We provide evidence for an essential role of chromatin-associated nucleoporins in gene expression of a multicellular organism that seems to be critical for establishing developmental transcription programs and transcriptional response to cellular stress. Surprisingly, the role of Nups in gene regulation is carried out to a great extent by an intranuclear, NE-independent pool of NPC components. This has major implications for our understanding of nuclear organization and its proposed roles in gene regulation. Our data suggest that in metazoa there is no strict requirement of genes to move to peripheral pores, because Nups have the capacity to interact with genomic sites independently of the NE. It remains to be determined whether these intranuclear chromatin-binding Nups shuttle between genomic sites and the nuclear periphery, which would be consistent with the dynamic behavior of some pore components (Rabut et al., 2004
). The former possibility is supported by previously reported observations that the dynamic shuttling of mammalian Nup98 and Nup153 is dependent on active transcription by RNAP II and I (Griffis et al., 2002
; Griffis et al., 2004
). It is tempting to speculate that the mobility of NPC components may establish a mechanism of communication between sites of production of mRNA and sites of its final exit.
Alternatively, the observed role of Nups in transcription could represent NPC-independent sub-complexes inside the nucleus and thus reflect NPC-independent functions of Nups, similar to those of the Nup107/160 complex in kinetochore function in mitosis (Belgareh et al., 2001
; Loiodice et al., 2004
). Importantly, the role of Sec13 at transcription sites is not linked to its role in secretion since the knockdown of COPII components did not affect transcription (not shown). Intranuclear Nups have been described in different cell types, however their role remained enigmatic. Recent reports of tissue-specific expression of certain Nups in post-mitotic cells, in which NPC assembly is generally absent (D’Angelo et al., 2009
), might reflect cell type specific differences in Nup-chromatin interactions.
Given their early recruitment that either precedes or coincides with transcriptional initiation, Sec13 and Nup98 appear to be instrumental in establishing a transcriptionally active state of a particular subset of genes. This novel role of Sec13 and Nup98 in transcriptional induction agrees with a previous study in yeast, which reported an interaction between Nups and promoters of genes during transcriptional initiation and proposed that transient contact with pore proteins may be a general feature of gene activation (Schmid et al., 2006
). On the other hand, other binding sites of NPC identified by the ChIP approach (Casolari et al., 2005
; Casolari et al., 2004
) were preferentially located at the 3′ ends of active genes in yeast. This function is likely reflected in the differential recruitment and transcription elongation dependency of FG-repeat Nups, which seem to be involved in later events of transcription.
In yeast, one explanation for the dual 5′ and 3′ end binding was suggested to be the presence of both gene ends at the NPC through interactions with different Nups, which would result in DNA looping. Such chromatin loops at the NPC were projected to serve as sites of assembly for transcription and mRNA processing machinery (Blobel, 1985
; Menon et al., 2005
) and/or to delimit active chromatin domains (Casolari et al., 2005
). This role may be conserved by metazoan Nups at intranuclear active sites, where they can serve as a platform for co-regulated assembly of transcription machinery and mRNA export factors. The FG repeats, found in Nup98 and the mAb414-reactive Nups, are known to interact with mRNA export receptors, such as Mex67/TAP (Strasser et al., 2000
), while the particular GLFG type of FG domain of Nup98 can associate with the CBP/p300 histone acetyltransferase (Kasper et al., 1999
). Since Sec13 has been found to be very stable at the NPC (Rabut et al., 2004
), it is unlikely that Sec13 itself shuttles but rather may exist in a separate intranuclear population. It is possible then that Sec13 either at the pore or at a transcribing locus inside the nucleus serves as the docking point for Nup98, which in turn brings about further recruitment of transcriptional regulators or mRNA export or other RNA processing factors. In this manner, the communication between intranuclear transcription sites and the NE-embedded nuclear pores, suggested above, may be carried out by a shuttling Nup98 component.
Furthermore, other reports revealed that Nups are able to interact with chromatin regions enriched in repressive histone modifications or in non-active genes (Brown et al., 2008
; Casolari et al., 2004
). In support of these observations, our findings demonstrate that other nucleoporins such as Nup88 may be targeted to regions in the genome where they may participate in alternative gene regulatory processes. Significantly, both Nup88 (Agudo et al., 2004
) and Nup98 have been implicated in tumorigenesis. Particularly, genomic fusions of Nup98 to transcription regulators have been shown to be the underlying mutations behind multiple types of leukemia (Slape and Aplan, 2004
). The uncovered association of Nup88 and Nup98 with chromatin opens new directions for understanding the roles of NPC components in cancer.