Single-molecule imaging of transcriptionally coupled and uncoupled splicing.

Introns are removed from pre-mRNAs during transcription while the pre-mRNA is still tethered to the gene locus via RNA polymerase. However, during alternative splicing, it is important that splicing be deferred until all of the exons and introns involved in the choice have been synthesized. We have developed an in situ RNA imaging method with single-molecule sensitivity to define the intracellular sites of splicing. Using this approach, we found that the normally tight coupling between transcription and splicing is broken in situations where the intron's polypyrimidine tract is sequestered within strong secondary structures. We also found that in two cases of alternative splicing, in which certain exons are skipped due to the activity of the RNA-binding proteins Sxl and PTB, splicing is uncoupled from transcription. This uncoupling occurs only on the perturbed introns, whereas the preceding and succeeding introns are removed cotranscriptionally. PAPERCLIP:

The role of secondary structures in the functioning of 3' untranslated regions of mRNA: A review of functions of 3' UTRs' secondary structures and hypothetical involvement of secondary structures in cytoplasmic polyadenylation in Drosophila.

3' untranslated regions (3' UTRs) of mRNAs have many functions, including mRNA processing and transport, translational regulation, and mRNA degradation and stability. These different functions require cis-elements in 3' UTRs that can be either sequence motifs or RNA structures. Here we review the role of secondary structures in the functioning of 3' UTRs and discuss some of the trans-acting factors that interact with these secondary structures in eukaryotic organisms. We propose potential participation of 3'-UTR secondary structures in cytoplasmic polyadenylation in the model organism Drosophila melanogaster. Because the secondary structures of 3' UTRs are essential for post-transcriptional regulation of gene expression, their disruption leads to a wide range of disorders, including cancer and cardiovascular diseases. Trans-acting factors, such as STAU1 and nucleolin, which interact with 3'-UTR secondary structures of target transcripts, influence the pathogenesis of neurodegenerative diseases and tumor metastasis, suggesting that they are possible therapeutic targets.

Crol contributes to PRE-mediated repression and Polycomb group proteins recruitment in Drosophila.

The Polycomb group (PcG) proteins are fundamental epigenetic regulators that control the repressive state of target genes in multicellular organisms. One of the open questions is defining the mechanisms of PcG recruitment to chromatin. In Drosophila, the crucial role in PcG recruitment is thought to belong to DNA-binding proteins associated with Polycomb response elements (PREs). However, current data suggests that not all PRE-binding factors have been identified. Here, we report the identification of the transcription factor Crooked legs (Crol) as a novel PcG recruiter. Crol is a C2H2-type Zinc Finger protein that directly binds to poly(G)-rich DNA sequences. Mutation of Crol binding sites as well as crol CRISPR/Cas9 knockout diminish the repressive activity of PREs in transgenes. Like other PRE-DNA binding proteins, Crol co-localizes with PcG proteins inside and outside of H3K27me3 domains. Crol knockout impairs the recruitment of the PRC1 subunit Polyhomeotic and the PRE-binding protein Combgap at a subset of sites. The decreased binding of PcG proteins is accompanied by dysregulated transcription of target genes. Overall, our study identified Crol as a new important player in PcG recruitment and epigenetic regulation.

Preformation and epigenesis converge to specify primordial germ cell fate in the early Drosophila embryo.

A critical step in animal development is the specification of primordial germ cells (PGCs), the precursors of the germline. Two seemingly mutually exclusive mechanisms are implemented across the animal kingdom: epigenesis and preformation. In epigenesis, PGC specification is non-autonomous and depends on extrinsic signaling pathways. The BMP pathway provides the key PGC specification signals in mammals. Preformation is autonomous and mediated by determinants localized within PGCs. In Drosophila, a classic example of preformation, constituents of the germ plasm localized at the embryonic posterior are thought to be both necessary and sufficient for proper determination of PGCs. Contrary to this longstanding model, here we show that these localized determinants are insufficient by themselves to direct PGC specification in blastoderm stage embryos. Instead, we find that the BMP signaling pathway is required at multiple steps during the specification process and functions in conjunction with components of the germ plasm to orchestrate PGC fate.

Redundant enhancers in the iab-5 domain cooperatively activate Abd-B in the A5 and A6 abdominal segments of Drosophila.

The Abdominal-B (Abd-B) gene belongs to the bithorax complex and its expression is controlled by four regulatory domains, iab-5, iab-6, iab-7 and iab-8, each of which is thought to be responsible for directing the expression of Abd-B in one of the abdominal segments from A5 to A8. A variety of experiments have supported the idea that BX-C regulatory domains are functionally autonomous and that each domain is both necessary and sufficient to orchestrate the development of the segment they specify. Unexpectedly, we discovered that this model does not always hold. Instead, we find that tissue-specific enhancers located in the iab-5 domain are required for the proper activation of Abd-B not only in A5 but also in A6. Our findings indicate that the functioning of the iab-5 and iab-6 domains in development of the adult cuticle A5 and A6 in males fit better with an additive model, much like that first envisioned by Ed Lewis.

The 3'UTR of the Drosophila CPEB translation factor gene orb2 plays a crucial role in spermatogenesis.

CPEB proteins are conserved translation regulators involved in multiple biological processes. One of these proteins in Drosophila, Orb2, is a principal player in spermatogenesis. It is required for meiosis and spermatid differentiation. During the later process, orb2 mRNA and protein are localized within the developing spermatid. To evaluate the role of the orb2 mRNA 3'UTR in spermatogenesis, we used the CRISPR/Cas9 system to generate a deletion of the orb2 3'UTR, orb2R. This deletion disrupts the process of spermatid differentiation but has no apparent effect on meiosis. Differentiation abnormalities include defects in the initial polarization of the 64-cell spermatid cysts, mislocalization of mRNAs and proteins in the elongating spermatid tails, altered morphology of the elongating spermatid tails, and defects in the assembly of the individualization complex. These disruptions in differentiation appear to arise because orb2 mRNA and protein are not properly localized within the 64-cell spermatid cyst.

Transcriptional Readthrough Interrupts Boundary Function in Drosophila.

In higher eukaryotes, distance enhancer-promoter interactions are organized by topologically associated domains, tethering elements, and chromatin insulators/boundaries. While insulators/boundaries play a central role in chromosome organization, the mechanisms regulating their functions are largely unknown. In the studies reported here, we have taken advantage of the well-characterized Drosophila bithorax complex (BX-C) to study one potential mechanism for controlling boundary function. The regulatory domains of BX-C are flanked by boundaries, which block crosstalk with their neighboring domains and also support long-distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, we asked whether readthrough transcription (RT) can impact boundary function. For this purpose, we took advantage of two BX-C boundary replacement platforms, Fab-7attP50 and F2attP, in which the Fab-7 and Fub boundaries, respectively, are deleted and replaced with an attP site. We introduced boundary elements, promoters, and polyadenylation signals arranged in different combinations and then assayed for boundary function. Our results show that RT can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is therefore possible that RT may be a widely used mechanism to alter boundary function and regulation of gene expression.

Functional analysis of Niemann-Pick disease type C family protein, NPC1a, in Drosophila melanogaster.

During embryonic gonad coalescence, primordial germ cells (PGCs) follow a carefully choreographed migratory route circumscribed by guidance signals towards somatic gonadal precursor cells (SGPs). In Drosophila melanogaster, SGP-derived Hedgehog (Hh), which serves as a guidance cue for the PGCs, is potentiated by mesodermally restricted HMGCoA-reductase (Hmgcr) and the ABC transporter Multi-drug-resistant-49 (Mdr49). Given the importance of cholesterol modification in the processing and long-distance transmission of the Hh ligand, we have analyzed the involvement of the Niemann-Pick disease type C-1a (NPC1a) protein, a cholesterol transporter, in germ cell migration and Hedgehog signaling. We show that mesoderm-specific inactivation of Npc1a results in germ cell migration defects. Similar to Mdr49, PGC migration defects in the Npc1a embryos are ameliorated by a cholesterol-rich diet. Consistently, reduction in Npc1a weakens the ability of ectopic HMG Coenzyme A reductase (Hmgcr) to induce germ cell migration defects. Moreover, compromising Npc1a levels influences Hh signaling adversely during wing development, a process that relies upon long-range Hh signaling. Last, doubly heterozygous embryos (Mdr49/Npc1a) display enhanced germ cell migration defects when compared with single mutants (Npc1a/+ or Mdr49/+), supporting cooperative interaction between the two.

Mapping parameter spaces of biological switches.

Since the seminal 1961 paper of Monod and Jacob, mathematical models of biomolecular circuits have guided our understanding of cell regulation. Model-based exploration of the functional capabilities of any given circuit requires systematic mapping of multidimensional spaces of model parameters. Despite significant advances in computational dynamical systems approaches, this analysis remains a nontrivial task. Here, we use a nonlinear system of ordinary differential equations to model oocyte selection in Drosophila, a robust symmetry-breaking event that relies on autoregulatory localization of oocyte-specification factors. By applying an algorithmic approach that implements symbolic computation and topological methods, we enumerate all phase portraits of stable steady states in the limit when nonlinear regulatory interactions become discrete switches. Leveraging this initial exact partitioning and further using numerical exploration, we locate parameter regions that are dense in purely asymmetric steady states when the nonlinearities are not infinitely sharp, enabling systematic identification of parameter regions that correspond to robust oocyte selection. This framework can be generalized to map the full parameter spaces in a broad class of models involving biological switches.

GAGA factor: a multifunctional pioneering chromatin protein.

The Drosophila GAGA factor (GAF) is a multifunctional protein implicated in nucleosome organization and remodeling, activation and repression of gene expression, long distance enhancer-promoter communication, higher order chromosome structure, and mitosis. This broad range of activities poses questions about how a single protein can perform so many seemingly different and unrelated functions. Current studies argue that GAF acts as a "pioneer" factor, generating nucleosome-free regions of chromatin for different classes of regulatory elements. The removal of nucleosomes from regulatory elements in turn enables other factors to bind to these elements and carry out their specialized functions. Consistent with this view, GAF associates with a collection of chromatin remodelers and also interacts with proteins implicated in different regulatory functions. In this review, we summarize the known activities of GAF and the functions of its protein partners.

The CPEB translational regulator, Orb, functions together with Par proteins to polarize the Drosophila oocyte.

orb is a founding member of the CPEB family of translational regulators and is required at multiple steps during Drosophila oogenesis. Previous studies showed that orb is required during mid-oogenesis for the translation of the posterior/germline determinant oskar mRNA and the dorsal-ventral determinant gurken mRNA. Here, we report that orb also functions upstream of these axes determinants in the polarization of the microtubule network (MT). Prior to oskar and gurken translational activation, the oocyte MT network is repolarized. The MT organizing center at the oocyte posterior is disassembled, and a new MT network is established at the oocyte anterior. Repolarization depends upon cross-regulatory interactions between anterior (apical) and posterior (basal) Par proteins. We show that repolarization of the oocyte also requires orb and that orb is needed for the proper functioning of the Par proteins. orb interacts genetically with aPKC and cdc42 and in egg chambers compromised for orb activity, Par-1 and aPKC protein and aPKC mRNA are mislocalized. Moreover, like cdc42-, the defects in Par protein localization appear to be connected to abnormalities in the cortical actin cytoskeleton. These abnormalities also disrupt the localization of the spectraplakin Shot and the microtubule minus-end binding protein Patronin. These two proteins play a critical role in the repolarization of the MT network.

Complete reconstitution of bypass and blocking functions in a minimal artificial Fab-7 insulator from Drosophila bithorax complex.

Boundaries in the bithorax complex (BX-C) delimit autonomous regulatory domains that drive parasegment-specific expression of the Hox genes Ubx, abd-A, and Abd-B The Fab-7 boundary is located between the iab-6 and iab-7 domains and has two key functions: blocking cross-talk between these domains and at the same time promoting communication (boundary bypass) between iab-6 and the Abd-B promoter. Using a replacement strategy, we found that multimerized binding sites for the architectural proteins Pita, Su(Hw), and dCTCF function as conventional insulators and block cross-talk between the iab-6 and iab-7 domains; however, they lack bypass activity, and iab-6 is unable to regulate Abd-B Here we show that an ∼200-bp sequence of dHS1 from the Fab-7 boundary rescues the bypass defects of these multimerized binding sites. The dHS1 sequence is bound in embryos by a large multiprotein complex, Late Boundary Complex (LBC), that contains the zinc finger proteins CLAMP and GAF. Using deletions and mutations in critical GAGAG motifs, we show that bypass activity correlates with the efficiency of recruitment of LBC components CLAMP and GAF to the artificial boundary. These results indicate that LBC orchestrates long-distance communication between the iab-6 regulatory domain and the Abd-B gene, while the Pita, Su(Hw), and dCTCF proteins function to block local cross-talk between the neighboring regulatory domains iab-6 and iab-7.

Boundaries potentiate polycomb response element-mediated silencing.

BACKGROUND: Epigenetic memory plays a critical role in the establishment and maintenance of cell identities in multicellular organisms. Polycomb and trithorax group (PcG and TrxG) proteins are responsible for epigenetic memory, and in flies, they are recruited to specialized DNA regulatory elements termed polycomb response elements (PREs). Previous transgene studies have shown that PREs can silence reporter genes outside of their normal context, often by pairing sensitive (PSS) mechanism; however, their silencing activity is non-autonomous and depends upon the surrounding chromatin context. It is not known why PRE activity depends on the local environment or what outside factors can induce silencing. RESULTS: Using an attP system in Drosophila, we find that the so-called neutral chromatin environments vary substantially in their ability to support the silencing activity of the well-characterized bxdPRE. In refractory chromosomal contexts, factors required for PcG-silencing are unable to gain access to the PRE. Silencing activity can be rescued by linking the bxdPRE to a boundary element (insulator). When placed next to the PRE, the boundaries induce an alteration in chromatin structure enabling factors critical for PcG silencing to gain access to the bxdPRE. When placed at a distance from the bxdPRE, boundaries induce PSS by bringing the bxdPREs on each homolog in close proximity. CONCLUSION: This proof-of-concept study demonstrates that the repressing activity of PREs can be induced or enhanced by nearby boundary elements.

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

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

The bithorax complex iab-7 Polycomb response element has a novel role in the functioning of the Fab-7 chromatin boundary.

Expression of the three bithorax complex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used an in situ replacement strategy to reanalyze the sequences required for the functioning of one of the best-characterized fly boundaries, Fab-7. It was initially identified by a deletion, Fab-71, that transformed parasegment (PS) 11 into a duplicate copy of PS12. Fab-71 deleted four nuclease hypersensitive sites, HS*, HS1, HS2, and HS3, located between the iab-6 and iab-7 regulatory domains. Transgenic and P-element excision experiments mapped the boundary to HS*+HS1+HS2, while HS3 was shown to be the iab-7 Polycomb response element (PRE). Recent replacement experiments showed that HS1 is both necessary and sufficient for boundary activity when HS3 is also present in the replacement construct. Surprisingly, while HS1+HS3 combination has full boundary activity, we discovered that HS1 alone has only minimal function. Moreover, when combined with HS3, only the distal half of HS1, dHS1, is needed. A ~1,000 kD multiprotein complex containing the GAF protein, called the LBC, binds to the dHS1 sequence and we show that mutations in dHS1, that disrupt LBC binding in nuclear extracts, eliminate boundary activity and GAF binding in vivo. HS3 has binding sites for GAF and Pho proteins that are required for PRE silencing. In contrast, HS3 boundary activity only requires the GAF binding sites. LBC binding with HS3 in nuclear extracts, and GAF association in vivo, depend upon the HS3 GAF sites, but not the Pho sites. Consistent with a role for the LBC in HS3 boundary activity, the boundary function of the dHS1+HS3mPho combination is lost when the flies are heterozygous for a mutation in the GAF gene. Taken together, these results reveal a novel function for the iab-7 PREs in chromosome architecture.

Subunits of the PBAP Chromatin Remodeler Are Capable of Mediating Enhancer-Driven Transcription in Drosophila.

The chromatin remodeler SWI/SNF is an important participant in gene activation, functioning predominantly by opening the chromatin structure on promoters and enhancers. Here, we describe its novel mode of action in which SWI/SNF factors mediate the targeted action of an enhancer. We studied the functions of two signature subunits of PBAP subfamily, BAP170 and SAYP, in Drosophila. These subunits were stably tethered to a transgene reporter carrying the hsp70 core promoter. The tethered subunits mediate transcription of the reporter in a pattern that is generated by enhancers close to the insertion site in multiple loci throughout the genome. Both tethered SAYP and BAP170 recruit the whole PBAP complex to the reporter promoter. However, we found that BAP170-dependent transcription is more resistant to the depletion of other PBAP subunits, suggesting that BAP170 may play a more critical role in establishing enhancer-dependent transcription.

Transcriptional quiescence in primordial germ cells.

In most animal species, newly formed primordial germ cells (PGCs) acquire the special characteristics that distinguish them from the surrounding somatic cells. Proper fate specification of the PGCs is coupled with transcriptional quiescence, whether they are segregated by determinative or inductive mechanisms. Inappropriate differentiation of PGCs into somatic cells is thought to be prevented due to repression of RNA polymerase (Pol) II-dependent transcription. In the case of a determinative mode of PGC formation (Drosophila, Caenorhabditis elegans, etc.), there is a broad downregulation of Pol II activity. By contrast, PGCs display only gene-specific repression in organisms that rely on inductive signaling-based mechanism (e.g., mice). In addition to the global block of Pol II activity in PGCs, gene expression can be suppressed in other ways, such as chromatin remodeling and Piwi-mediated RNAi. Here, we discuss the mechanisms responsible for the transcriptionally silent state of PGCs in common experimental animals, such as Drosophila, C. elegans, Danio rerio, Xenopus, and mouse. While a PGC-specific downregulation of transcription is a common feature among these organisms, the diverse nature of underlying mechanisms suggests that this functional trait likely evolved independently on several instances. We discuss the possible biological relevance of these silencing mechanisms vis-a-vis fate determination of PGCs.

Architectural protein Pita cooperates with dCTCF in organization of functional boundaries in Bithorax complex.

Boundaries in the Bithorax complex (BX-C) of Drosophila delimit autonomous regulatory domains that drive parasegment-specific expression of homeotic genes. BX-C boundaries have two crucial functions: they must block crosstalk between adjacent regulatory domains and at the same time facilitate boundary bypass. The C2H2 zinc-finger protein Pita binds to several BX-C boundaries, including Fab-7 and Mcp To study Pita functions, we have used a boundary replacement strategy by substituting modified DNAs for the Fab-7 boundary, which is located between the iab-6 and iab-7 regulatory domains. Multimerized Pita sites block iab-6↔iab-7 crosstalk but fail to support iab-6 regulation of Abd-B (bypass). In the case of Fab-7, we used a novel sensitized background to show that the two Pita-binding sites contribute to its boundary function. Although Mcp is from BX-C, it does not function appropriately when substituted for Fab-7: it blocks crosstalk but does not support bypass. Mutation of the Mcp Pita site disrupts blocking activity and also eliminates dCTCF binding. In contrast, mutation of the Mcp dCTCF site does not affect Pita binding, and this mutant boundary retains partial function.

Role of the ABC transporter Mdr49 in Hedgehog signaling and germ cell migration.

Coalescence of the embryonic gonad in Drosophila melanogaster requires directed migration of primordial germ cells (PGCs) towards somatic gonadal precursor cells (SGPs). It was recently proposed that the ATP-binding cassette (ABC) transporter Mdr49 functions in the embryonic mesoderm to facilitate the transmission of the PGC attractant from the SGPs; however, the precise molecular identity of the Mdr49-dependent guidance signal remained elusive. Employing the loss- and gain-of-function strategies, we show that Mdr49 is a component of the Hedgehog (hh) pathway and it potentiates the signaling activity. This function is direct because in Mdr49 mutant embryos the Hh ligand is inappropriately sequestered in the hh-expressing cells. Our data also suggest that the role of Mdr49 is to provide cholesterol for the correct processing of the Hh precursor protein. Supporting this conclusion, PGC migration defects in Mdr49 embryos are substantially ameliorated by a cholesterol-rich diet.

Boundaries of loop domains (insulators): Determinants of chromosome form and function in multicellular eukaryotes.

Chromosomes in multicellular animals are subdivided into a series of looped domains. In addition to being the underlying principle for organizing the chromatin fiber, looping is critical for processes ranging from gene regulation to recombination and repair. The subdivision of chromosomes into looped domains depends upon a special class of architectural elements called boundaries or insulators. These elements are distributed throughout the genome and are ubiquitous building blocks of chromosomes. In this review, we focus on features of boundaries that are critical in determining the topology of the looped domains and their genetic properties. We highlight the properties of fly boundaries that are likely to have an important bearing on the organization of looped domains in vertebrates, and discuss the functional consequences of the observed similarities and differences.