Core Components of the Nuclear Pore Bind Distinct States of Chromatin and Contribute to Polycomb Repression.

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. Here, we report high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. Our 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, we identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. Our 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.

Metazoan Nuclear Pores Provide a Scaffold for Poised Genes and Mediate Induced Enhancer-Promoter Contacts.

Nuclear pore complex components (Nups) have been implicated in transcriptional regulation, yet what regulatory steps are controlled by metazoan Nups remains unclear. We identified the presence of multiple Nups at promoters, enhancers, and insulators in the Drosophila genome. In line with this binding, we uncovered a functional role for Nup98 in mediating enhancer-promoter looping at ecdysone-inducible genes. These genes were found to be stably associated with nuclear pores before and after activation. Although changing levels of Nup98 disrupted enhancer-promoter contacts, it did not affect ongoing transcription but instead compromised subsequent transcriptional activation or transcriptional memory. In support of the enhancer-looping role, we found Nup98 to gain and retain physical interactions with architectural proteins upon stimulation with ecdysone. Together, our data identify Nups as a class of architectural proteins for enhancers and supports a model in which animal genomes use the nuclear pore as an organizing scaffold for inducible poised genes.

Mip6 binds directly to the Mex67 UBA domain to maintain low levels of Msn2/4 stress-dependent mRNAs.

RNA-binding proteins (RBPs) participate in all steps of gene expression, underscoring their potential as regulators of RNA homeostasis. We structurally and functionally characterize Mip6, a four-RNA recognition motif (RRM)-containing RBP, as a functional and physical interactor of the export factor Mex67. Mip6-RRM4 directly interacts with the ubiquitin-associated (UBA) domain of Mex67 through a loop containing tryptophan 442. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci under stress. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation experiments show preferential binding of Mip6 to mRNAs regulated by the stress-response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affects their export and expression levels. Additionally, Mip6 interacts physically and/or functionally with proteins with a role in mRNA metabolism and transcription such as Rrp6, Xrn1, Sgf73, and Rpb1. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4-dependent transcripts through its direct interaction with the Mex67 UBA domain.

Nup98 promotes antiviral gene expression to restrict RNA viral infection in Drosophila.

In response to infection, the innate immune system rapidly activates an elaborate and tightly orchestrated gene expression program to induce critical antimicrobial genes. While many key players in this program have been identified in disparate biological systems, it is clear that there are additional uncharacterized mechanisms at play. Our previous studies revealed that a rapidly-induced antiviral gene expression program is active against disparate human arthropod-borne viruses in Drosophila. Moreover, one-half of this program is regulated at the level of transcriptional pausing. Here we found that Nup98, a virus-induced gene, was antiviral against a panel of viruses both in cells and adult flies since its depletion significantly enhanced viral infection. Mechanistically, we found that Nup98 promotes antiviral gene expression in Drosophila at the level of transcription. Expression profiling revealed that the virus-induced activation of 36 genes was abrogated upon loss of Nup98; and we found that a subset of these Nup98-dependent genes were antiviral. These Nup98-dependent virus-induced genes are Cdk9-dependent and translation-independent suggesting that these are rapidly induced primary response genes. Biochemically, we demonstrate that Nup98 is directly bound to the promoters of virus-induced genes, and that it promotes occupancy of the initiating form of RNA polymerase II at these promoters, which are rapidly induced on viral infection to restrict human arboviruses in insects.

A novel role for Sem1 and TREX-2 in transcription involves their impact on recruitment and H2B deubiquitylation activity of SAGA.

Transcription and mRNA export are linked processes. However, the molecular mechanisms of this coordination are not clear. Sus1 (hENY2) participates in this coordination as part of two protein complexes: SAGA, a transcriptional co-activator; TREX-2, which functions in mRNA biogenesis and export. Here, we investigate the coordinated action of SAGA and TREX-2 required for gene expression. We demonstrate that TREX-2 subunit Sem1 also participates in transcription activation. Like Sus1, Sem1 is required for the induction of ARG1 and GAL1, these being SAGA-regulated genes. Chromatin immunoprecipitations show that proper recruitment of certain SAGA subunits to the GAL1 promoter depends on Sem1. Notably, both in vivo and in vitro analyses reveal that Sem1 influences SAGA-dependent histone H2B deubiquitylation. Most of these phenotypes are also found to depend on another TREX-2 subunit, Thp1. These results unveil a new role for Sem1 in the activation of the SAGA-dependent gene GAL1 and influencing H2B deubiquitylation. Our work provides insights into a novel functional relationship between Sem1 and the SAGA complex.

SUS1 introns are required for efficient mRNA nuclear export in yeast.

Efficient coupling between mRNA synthesis and export is essential for gene expression. Sus1/ENY2, a component of the SAGA and TREX-2 complexes, is involved in both transcription and mRNA export. While most yeast genes lack introns, we previously reported that yeast SUS1 bears two. Here we show that this feature is evolutionarily conserved and critical for Sus1 function. We determine that while SUS1 splicing is inefficient, it responds to cellular conditions, and intronic mutations either promoting or blocking splicing lead to defects in mRNA export and cell growth. Consistent with this, we find that an intron-less SUS1 only partially rescues sus1Δ phenotypes. Remarkably, splicing of each SUS1 intron is also affected by the presence of the other and by SUS1 exonic sequences. Moreover, by following SUS1 RNA and protein levels we establish that nonsense-mediated decay (NMD) pathway and the splicing factor Mud2 both play a role in SUS1 expression. Our data (and those of the accompanying work by Hossain et al.) provide evidence of the involvement of splicing, translation, and decay in the regulation of early events in mRNP biogenesis; and imply the additional requirement for a balance in splicing isoforms from a single gene.

Nup98-dependent transcriptional memory is established independently of transcription.

Cellular ability to mount an enhanced transcriptional response upon repeated exposure to external cues is termed transcriptional memory, which can be maintained epigenetically through cell divisions and can depend on a nuclear pore component Nup98. The majority of mechanistic knowledge on transcriptional memory has been derived from bulk molecular assays. To gain additional perspective on the mechanism and contribution of Nup98 to memory, we used single-molecule RNA FISH (smFISH) to examine the dynamics of transcription in Drosophila cells upon repeated exposure to the steroid hormone ecdysone. We combined smFISH with mathematical modeling and found that upon hormone exposure, cells rapidly activate a low-level transcriptional response, but simultaneously begin a slow transition into a specialized memory state characterized by a high rate of expression. Strikingly, our modeling predicted that this transition between non-memory and memory states is independent of the transcription stemming from initial activation. We confirmed this prediction experimentally by showing that inhibiting transcription during initial ecdysone exposure did not interfere with memory establishment. Together, our findings reveal that Nup98's role in transcriptional memory is to stabilize the forward rate of conversion from low to high expressing state, and that induced genes engage in two separate behaviors - transcription itself and the establishment of epigenetically propagated transcriptional memory.

The nuclear pore complex and the genome: organizing and regulatory principles.

The nuclear pore complex (NPC) is a massive nuclear envelope-embedded protein complex, the canonical function of which is to mediate selective nucleocytoplasmic transport. In addition to its transport function, the NPC has been shown to interact with the underlying chromatin and to influence both activating and repressive gene regulatory processes, contributing to the establishment and the epigenetic maintenance of cell identity. In this review, we discuss diverse gene regulatory functions of NPC components and emerging mechanisms underlying these functions, including roles in genome architecture, transcription complex assembly, chromatin remodeling, and coordination of transcription and mRNA export. These functional roles highlight the importance of the NPC as a nuclear scaffold directing genome organization and function.

Correct dosage of X chromosome transcription is controlled by a nuclear pore component.

Dosage compensation in Drosophila melanogaster involves a 2-fold transcriptional upregulation of the male X chromosome, which relies on the X-chromosome-binding males-specific lethal (MSL) complex. However, how such 2-fold precision is accomplished remains unclear. Here, we show that a nuclear pore component, Mtor, is involved in setting the correct levels of transcription from the male X chromosome. Using larval tissues, we demonstrate that the depletion of Mtor results in selective upregulation at MSL targets of the male X, beyond the required 2-fold. Mtor and MSL components interact genetically, and depletion of Mtor can rescue the male lethality phenotype of MSL components. Using RNA fluorescence in situ hybridization (FISH) analysis and nascent transcript sequencing, we find that the effect of Mtor is not due to defects in mRNA export but occurs at the level of nascent transcription. These findings demonstrate a physiological role for Mtor in the process of dosage compensation, as a transcriptional attenuator of X chromosome gene expression.

A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism.

BACKGROUND: Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts. RESULTS: In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'-->3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association. CONCLUSIONS: In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function.

A tale of coupling, Sus1 function in transcription and mRNA export.

Eukaryotic cells have evolved unveiling a tightly coupled scenario between transcription, RNA biogenesis and mRNA transport out of the nucleus. Gene expression regulation relies partially on the action of some protein complexes, which ensure that all these processes work co-ordinately in order to achieve an optimal performance. The evolutionarily conserved export factor Sus1 plays a key role in coupling transcription activation with mRNA export as part of the nuclear pore associated complex TREX2 and the transcriptional coactivator SAGA. Being a factor shared by these two complexes, Sus1 physically link the activated GAL1 gene to the nuclear periphery, which constitute a determinant of regulation. An exciting new study has expanded our knowledge of Sus1 function. Sus1 is required for transcription elongation, it's recruited to coding regions and interacts with different factors during mRNA biogenesis. Moreover, SAGA and TREX2 are necessary for efficient Sus1 recruitment to chromatin. These results underscore the key role of Sus1 coordinating gene transcription coupled to mRNA export.

The mRNA export factor Sus1 is involved in Spt/Ada/Gcn5 acetyltransferase-mediated H2B deubiquitinylation through its interaction with Ubp8 and Sgf11.

Sus1 acts in nuclear mRNA export via its association with the nuclear pore-associated Sac3-Thp1-Cdc31 complex. In addition, Sus1 plays a role in transcription through its interaction with the Spt/Ada/Gcn5 acetyltransferase (SAGA) complex. Here, we have analyzed function and interaction of Sus1 within the SAGA complex. We demonstrate that Sus1 is involved in the SAGA-dependent histone H2B deubiquitinylation and maintenance of normal H3 methylation levels. By deletion analyses, we show that binding of Sus1 to SAGA depends on the deubiquitinylating enzyme Ubp8 and Sgf11. Moreover, a stable subcomplex between Sus1, Sgf11, and Ubp8 could be dissociated from SAGA under high salt conditions. In vivo recruitment of Sus1 to the activated GAL1 promoter depends on Ubp8 and vice versa. In addition, histones coenrich during SAGA purification in a Sus1-Sgf11-Ubp8-dependent way. Interestingly, sgf11 deletion enhances the mRNA export defect observed in sus1delta cells. Thus, the Sus1-Sgf11-Ubp8 module could work at the junction between SAGA-dependent transcription and nuclear mRNA export.

Nuclear pores in genome architecture and enhancer function.

Nuclear genome architecture relies on interactions between the genome and various nuclear scaffolds. One such a nuclear scaffold is the nuclear pore complex (NPC), which in addition to its nuclear transport function, can interact with underlying chromatin. In particular, NPCs have been recently reported to associate with a number of enhancers and superenhancers in metazoan genomes, and select NPC components have been shown to promote the formation of specific genomic loops. Here, we provide a brief overview of current models of enhancer function, and discuss recent evidence that NPCs bind enhancers and contribute to topological genome organization. We also examine possible models of how gene and enhancer targeting to NPCs may contribute to tissue-specific genome architecture and expression programs, including the possibility that NPCs may promote phase separation of transcriptional compartments.

Chromatin targeting of nuclear pore proteins induces chromatin decondensation.

Nuclear pore complexes have emerged in recent years as chromatin-binding nuclear scaffolds, able to influence target gene expression. However, how nucleoporins (Nups) exert this control remains poorly understood. Here we show that ectopically tethering Drosophila Nups, especially Sec13, to chromatin is sufficient to induce chromatin decondensation. This decondensation is mediated through chromatin-remodeling complex PBAP, as PBAP is both robustly recruited by Sec13 and required for Sec13-induced decondensation. This phenomenon is not correlated with localization of the target locus to the nuclear periphery, but is correlated with robust recruitment of Nup Elys. Furthermore, we identified a biochemical interaction between endogenous Sec13 and Elys with PBAP, and a role for endogenous Elys in global as well as gene-specific chromatin decompaction. Together, these findings reveal a functional role and mechanism for specific nuclear pore components in promoting an open chromatin state.

Nuclear pores as versatile platforms for gene regulation.

Functional compartmentalization of the genome relies on interactions between genomic regions and various nuclear scaffolds and macro-complexes. The Nuclear Pore Complex (NPC) is a large nuclear envelope-embedded protein complex, which creates a highly regulated transport channel between the nucleus and the cytoplasm. In addition to its central role in transport, the NPC has been linked to genome compartmentalization via binding to specific regions of the genome and association with gene regulatory machinery. Although originally proposed to preferentially associate with active genes, the NPC has now been implicated in both gene activating and gene silencing processes. Here, we review recent findings that highlight the roles of various components of the NPC in transcriptional activation, transcriptional memory, heterochromatin formation, post-transcriptional gene silencing and RNA processing. Together, these findings suggest that the nuclear pore is utilized as a regulatory platform for a number of distinct gene expression processes and further point to its central role in setting up particular expression environments on the genomic template.

Nucleoporin Nup98 associates with Trx/MLL and NSL histone-modifying complexes and regulates Hox gene expression.

The nuclear pore complex is a transport channel embedded in the nuclear envelope and made up of 30 different components termed nucleoporins (Nups). In addition to their classical role in transport, a subset of Nups has a conserved role in the regulation of transcription via direct binding to chromatin. The molecular details of this function remain obscure, and it is unknown how metazoan Nups are recruited to their chromatin locations or what transcription steps they regulate. Here, we demonstrate genome-wide and physical association between Nup98 and histone-modifying complexes MBD-R2/NSL [corrected] and Trx/MLL. Importantly, we identify a requirement for MBD-R2 in recruitment of Nup98 to many of its genomic target sites. Consistent with its interaction with the Trx/MLL complex, Nup98 is shown to be necessary for Hox gene expression in developing fly tissues. These findings introduce roles of Nup98 in epigenetic regulation that may underlie the basis of oncogenicity of Nup98 fusions in leukemia.

Sus1 is recruited to coding regions and functions during transcription elongation in association with SAGA and TREX2.

Gene transcription, RNA biogenesis, and mRNA transport constitute a complicated process essential for all eukaryotic cells. The transcription/export factor Sus1 plays a key role in coupling transcription activation with mRNA export, and it resides in both the SAGA and TREX2 complexes. Moreover, Sus1 is responsible for GAL1 gene gating at the nuclear periphery, which is important for its transcriptional status. Here, we show that Sus1 is required during transcription elongation and is associated with the elongating form of RNA Polymerase II (RNAP II) phosphorylated on Ser5 and Ser2 of the C-terminal domain (CTD). In addition, Sus1 copurifies with the essential mRNA export factors Yra1 and Mex67, which bind to the mRNA cotranscriptionally. Consistently, ChIP analysis reveals that Sus1 is present at coding regions dependent on transcription in a manner stimulated by Kin28-dependent CTD phosphorylation. Strikingly, eliminating the TREX2 component Sac3 or the SAGA subunit Ubp8 partially impairs Sus1 targeting to coding sequences and upstream activating sequences (UAS). We found, unexpectedly, that Sgf73 is necessary for association of Sus1 with both SAGA and TREX2, and that its absence dramatically reduces Sus1 occupancy of UAS and ORF sequences. Our results reveal that Sus1 plays a key role in coordinating gene transcription and mRNA export by working at the interface between the SAGA and TREX2 complexes during transcription elongation.