectrum disorders (ASD) by whole-exome sequencing. Given the strong connection between PcG and ASD, Stx may play a role in ASD through its regulation of PcG activity. Thus, the identification of regulators of PcG activity, such as Stx, may provide additional therapeutic targets for relevant diseases (Du, 2016).

GENE STRUCTURE

Bases in 5' UTR - 107

Exons - two - The intron is centered in the chromo domain (Paro, 1993).

Bases in 3' UTR - 1170

PROTEIN STRUCTURE

Amino Acids - 390

Structural Domains

The PC protein exhibits homology to the heterochromatin associated protein HP1 (Platero, 1995). The homology is confined to a 37 amino acid domain in the N-terminal part of the two proteins. This region is termed the chromo domain, standing for chromatin organization modifier (Paro, 1991 and Messmer, 1992). Carboxy-terminal truncations of the PC protein do not affect chromosomal binding of PC. However, mutations affecting only the chromo domain (including in vitro generated deletions, as well as point mutations) abolish chromosomal binding. Thus the chromo domain is important for the function of PC and it is absolutely required for binding of PC protein to chromatin. Some of the nuclear patterns generated by the mutated forms of the fusion proteins suggest that the chromo domain could be involved in a packaging mechanism, essential for compacting chromosomal proteins within heterochromatin or heterochromatin-like complexes (Messmer, 1992).

Progressive polycomb assembly on H3K27me3 compartments generates polycomb bodies with developmentally regulated motion

Polycomb group (PcG) proteins are conserved chromatin factors that maintain silencing of key developmental genes outside of their expression domains. Recent genome-wide analyses showed a Polycomb (PC) distribution with binding to discrete PcG response elements (PREs). Within the cell nucleus, PcG proteins localize in structures called PC bodies that contain PcG-silenced genes, and it has been recently shown that PREs form local and long-range spatial networks. The nuclear distribution of two PcG proteins, PC and Polyhomeotic (PH) was examined in this study. Thanks to a combination of immunostaining, immuno-FISH, and live imaging of GFP fusion proteins, it was possible to analyze the formation and the mobility of PC bodies during fly embryogenesis as well as compare their behavior to that of the condensed fraction of euchromatin. Immuno-FISH experiments show that PC bodies mainly correspond to 3D structural counterparts of the linear genomic domains identified in genome-wide studies. During early embryogenesis, PC and PH progressively accumulate within PC bodies, which form nuclear structures localized on distinct euchromatin domains containing histone H3 tri-methylated on K27. Time-lapse analysis indicates that two types of motion influence the displacement of PC bodies and chromatin domains containing H2Av-GFP. First, chromatin domains and PC bodies coordinately undergo long-range motions that may correspond to the movement of whole chromosome territories. Second, each PC body and chromatin domain has its own fast and highly constrained motion. In this motion regime, PC bodies move within volumes slightly larger than those of condensed chromatin domains. Moreover, both types of domains move within volumes much smaller than chromosome territories, strongly restricting their possibility of interaction with other nuclear structures. The fast motion of PC bodies and chromatin domains observed during early embryogenesis strongly decreases in late developmental stages, indicating a possible contribution of chromatin dynamics in the maintenance of stable gene silencing (Cheutin, 2012).

This study showed that PC bodies co-localize with H3K27me3 and form small nuclear domains of heterogeneous intensity. Surprisingly, PC bodies are found in DAPI poor regions, often adjacent to DAPI and histone-dense euchromatic regions. This result thus indicates that PC bodies are not among the most condensed chromatin portions of the euchromatic part of the genome. This localization of PC bodies is consistent with a previous study with electron microscopy, which has shown that PC is concentrated in the perichromatin compartment of the mammalian nucleus. In contrast, these data are in apparent contrast with a series of papers reporting PcG protein-dependent chromatin condensation. PcG complexes have been shown to compact chromatin in vitro and reduce DNA accessibility in vivo. Moreover, recent works show that PcG proteins are required to maintain compaction of Hox loci in mammalian embryonic stem cells and of the mouse Kcnq1 imprinted cluster. In those studies, condensation has been addressed by measuring either the compaction of nucleosomal fibers in electron microscopy, or the distance between close genomic loci by FISH. It is difficult to relate in vitro data to the current in vivo analysis. In particular, FISH analyses do not directly distinguish between a truly dense 3D organization and other types of conformations, such as a multi-looped architecture that would not necessarily induce an increase in chromatin density. Therefore, PcG target chromatin is probably organized in higher-order 3D structures that involve nucleosome-nucleosome and protein-protein interactions, but the net density of DNA (as seen by DAPI) or histones (as seen by tagged-histone microscopy) is not particularly high in these structures (Cheutin, 2012).

Earlier studies indicated that PcG proteins rapidly exchange between the nucleoplasm and PC bodies, suggesting that PC bodies consist of a local transient accumulation of PcG proteins in the cell nucleus. Earlier studies have detected the same number of PC bodies inside the nucleus as the number of bands observed on polytene chromosomes, suggesting that PC bodies are formed by PcG proteins binding to their target chromatin. The observed colocalization of PcG target genes with PC bodies in diploid cells confirms this view. An alternative scenario posits that PC bodies could form nucleation sites onto which PcG-target genes move to become silenced. Two lines of evidence from this work suggest the first scenario to be closer to reality. Firstly, it was found that the amount of PC within a PC body depends on the linear size of the genomic region coated by PC and H3K27me3. Secondly, the higher enrichment of PC in PC bodies after homologous chromosome pairing strongly suggests that PC bodies are the nuclear counterparts of linear genomic domains identified in genome-wide studies rather than nuclear structures to which Polycomb target genes have to be localized for their silencing (Cheutin, 2012).

In the head of embryos, where the Antp and Abd-B genes are silenced, they localize in large PC bodies in all cell nuclei. In contrast, loci where PC coating is restricted to smaller genomic regions do not always localize within PC bodies in interphase cell nuclei. Interestingly, time-lapse imaging shows that large PC bodies are stable structures that can be visualized in all frames of time series, whereas small PC bodies are apparently less stable because they are not visible in all of the frames. One possible explanation for the lack of colocalization between PC target genes and PC bodies is that small genomic regions may not be coated by PC in every cell. Alternatively, the amount of PC within the PC body in which small genomic regions localize might be too small to be directly observed, and only become visible when several small PC bodies interact together. For instance a previous study showed that a transgene containing only two copies of a PRE could be detected in about 50% of cell nuclei (Cheutin, 2012).

Intense PC bodies can be visualized during entire time-lapse experiments, allowing the study of their motion. The interpretation of these time-lapse experiments is not straightforward because the MSD of PC bodies only weakly correlates with the MSC. Interestingly, tracks of PC bodies are mainly composed of narrow angles. The analysis of the motion of chromatin domains containing H2Av-GFP gave similar results, but gave unambiguous evidence for the coordinated motion of several chromatin domains. By using the Lac repressor/lac operator system, two components of chromatin motion in early G2 Drosophila spermatocyte nuclei have been reported: a short range motion which occurs in approximately 0.5 µm radius domains, and long-range motion confined to a large, chromosome-sized domain. Another study has also identified a two-regime motion of a chromatin locus inside mammalian nucleus by using a two-photon microscope, which provides high spatial and temporal resolution. This work indicated that chromatin loci undergo apparent constrained diffusion during long periods, interrupted by jumps of 150 nm lasting less than 2 s. However, none of these previous works reported any coordinated motion of adjacent chromatin domains, and therefore they both described the motion of chromatin as being consistent with a random walk (Cheutin, 2012).

In tracking experiments, it was realized that the fast regime of motion is tightly constrained within volumes much smaller than chromosome territories. This suggests that any given locus will normally explore a restricted three-dimensional environment in the cell nucleus. Since this applies generally to chromatin at all developmental stages, one can deduce that each genomic locus is likely to locate in the vicinity of neighboring loci in the three-dimensional nuclear space. The prediction is thus that each locus should most frequently contact other loci that are in its linear neighborhood along the chromosome. This behavior matches the results observed in chromosome conformation capture on chip (4C) experiments, where each 4C bait had most contacts within few hundred kb to a few Mb of surrounding chromatin. Thus, the current results provide a possible scenario for the explanation of these results obtained from large cell populations. Recent studies showed that homeotic gene clusters form an extensive network of contacts with other PcG target loci. This is consistent with the observation of multiple PC body collisions that can be stable for prolonged times in the nucleus. In contrast, the fact that PC intensity correlates with the linear extension of genomic PC and H3K27me3 domains suggests that PC-mediated associations are relatively rare, at least during embryogenesis (Cheutin, 2012).

The slower regime of long-range motion depends on coordinated large-scale chromatin movements that were not documented before. This may depend on the tools used in previous studies. Time-lapse experiments performed by using the Lac repressor/lac operator system only follow one or a few points inside the cell nucleus, limiting the probability to observe coordinated motions, especially in species containing many chromosomes. In contrast, this study followed many chromatin domains inside Drosophila nuclei and long-range coordinated motions were easily identified when at least two distinct nuclear structures moved simultaneously with a similar trajectory. This motion is directional and chromatin domains and PC bodies can cover up to 1 µm in 10 sec. Different objects having coordinated motion probably belong to the same structure, which suggests that the ensemble of chromatin domains and PC bodies displaying a similar coordinated motion forms a single higher-order nuclear structure. This kind of motion is perfectly consistent with the observation of a chromosome territory, which implies that chromosomes form distinct nuclear structures in interphase cells. A displacement of an entire chromosome, or of a chromosome arm, or a large part thereof, would induce the coordinated motion of all chromatin domains and PC bodies associated to the corresponding chromosome portion (Cheutin, 2012).

The few association and dissociation events of PC bodies observed during this work are related to long-range coordinated motion events that affect both chromatin domains and PC bodies. Therefore, gene kissing depending on PcG proteins could rely on large scale chromatin movements which lead to transient fusion of PC bodies, and may be in turn specifically stabilized by interactions among PcG proteins. Moreover, the association and dissociation of PC bodies seems to be developmentally regulated, because dynamic associations and dissociations were observed during early embryogenesis, but are strongly reduced later in development (Cheutin, 2012).

Condensed chromatin domains and PC bodies move in confined volumes much smaller than chromosome territories. This highly constrained motion prevents chromatin domains from dispersing inside the cell nucleus and can explain why chromosomes form chromosome territories in interphase cells. This movement within highly confined volumes implies that some forces prevent chromatin from diffusing within entire chromosome territories. Interestingly, it was shown before that chromatin loci localized in peri-nucleolar areas or within heterochromatin move less than the ones included in euchromatin, and it was concluded that association of chromatin loci with different nuclear compartments induces specific constraints on their motion. Another time-lapse experiment performed on one Drosophila locus flanking a large block of heterochromatin showed that random association of this locus with pericentric heterochromatin is quite stable and decreases its motion. The motion of larger chromatin structures such as heterochromatin or euchromatin domains cannot be addressed by tracking single loci. By analyzing structures larger than individual chromatin loci, the motion of both bulk chromatin domains and of PC bodies seems to be influenced by their respective local enrichment of histone and PC proteins. Therefore, one key determinant of the motion constraint is an inner property of these structures, which is coherent with the concept of self-organization (Cheutin, 2012).

The most dramatic change of PC body motion occurs during embryogenesis when nuclear volumes strongly decrease, concomitant with a decrease in bulk chromatin motion. Comparison of chromatin motion between early and late G2 Drosophila spermatocytes or between undifferentiated and differentiated cells of eye imaginal discs indicated that the volume in which chromatin loci move decreases during differentiation. However, because of the particularly rapid motion of chromatin domains and PC bodies during early embryogenesis, the slowdown of chromatin motion occurring during embryogenesis is higher than the ones previously described during differentiation. Interestingly, the reduction of the volume of constraint during developmental progression suggests a correlation between the flexibility of chromatin structures and the potential for cell differentiation (Cheutin, 2012).

It is interesting to note that the motion of PC bodies appears less sensitive to temperature than chromatin domains in late embryos, suggesting that Polycomb proteins may specifically buffer environmental effects such as temperature change. This buffering may be an important determinant of the stability of Polycomb-dependent gene silencing during development. During this work, no other fundamental difference was observed between the motion of condensed chromatin domains and of PC bodies. This apparent absence in specificity is coherent with data implying that PC bodies form molecularly specialized chromatin regions, but suggests that the molecular identity of these structures is not the main determinant of their motion. Interestingly, a previous study has shown that the artificial Mx1-YFP nuclear body exhibits a very similar mobility compared with Promyelocytic leukemia and Cajal bodies. Although being molecularly different, no specific motion of these nuclear bodies was observed, indicating that the motion of nuclear bodies mainly depends on structural issues such as their size and the nuclear volume. During fly embryogenesis, PC bodies and condensed chromatin domains move similarly, but PC bodies move in a larger volume than chromatin domains. To explain this difference, one might argue that condensed chromatin domains would form much larger structures than PC bodies. This is difficult to ascertain until the identity of these DAPI- and histone-dense regions is better understood. Genome-wide analysis of chromatin components has recently identified five different types of chromatin in Drosophila cells, among which three contained silent genes (Filion, 2010). In addition to heterochromatin and Polycomb-repressed chromatin, a third type of silent chromatin was uncovered, which is composed of very large genomic domains encompassing half of the genomic euchromatin. It is proposed that this silent chromatin portion of the genome is physically manifested as the DAPI- and histone-dense chromatin that this study has identified to be distinct from PC bodies (Cheutin, 2012).

Polycomb-mediated chromatin loops revealed by a subkilobase-resolution chromatin interaction map

The locations of chromatin loops in Drosophila were determined by Hi-C (chemical cross-linking, restriction digestion, ligation, and high-throughput DNA sequencing). Whereas most loop boundaries or 'anchors' are associated with CTCF protein in mammals, loop anchors in Drosophila were found most often in association with the polycomb group (PcG) protein Polycomb (Pc), a subunit of polycomb repressive complex 1 (PRC1). Loops were frequently located within domains of PcG-repressed chromatin. Promoters located at PRC1 loop anchors regulate some of the most important developmental genes and are less likely to be expressed than those not at PRC1 loop anchors. Although DNA looping has most commonly been associated with enhancer-promoter communication, the results indicate that loops are also associated with gene repression (Eagen, 2017).

Active and inactive genes are folded differently and located in different regions of the nucleus, but the molecular basis of chromosome folding and nuclear architecture remains to be determined. One folding paradigm is the formation of protein-mediated DNA loops, which are most commonly associated with enhancer-promoter communication. On the other hand, gene repression is most often associated with heterochromatin formation and chromosome condensation (Eagen, 2017).

Chromosome folding can be revealed by chemical cross-linking, followed by restriction digestion, ligation, and high-throughput DNA sequencing. Such 'Hi-C' analysis has revealed intrachromosomal folding on multiple length scales. From lengths of 1 kb, the smallest so far examined, to hundreds of kilobases, two features are observed, loops and so-called topologically associating domains, or 'TADs' (which have also been referred to as A/B domains, physical domains, topological domains, or contact domains). Loops bring a pair of loci into close physical proximity; TADs represent genomic intervals in which all pairs of loci exhibit an enhanced frequency of contact, and correspond to stably condensed chromosomal regions. On a larger scale, extending to whole chromosomes, TADs interact with one another to form 'compartments' (Eagen, 2017).

Hi-C, on the smallest length scale, based on the most extensive sequencing, is needed for unambiguous identification of chromatin loops and has been reported thus far only for the mouse and human genomes. This limitation has also been overcome Drosophila by Hi-C analysis at subkilobase resolution. An unanticipated correlation was found with results of ChIP-seq analysis in Drosophila, with an important functional correlate (Eagen, 2017).

The locations of loop anchors in Drosophila determined in this study are notable both for correlations with ChIP-seq data and for the lack thereof. The lack of correlation with locations of CTCF protein was unexpected, inasmuch as most loop anchors in mammals are associated with CTCF protein, apparently bound to CTCF sequence motifs in a convergent orientation. There are evidently multiple patterns of protein association with loop anchors in metazoans. The association of loop anchors in Drosophila with Pc protein is noteworthy because it points to a role of looping not only in gene activation, as widely observed in the past, but in gene repression as well (Eagen, 2017).

Regions of PcG-repressed chromatin ('PcG domains') that are separated by hundreds of kilobases to megabases are known to be in enhanced spatial proximity, but details of their internal organization have only been investigated by averaging over many PcG domains. The high resolution of the Hi-C contact maps revealed chromatin loops within individual PcG domains, giving insight into their internal organization. PRC1 is known to compact nucleosome arrays in vitro. Knockdown of the PRC1 subunit, Polyhomeotic (Ph), in vivo decompacts PcG-repressed chromatin, and Ph that is unable to polymerize impairs the ability of PRC1 to form clusters. Together with these findings, the results suggest that PRC1-bound chromatin loops within PcG-repressed domains either establish or maintain a condensed state (Eagen, 2017).

Previous analyses by 3C have pointed to associations of PcG proteins with chromatin loops for the Bithorax complex (BX-C) in S2 cells; for inv and en in BG3 and Sg4 cells; and for an embryonic, pupae, and adult transgenic reporter system. The Hi-C data are, however, at higher resolution and genome-wide. Higher resolution allowed more comprehensive analysis, such as the unambiguous identification of loops and the segmentation of ANT-C into a series of TADs with one or two homeotic gene promoters per TAD. Genome-wide analysis revealed both the pervasive nature of Pc protein association and the absence of significant CTCF protein association, despite conservation of CTCF from Drosophila to human (Eagen, 2017).

A report by Cubeñas-Potts (2017) on Drosophila chromatin loops in Kc cells appeared while this manuscript was in preparation. Cubenas-Potts noted an enrichment of cohesin but a lack of Drosophila CTCF at loop anchors, consistent with the current observations. Cubenas-Potts did not mention Pc, but the current analysis of their data revealed an enrichment of loop anchors at Pc ChIP peaks and an enrichment of Pc ChIP peaks at loop anchors. A likelihood was found of repression of promoters at Pc-bound loop anchors, especially for developmental genes; Cubenas-Potts et al. observed an enrichment of active developmental enhancers at loop anchors, possibly because these are among the many loop anchors not bound by Pc, or because the chromatin at these loop anchors is bivalent, bound by nonhistone proteins and histone posttranslational modifications associated with both gene activation and repression (Eagen, 2017).

The occurrence of PRC1 at loop anchors could reflect a role in loop formation similar to that proposed for CTCF in mammals, wherein cohesion complexes extrude loops, in a process halted upon reaching bound CTCF. Consistent with this model, a large majority (72.8%) of Drosophila loop anchors are bound by the Rad21 subunit of cohesin. Regardless of whether PRC1 performs such a role, additional proteins must be involved, because PRC1 is present at only 26% of Drosophila loop anchors (Eagen, 2017).

Anterior CNS expansion driven by brain transcription factors

During CNS development, there is prominent expansion of the anterior region of the brain. In Drosophila, anterior CNS expansion emerges from three rostral features: (1) increased progenitor cell generation, (2) extended progenitor cell proliferation, (3) more proliferative daughters. This study finds that tailless (mouse Nr2E1/Tlx), otp/Rx/hbn (Otp/Arx/Rax) and Doc1/2/3 (Tbx2/3/6) are important for brain progenitor generation. These genes, and earmuff (FezF1/2), are also important for subsequent progenitor and/or daughter cell proliferation in the brain. Brain TF co-misexpression can drive brain-profile proliferation in the nerve cord, and can reprogram developing wing discs into brain neural progenitors. Brain TF expression is promoted by the PRC2 complex, acting to keep the brain free of anti-proliferative and repressive action of Hox homeotic genes. Hence, anterior expansion of the Drosophila CNS is mediated by brain TF driven 'super-generation' of progenitors, as well as 'hyper-proliferation' of progenitor and daughter cells, promoted by PRC2-mediated repression of Hox activity (Curt, 2019).

Detailed analysis of Drosophila CNS development has revealed that there is 'super-generation' of NBs in the B1 segment; ~160 NBs in B1 compared to 28-70 NBs/segment for each of the 18 posterior segments (B2-A10). In the ventral neurogenic regions (generating the nerve cord) a single NB delaminates from each proneural cluster. In contrast, the NB super-generation in B1 stems, at least in part, from group delamination of NBs. The specification of NB cell fate depends upon low, or no, Notch activity. In line with this notion, evidence points to reduced Notch signalling in the procephalic neuroectoderm (Curt, 2019).

Head gap genes, such as tll, were previously shown to be important for B1 NB generation, and in line with this strikingly reduced NB generation was observed in tll. Does tll intersect with Notch signalling? tll mutants show loss of expression of the proneural gene l'sc, which is negatively regulated by Notch. Recent studies furthermore reveal an intimate interplay between tll and Notch signalling in the developing Drosophila embryonic optic placodes. In addition, the C. elegans tll orthologue nhr-67 regulates both lin-12 (Notch) and lag-2 (Delta) during uterus development. Strikingly, in the mouse brain, the tll orthologue Nr2E1 (aka Tlx) was recently shown to negatively regulate the canonical Notch target gene Hes1. Against this backdrop, it is tempting to speculate that the group NB delamination normally observed in the procephalic region results, at least in part from tll repression of the Notch pathway. Indeed, tll was the only one of the four TFs that could act alone to trigger ectopic NBs in the wing disc (Curt, 2019).

Other previously identified head gap genes are oc (also known as orthodenticle: otd), buttonhead (btd) and ems. However, it was not observed that misexpression of oc or ems from elav-Gal4 efficiently drove ectopic proliferation in the nerve cord. Moreover, oc acts both in B1-B2, ems in B2-B3, being repressed from B1 by tll, and btd acts in B2-B3. Because B2 and B3 segments do not display super-generation of NBs these findings point to tll as the key head gap gene driving the super-generation of NBs specifically observed in the B1 segment (Curt, 2019).

Reduced NB generation was observed in the triple otp/Rx/hbn and Doc1/2/3 mutants. This would tentatively place them in the category of head gap genes, at least as far as being important for NB generation. However, their effects on NB generation is weaker than that observed in tll mutants. In addition, otp/Rx/hbn and Doc1/2/3 show genetic redundancy. The combination of genetic redundancy and their weaker effects on NB generation, likely explain why they were not previously categorised as head gap genes (Curt, 2019).

The connection between the brain TFs examined in this study herein and NB super-generation is not only evident from the mutant phenotypes, but also from their potent gain-of-function effects. Strikingly, it was found that brain TF co-misexpression was sufficient to generate ectopic NBs in the embryonic ectoderm and developing wing discs. A number of markers indicate that these ectopic NBs undergo normal CNS NB lineage progression, generating neurons and glia. Moreover, the ectopic expression of the brain-specific factors Rx and Hbn, the apparently higher neuron/glia ratio, the reduced GsbN expression, the generation of Dpn+/Ase- NBs (Type II-like) in both the embryonic ectoderm and wing discs, in combination suggest that brain TF co-misexpression specifically triggered reprogramming towards a B1 brain-like phenotype (Curt, 2019).

One surprising finding pertains to the clear difference between the potency of the tll,erm double and the Tetra (tll, erm, Doc2 and otp) in the embryonic ectoderm versus the wing disc, with the double being more potent in the wing disc and the Tetra more potent in the embryo. Indeed, in the wing disc the strong effect of tll,erm is suppressed by the addition of any combination of otp and Doc2. There is no obvious explanation for the different responsiveness to brain TF misexpression in the two tissues, but it may reflect the fact the embryonic neuroectoderm is already primed for the generation of NBs (Curt, 2019).

Another surprising finding pertains to the role of erm in embryonic versus larva Type II NBs. Previous studies of erm function in the larvae found that erm mutants displayed more Type II NBs. Larval MARCM clone induction and marker analysis demonstrate that this is due to de-differentiation of INPs back to type II NBs, rather than excess generation of Type II NBs in the embryo. No extra Type II or Type I NBs were found in erm mutants but rather reduced number of cells generated in the embryonic Type II lineages, showing that erm is important for lineage progression. Hence, the role of erm appears to be different in the embryonic versus larval Type II lineages (Curt, 2019).

In addition to the NB super-generation in B1, recent studies reveal that three different lineage topology mechanisms underlie the hyper-proliferation of the brain. First, the majority of NBs (136 out 160 NB) display a protracted phase of NB proliferation, and do not show evidence of switching from Type I to Type 0 daughter proliferation (Yaghmaeian Salmani, 2018). Second, the eight MBNBs, which appear to divide in the Type I mode and never enter quiescence, also generate large lineages. Third, the 16 Type II NBs progress by budding off INP daughter cells, which divide multiple times to generate daughter cells that in turn divide once, hence resulting in lineage expansion. In contrast, in the nerve cord many NBs switch from Type I to Type 0, and all halt neurogenesis by mid-embryogenesis. The Hox anti-proliferation gradient further results in a gradient of the Type I-->0 switch and NB exit along the nerve cord. The combined effects of these alternate lineage topology behaviours translate into striking differences in the average lineage size in the brain when compared to the nerve cord (Yaghmaeian Salmani, 2018) (see Mechanisms underlying the anterior expansion of the Drosophila CNS). Moreover, the three different modes of more extensive NB and daughter cell proliferation combine with the super-generation of NBs in B1 to generate many more cells in the B1 brain segment, when compared to all posterior segments (Curt, 2019).

The brain TFs examined in this study are expressed in several or all (Tll) of the three brain NB types, and are important for both NB and daughter cell proliferation. In line with this, brain TF ectopic expression, with the late neural driver elav-Gal4, drives aberrant nerve cord proliferation and blocks both the Type I-->0 daughter cell proliferation switch and NB cell cycle exit. This results in the generation of supernumerary cells, evident both by the expansion of specific lineages and an increase in overall nerve cord cell numbers. This study found that both the double and Tetra misexpression can trigger the ectopic generation of what appears to be a mix of Type I and Type II-like NBs. The mix of these two NB types may reflect that the misexpression scenario does not accurately and reproducibly recreate the temporal order of the brain TFs, with for example tll expressed prior to erm in the wild type (Curt, 2019).

The ectopic appearance of symmetrically dividing NBs in the brain TF co-misexpression nerve cords is more difficult to explain. However, since there normally are divisions of cells in the neuroectodermal layer prior to NB delamination, and given the early expression of the brain TFs (prior to NB delamination), it is tempting to speculate that brain TF co-misexpression to some extent can trigger an early neuroectodermal cell fate (Curt, 2019).

It was recently found that NB and daughter proliferation is also promoted by a set of early TFs expressed by most, if not all NBs. Strikingly, these TFs are expressed at higher levels in the brain, due to the lack of Hox expression therein, thereby contributing to the extended NB proliferation and more proliferative daughter cells observed in the brain. It will be interesting to address the possible regulatory interplay between these broadly expressed early NB factors and the brain TFs described in this study (Curt, 2019).

Gene expression studies have revealed the mutually exclusive territory of brain TF and Hox gene expression in the Drosophila CNS. In line with this notion, it was found that co-misexpression of brain TFs in the nerve cord repressed expression of the posterior Hox genes of the BX-C, and conversely that BX-C co-misexpression repressed several brain TFs; Bsh, Rx, Hbn, Tll and Doc2 (Curt, 2019).

A key 'gate-keeper' of the brain versus nerve cord territories appears to be the PRC2 epigenetic complex. Removing PRC2 function results in complete loss of the H3K27me3 repressive epigenetic mark and anterior expansion of the expression of all Hox genes. This furthermore results in repression of brain TF expression, that is Tll and Doc2, as well as Rx. Surprisingly, in spite of the many roles that PRC2 may play, this study found that transgenic brain TF co-expression could rescue the PRC2 mutant proliferation defects. Given the repressive action of BX-C Hox genes on brain TFs, this suggests that the principle role of PRC2 during early CNS development, at least regarding proliferation, is to ensure that Hox genes are prevented from being expressed in the brain, thus ensuring brain TF expression. Indeed, it was recently demonstrated that the reduced brain proliferation observed in esc mutants could also be fully rescued by the simultaneous removal of the posterior-most and most anti-proliferative Hox gene, Abd-B (Curt, 2019).

In mammals, the precise number of neural progenitors present at different axial levels during embryonic development has not yet been mapped. However, the wider expanse of the anterior embryonic neuroectoderm would suggest the generation of more progenitors anteriorly. There is also an extended phase of neurogenesis in the forebrain, when compared to the spinal cord. Dividing daughter cells (most often referred to as basal progenitors; bP) have been identified along the entire A-P axis of the mouse CNS. Intriguingly, the ratio of dividing bPs to apical progenitors (radial glial cells) was found to be higher in the telencephalon than in the hindbrain. Similarly, recent studies revealed a higher ratio of dividing cells in the outer layers than in the lumen, when comparing the developing telencephalon to the lumbo-sacral spinal cord. Albeit still limited in their scope, these studies suggest that a similar scenario is playing out along the A-P axis of mouse CNS as that observed in Drosophila, with an anteriorly extended phase of progenitor proliferation and a higher prevalence of proliferating daughter cells (Curt, 2019).

In addition to the similarities between Drosophila and mouse regarding progenitor generation, as well as progenitor and daughter cell proliferation, the genetic mechanisms controlling these events may also be conserved. Mouse orthologues of the Drosophila brain TFs examined in this study, that is Nr2E1/Tlx (Tll); Otp, Rax and Arx (Otp); Tbx2/3/6 (Doc1/2/3); and FezF1/2 (Erm), are restricted to the brain and are known to be critical for normal mouse brain development, and in several cases for promoting proliferation. Furthermore, Hox genes are not expressed in the mouse forebrain and there is a generally conserved feature of brain TFs expressed anteriorly and Hox genes posteriorly. Mutation and misexpression has revealed that Hox genes are anti-proliferative also in the vertebrate CNS. Moreover, PRC2 (EED) mouse mutants show extensive expression of Hox genes into the forebrain and reduced gene expression of for example Nr2E1, Fezf2 and Arx. This is accompanied by reduced proliferation in the telencephalon and a microcephalic brain, while the spinal cord does not appear effected (Curt, 2019).

Gene expression and phylogenetic consideration recently led to the proposal that the CNS may have evolved by 'fusion' of two separate nervous systems, the apical and basal nervous systems, present in the common ancestor. Interestingly, in arthropods for example Drosophila, the brain and nerve cord initially form in separate regions only to merge during subsequent development. Recent studies of the role of the PRC2 complex and Hox genes in controlling A-P differences in CNS proliferation, in both Drosophila and mouse, lend support for the notion of a 'fused' CNS (Yaghmaeian Salmani, 2018). This idea is further supported by recent studies of the epigenomic signature and early embryonic cell origins of the anterior versus posterior developing CNS. The findings outlined in this study, showing that brain hyperproliferation is driven not only by the lack of Hox homeotic gene expression, but also by the specific expression of highly conserved brain TFs, lend further support to the notion of a separate evolutionary origin of brain and nerve cord (Curt, 2019).

It is tempting to speculate that the possibly separate evolutionary origins of the brain and nerve cord may manifest not only as distinct modes of neurogenesis, but also be reflected by separate regulatory mechanisms. These would involve brain TFs acting anteriorly, generating an abundance of progenitors, as well as driving progenitor and daughter cell proliferation. Conversely, Hox genes would act posteriorly, counteracting progenitor generation, as well as tempering progenitor and daughter cell proliferation. In this model, PRC2 would act as a 'gate keeper', ensuring that Hox genes are restricted from the brain and thereby promoting brain TF expression. This model clearly represents an over-simplification, but may serve as a useful launching point for future comparative studies in many model systems (Curt, 2019).

date revised:  15 August 2023

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