Genome organization around nuclear speckles drives mRNA splicing efficiency.

The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.

SPIDR: a highly multiplexed method for mapping RNA-protein interactions uncovers a potential mechanism for selective translational suppression upon cellular stress.

RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know what specific RNAs most RBPs bind. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs split-pool barcoding coupled with antibody-bead barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR reliably identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. Using SPIDR, we explored changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5'-untranslated regions of specific translationally repressed mRNAs only upon mTOR inhibition. This observation provides a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR has the potential to revolutionize our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation by enabling rapid, de novo discovery of RNA-protein interactions at an unprecedented scale.

Aging-associated HELIOS deficiency in naive CD4+ T cells alters chromatin remodeling and promotes effector cell responses.

Immune aging combines cellular defects in adaptive immunity with the activation of pathways causing a low-inflammatory state. Here we examined the influence of age on the kinetic changes in the epigenomic and transcriptional landscape induced by T cell receptor (TCR) stimulation in naive CD4+ T cells. Despite attenuated TCR signaling in older adults, TCR activation accelerated remodeling of the epigenome and induced transcription factor networks favoring effector cell differentiation. We identified increased phosphorylation of STAT5, at least in part due to aberrant IL-2 receptor and lower HELIOS expression, as upstream regulators. Human HELIOS-deficient, naive CD4+ T cells, when transferred into human-synovium-mouse chimeras, infiltrated tissues more efficiently. Inhibition of IL-2 or STAT5 activity in T cell responses of older adults restored the epigenetic response pattern to the one seen in young adults. In summary, reduced HELIOS expression in non-regulatory naive CD4+ T cells in older adults directs T cell fate decisions toward inflammatory effector cells that infiltrate tissue.

ChIP-DIP: A multiplexed method for mapping hundreds of proteins to DNA uncovers diverse regulatory elements controlling gene expression.

Gene expression is controlled by the dynamic localization of thousands of distinct regulatory proteins to precise regions of DNA. Understanding this cell-type specific process has been a goal of molecular biology for decades yet remains challenging because most current DNA-protein mapping methods study one protein at a time. To overcome this, we developed ChIP-DIP (ChIP Done In Parallel), a split-pool based method that enables simultaneous, genome-wide mapping of hundreds of diverse regulatory proteins in a single experiment. We demonstrate that ChIP-DIP generates highly accurate maps for all classes of DNA-associated proteins, including histone modifications, chromatin regulators, transcription factors, and RNA Polymerases. Using these data, we explore quantitative combinations of protein localization on genomic DNA to define distinct classes of regulatory elements and their functional activity. Our data demonstrate that ChIP-DIP enables the generation of 'consortium level', context-specific protein localization maps within any molecular biology lab.

Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.

Mammalian genomes are organized into three-dimensional DNA structures called A/B compartments that are associated with transcriptional activity/inactivity. However, whether these structures are simply correlated with gene expression or are permissive/impermissible to transcription has remained largely unknown because we lack methods to measure DNA organization and transcription simultaneously. Recently, we developed RNA & DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial organization of RNA and DNA. Here we show that RD-SPRITE measures genomic structure surrounding nascent pre-mRNAs and maps their spatial contacts. We find that transcription occurs within B compartments-with multiple active genes simultaneously colocalizing within the same B compartment-and at genes proximal to nucleoli. These results suggest that localization near or within nuclear structures thought to be inactive does not preclude transcription and that active transcription can occur throughout the nucleus. In general, we anticipate RD-SPRITE will be a powerful tool for exploring relationships between genome structure and transcription.

The transcription factor RFX5 coordinates antigen-presenting function and resistance to nutrient stress in synovial macrophages.

Tissue macrophages (Mϕ) are essential effector cells in rheumatoid arthritis (RA), contributing to autoimmune tissue inflammation through diverse effector functions. Their arthritogenic potential depends on their proficiency to survive in the glucose-depleted environment of the inflamed joint. Here, we identify a mechanism that links metabolic adaptation to nutrient stress with the efficacy of tissue Mϕ to activate adaptive immunity by presenting antigen to tissue-invading T cells. Specifically, Mϕ populating the rheumatoid joint produce and respond to the small cytokine CCL18, which protects against cell death induced by glucose withdrawal. Mechanistically, CCL18 induces the transcription factor RFX5 that selectively upregulates glutamate dehydrogenase 1 (GLUD1), thus enabling glutamate utilization to support energy production. In parallel, RFX5 enhances surface expression of HLA-DR molecules, promoting Mϕ-dependent expansion of antigen-specific T cells. These data place CCL18 at the top of a RFX5-GLUD1 survival pathway and couple adaptability to nutrient conditions in the tissue environment to antigen-presenting function in autoimmune tissue inflammation.

RNA promotes the formation of spatial compartments in the nucleus.

RNA, DNA, and protein molecules are highly organized within three-dimensional (3D) structures in the nucleus. Although RNA has been proposed to play a role in nuclear organization, exploring this has been challenging because existing methods cannot measure higher-order RNA and DNA contacts within 3D structures. To address this, we developed RNA & DNA SPRITE (RD-SPRITE) to comprehensively map the spatial organization of RNA and DNA. These maps reveal higher-order RNA-chromatin structures associated with three major classes of nuclear function: RNA processing, heterochromatin assembly, and gene regulation. These data demonstrate that hundreds of ncRNAs form high-concentration territories throughout the nucleus, that specific RNAs are required to recruit various regulators into these territories, and that these RNAs can shape long-range DNA contacts, heterochromatin assembly, and gene expression. These results demonstrate a mechanism where RNAs form high-concentration territories, bind to diffusible regulators, and guide them into compartments to regulate essential nuclear functions.

Succinyl-CoA Ligase Deficiency in Pro-inflammatory and Tissue-Invasive T Cells.

Autoimmune T cells in rheumatoid arthritis (RA) have a defect in mitochondrial oxygen consumption and ATP production. Here, we identified suppression of the GDP-forming ß subunit of succinate-CoA ligase (SUCLG2) as an underlying abnormality. SUCLG2-deficient T cells reverted the tricarboxylic acid (TCA) cycle from the oxidative to the reductive direction, accumulated α-ketoglutarate, citrate, and acetyl-CoA (AcCoA), and differentiated into pro-inflammatory effector cells. In AcCoAhi RA T cells, tubulin acetylation stabilized the microtubule cytoskeleton and positioned mitochondria in a perinuclear location, resulting in cellular polarization, uropod formation, T cell migration, and tissue invasion. In the tissue, SUCLG2-deficient T cells functioned as cytokine-producing effector cells and were hyperinflammatory, a defect correctable by replenishing the enzyme. Preventing T cell tubulin acetylation by tubulin acetyltransferase knockdown was sufficient to inhibit synovitis. These data link mitochondrial failure and AcCoA oversupply to autoimmune tissue inflammation.

Early reprogramming regulators identified by prospective isolation and mass cytometry.

In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of non-productive and staggered productive intermediates arise at different reprogramming time points. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells, prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that, during reprogramming, cells progressively lose donor cell identity and gradually acquire iPS cell properties. Here we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen, we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200, that are absent in both fibroblasts and iPS cells. Single-cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic, reprogramming phase. Expression profiling reveals early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1 (also known as Zfp42), Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus represent some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.