SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs

摘要

class="head no_bottom_margin" id="sec1title">IntroductionMammalian interphase chromosomes are organized into topologically constrained chromatin domains (), which are responsive to transcription () and local gene density (). Gene-poor genomic regions have a compact large-scale chromatin structure, while regions rich in genes and transcriptional activity have a more decompacted structure (, , ). Previously, we suggested that transcription and topoisomerase activities, that occur at the gene level, alter local topology to form supercoiling domains () and these correspond to structures seen by Hi-C (). It is unclear, however, how these processes could impact on large-scale chromatin structures.In contrast to mitosis, where topoisomerases and condensin play a central role in scaffolding chromatin (), the molecular underpinnings of interphase domains are poorly characterized () and their functional impact remains unknown. A “nuclear matrix” consisting of insoluble proteins and RNA particles was proposed to organize interphase chromatin architecture (, ), maintain chromosome territories (), enhance gene expression, and provide a platform for nuclear processes, but chromatin mobility in vivo (, ) and the lack of a stable nucleoprotein structure in live cells undermined the concept (href="#bib29" rid="bib29" class=" bibr popnode">Hancock, 2000). However, the structural contribution of RNA to chromatin organization remains undisputed: a large proportion of chromatin by mass corresponds to RNA (href="#bib31" rid="bib31" class=" bibr popnode">Holmes et al., 1972), mostly belonging to the loosely termed chromatin-associated RNA (caRNA) class. The functional roles of caRNAs are hinted by the observation that they are stably associated with interphase chromosome territories (href="#bib20" rid="bib20" class=" bibr popnode">Fey et al., 1986, href="#bib27" rid="bib27" class=" bibr popnode">Hall and Lawrence, 2016) and their disruption leads to chromatin condensation (href="#bib28" rid="bib28" class=" bibr popnode">Hall et al., 2014). The molecular basis for this is unknown, but it is thought heterogeneous ribonucleoprotein particles (hnRNPs) provide a docking platform to associate with nascent transcripts (href="#bib41" rid="bib41" class=" bibr popnode">Melé and Rinn, 2016) while caRNAs might influence chromatin structure (href="#bib9" rid="bib9" class=" bibr popnode">Caudron-Herger and Rippe, 2012). Recent studies have been unable to find specific species of RNA, similar to XIST, which could regulate large-scale chromatin structure, suggesting instead that diverse caRNAs transiently interact with chromatin, forming a dynamic compartment (href="#bib41" rid="bib41" class=" bibr popnode">Melé and Rinn, 2016). However, a refinement of this model would require insight into how the interaction of proteins with caRNAs can regulate chromatin structure.Scaffold attachment factor A (SAF-A), also known as heterogeneous ribonucleoprotein U (HNRNP-U) (href="#bib35" rid="bib35" class=" bibr popnode">Kiledjian and Dreyfuss, 1992, href="#bib50" rid="bib50" class=" bibr popnode">Romig et al., 1992), is an abundant protein reported to bind scaffold attachment regions (href="#bib24" rid="bib24" class=" bibr popnode">Göhring et al., 1997) and involved in several cellular processes such as pre-mRNA splicing (href="#bib59" rid="bib59" class=" bibr popnode">Xiao et al., 2012), accumulation at DNA damage sites (href="#bib8" rid="bib8" class=" bibr popnode">Britton et al., 2014) and Xist-mediated transcriptional silencing (href="#bib40" rid="bib40" class=" bibr popnode">McHugh et al., 2015). Structurally, SAF-A contains a low complexity RNA-binding RGG repeat and an ATP-binding AAA⁺ domain, known to facilitate the assembly (href="#bib17" rid="bib17" class=" bibr popnode">Erzberger and Berger, 2006) and operation of diverse protein and nucleoprotein machines. Other well-characterized AAA⁺ domain-containing proteins, such as replication factor C (RFC) and DnaA, oligomerize through their AAA domains, often with nucleic acids, to form higher molecular weight structures. We characterized SAF-A activity to understand the relationship between its structure and function in regulating chromatin architecture.We are able to demonstrate that SAF-A regulates transcriptionally active large-scale chromatin structures in human cells. Using functional mutants of SAF-A, we dissect the underlying molecular mechanisms to show that SAF-A can cycle from a monomeric to a homo-oligomeric state through ATP binding and caRNAs; concomitantly, SAF-A oligomerization drives chromatin decompaction while monomerization compacts large-scale chromatin organization.We suggest that SAF-A interacts with caRNAs to form a chromatin mesh (href="#bib45" rid="bib45" class=" bibr popnode">Nozawa and Gilbert, 2014), and unlike the historical concept of a nuclear matrix, is highly responsive to ongoing transcription and can undergo dynamic cycles of assembly and disassembly. Surprisingly, a gross change in chromatin structure has limited effect on transcription indicating that gene-level chromatin structure is important for regulating transcription, rather than large-scale chromatin architecture. Although changes in large-scale chromatin structure do not influence gene expression, an alteration in the chromatin landscape had a dramatic effect on genome stability; loss or mutation of SAF-A triggered a DNA damage response and chromosomal instability.To unify the structural and enzymatic aspects of SAF-A function, we speculate that SAF-A/RNA interactions will drive the formation of local chromatin domains or micro-bodies (href="#bib7" rid="bib7" class=" bibr popnode">Brackley et al., 2016), while its role in regulating large-scale chromatin structures will partition the genome into functionally diverse segments. This process is essential for maintaining genome stability and could provide a constraint to maintain clusters of genes together in the genome during evolution (href="#bib21" rid="bib21" class=" bibr popnode">Ghanbarian and Hurst, 2015).