Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates ...biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs-mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)-that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation.
•Recent RNAi screens have identified CAF-1 as a barrier to cell fate change in various cellular and developmental systems.•Effects of CAF-1 suppression are cell context-dependent, facilitating either ...differentiation, dedifferentiation or lineage conversion.•CAF-1 suppression influences cellular plasticity by altering local or global chromatin states.
During embryonic development, cells become progressively restricted in their differentiation potential. This is thought to be regulated by dynamic changes in chromatin structure and associated modifications, which act together to stabilize distinct specialized cell lineages. Remarkably, differentiated cells can be experimentally reprogrammed to a stem cell-like state or to alternative lineages. Thus, cellular reprogramming provides a valuable platform to study the mechanisms that normally safeguard cell identity and uncover factors whose manipulation facilitates cell fate transitions. Recent work has identified the chromatin assembly factor complex CAF-1 as a potent barrier to cellular reprogramming. In addition, CAF-1 has been implicated in the reversion of pluripotent cells to a totipotent-like state and in various lineage conversion paradigms, suggesting that modulation of CAF-1 levels may endow cells with a developmentally more plastic state. Here, we review these exciting results, discuss potential mechanisms and speculate on the possibility of exploiting chromatin assembly pathways to manipulate cell identity.
Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further ...elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.
Cell fate commitment is driven by dynamic changes in chromatin architecture and activity of lineage-specific transcription factors (TFs). The chromatin assembly factor-1 (CAF-1) is a histone ...chaperone that regulates chromatin architecture by facilitating nucleosome assembly during DNA replication. Accumulating evidence supports a substantial role of CAF-1 in cell fate maintenance, but the mechanisms by which CAF-1 restricts lineage choice remain poorly understood. Here, we investigate how CAF-1 influences chromatin dynamics and TF activity during lineage differentiation. We show that CAF-1 suppression triggers rapid differentiation of myeloid stem and progenitor cells into a mixed lineage state. We find that CAF-1 sustains lineage fidelity by controlling chromatin accessibility at specific loci, and limiting the binding of ELF1 TF at newly-accessible diverging regulatory elements. Together, our findings decipher key traits of chromatin accessibility that sustain lineage integrity and point to a powerful strategy for dissecting transcriptional circuits central to cell fate commitment.
Dicer is an RNase III-family nuclease that initiates RNA interference (RNAi) and related phenomena by generation of the small RNAs that determine the specificity of these gene silencing pathways. We ...have previously shown that Dicer is essential for mammalian development, with Dicer-deficient mice dying at embryonic day 7.5 with a lack of detectable multipotent stem cells. To permit a more detailed investigation of the biological roles of Dicer, we have generated embryonic stem cell lines in which their single Dicer gene can be conditionally inactivated. As expected, Dicer loss compromises maturation of microRNAs and leads to a defect in gene silencing triggered by long dsRNAs. However, the absence of Dicer does not affect the ability of small interfering RNAs to repress gene expression. Of interest, Dicer loss does compromise the proliferation of ES cells, possibly rationalizing the phenotype previously observed in Dicer-null animals. Dicer loss also affects the abundance of transcripts from mammalian centromeres but does so without a pronounced affect on histone modification status at pericentric repeats or methylation of centromeric DNA. These studies provide a conditional model of RNAi deficiency in mammals that will permit the dissection of the biological roles of the RNAi machinery in cultured mammalian cells.
The canonical microRNA (miRNA) biogenesis pathway requires two RNaseIII enzymes: Drosha and Dicer. To understand their functions in mammals in vivo, we engineered mice with germline or ...tissue-specific inactivation of the genes encoding these two proteins. Changes in proteomic and transcriptional profiles that were shared in Dicer- and Drosha-deficient mice confirmed the requirement for both enzymes in canonical miRNA biogenesis. However, deficiency in Drosha or Dicer did not always result in identical phenotypes, suggesting additional functions. We found that, in early-stage thymocytes, Drosha recognizes and directly cleaves many protein-coding messenger RNAs (mRNAs) with secondary stem-loop structures. In addition, we identified a subset of miRNAs generated by a Dicer-dependent but Drosha-independent mechanism. These were distinct from previously described mirtrons. Thus, in mammalian cells, Dicer is required for the biogenesis of multiple classes of miRNAs. Together, these findings extend the range of function of RNaseIII enzymes beyond canonical miRNA biogenesis, and help explain the nonoverlapping phenotypes caused by Drosha and Dicer deficiency.
The chromatin state of pluripotency genes has been studied extensively in embryonic stem cells (ESCs) and differentiated cells, but their potential interactions with other parts of the genome remain ...largely unexplored. Here, we identified a genome-wide, pluripotency-specific interaction network around the Nanog promoter by adapting circular chromosome conformation capture sequencing. This network was rearranged during differentiation and restored in induced pluripotent stem cells. A large fraction of Nanog-interacting loci were bound by Mediator or cohesin in pluripotent cells. Depletion of these proteins from ESCs resulted in a disruption of contacts and the acquisition of a differentiation-specific interaction pattern prior to obvious transcriptional and phenotypic changes. Similarly, the establishment of Nanog interactions during reprogramming often preceded transcriptional upregulation of associated genes, suggesting a causative link. Our results document a complex, pluripotency-specific chromatin “interactome” for Nanog and suggest a functional role for long-range genomic interactions in the maintenance and induction of pluripotency.
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•The Nanog promoter forms a pluripotency-specific, genome-wide chromatin interactome•Nanog interactions in ESCs are enriched for active marks and pluripotency factors•Mediator and cohesin are essential for the maintenance of the Nanog interactome•Many Nanog interactions form before increased gene expression during reprogramming
The Nanog promoter interacts with the rest of the genome in an unexpectedly complex pluripotency-specific chromatin interaction network that depends on Mediator and cohesin.
How RNA-binding proteins (RBPs) convey regulatory instructions to the core effectors of RNA processing is unclear. Here, we document the existence and functions of a multivalent RBP-effector ...interface. We show that the effector interface of a conserved RBP with an essential role in metazoan development, Unkempt, is mediated by a novel type of 'dual-purpose' peptide motifs that can contact two different surfaces of interacting proteins. Unexpectedly, we find that the multivalent contacts do not merely serve effector recruitment but are required for the accuracy of RNA recognition by Unkempt. Systems analyses reveal that multivalent RBP-effector contacts can repurpose the principal activity of an effector for a different function, as we demonstrate for the reuse of the central eukaryotic mRNA decay factor CCR4-NOT in translational control. Our study establishes the molecular assembly and functional principles of an RBP-effector interface.
The life span of a mammalian mRNA is determined, in part, by the binding of regulatory proteins and small RNA-guided complexes. The conserved endonuclease activity of Argonaute2 requires extensive ...complementarity between a small RNA and its target and is not used by animal microRNAs, which pair with their targets imperfectly. Here we investigate the endonucleolytic function of Ago2 and other nucleases by transcriptome-wide profiling of mRNA cleavage products retaining 5
′ phosphate groups in mouse embryonic stem cells (mESCs). We detect a prominent signature of Ago2-dependent cleavage events and validate several such targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in site-specific mRNA cleavage. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events that functionally regulate mRNA levels in mESCs, including one in the Dgcr8 mRNA. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals.
► Transcriptome-wide RACE identifies sites of endonucleolytic cleavage in mRNAs ► Ago2 catalyzes numerous miRNA-guided mRNA cleavages ► Drosha appears to have many mRNA substrates, which it may cleave directly ► Ago2- and Drosha-independent cleavages imply involvement of additional nucleases
An expanding repertoire of histone variants and specialized histone chaperone partners showcases the versatility of nucleosome assembly during different cellular processes. Recent research has ...suggested an integral role of nucleosome assembly pathways in both maintaining cell identity and influencing cell fate decisions during development and normal homeostasis. Mutations and altered expression profiles of histones and corresponding histone chaperone partners are associated with developmental defects and cancer. Here, we discuss the spatiotemporal deposition mechanisms of the Histone H3 variants and their influence on mammalian cell fate during development. We focus on H3 given its profound effect on nucleosome stability and its recently characterized deposition pathways. We propose that differences in deposition of H3 variants are largely dependent on the phase of the cell cycle and cellular potency but are also affected by cellular stress and changes in cell fate. We also discuss the utility of modern technologies in dissecting the spatiotemporal control of H3 variant deposition, and how this could shed light on the mechanisms of cell identity maintenance and lineage commitment. The current knowledge and future studies will help us better understand how organisms employ nucleosome dynamics in health, disease, and aging. Ultimately, these pathways can be manipulated to induce cell fate change in a therapeutic setting depending on the cellular context.
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