The histone chaperone CAF-1 safeguards somatic 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

Control of a neuronal morphology program by an RNA-binding zinc finger protein, Unkempt

Cellular morphology is an essential determinant of cellular function in all kingdoms of life, yet little is known about how cell shape is controlled. Here we describe a molecular program that controls the early morphology of neurons through a metazoan-specific zinc finger protein, Unkempt. Depletion of Unkempt in mouse embryos disrupts the shape of migrating neurons, while ectopic expression confers neuronal-like morphology to cells of different nonneuronal lineages. We found that Unkempt is a sequence-specific RNA-binding protein and identified its precise binding sites within coding regions of mRNAs linked to protein metabolism and trafficking. RNA binding is required for Unkempt-induced remodeling of cellular shape and is directly coupled to a reduced production of the encoded proteins. These findings link post-transcriptional regulation of gene expression with cellular shape and have general implications for the development and disease of multicellular organisms

A Serial shRNA Screen for Roadblocks to Reprogramming Identifies the Protein Modifier SUMO2

Summary The generation of induced pluripotent stem cells (iPSCs) from differentiated cells following forced expression of OCT4, KLF4, SOX2, and C-MYC (OKSM) is slow and inefficient, suggesting that transcription factors have to overcome somatic barriers that resist cell fate change. Here, we performed an unbiased serial shRNA enrichment screen to identify potent repressors of somatic cell reprogramming into iPSCs. This effort uncovered the protein modifier SUMO2 as one of the strongest roadblocks to iPSC formation. Depletion of SUMO2 both enhances and accelerates reprogramming, yielding transgene-independent, chimera-competent iPSCs after as little as 38 hr of OKSM expression. We further show that the SUMO2 pathway acts independently of exogenous C-MYC expression and in parallel with small-molecule enhancers of reprogramming. Importantly, suppression of SUMO2 also promotes the generation of human iPSCs. Together, our results reveal sumoylation as a crucial post-transcriptional mechanism that resists the acquisition of pluripotency from fibroblasts using defined factors

PANDORA-seq expands the repertoire of regulatory small RNAs by overcoming RNA modifications

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

Cell Fate Decisions in the Wake of Histone H3 Deposition.

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

The nexus between RNA-binding proteins and their effectors

RNA-binding proteins (RBPs) regulate essentially every event in the lifetime of an RNA molecule, from its production to its destruction. Whereas much has been learned about RNA sequence specificity and general functions of individual RBPs, the ways in which numerous RBPs instruct a much smaller number of effector molecules, that is, the core engines of RNA processing, as to where, when and how to act remain largely speculative. Here, we survey the known modes of communication between RBPs and their effectors with a particular focus on converging RBP-effector interactions and their roles in reducing the complexity of RNA networks. We discern the emerging unifying principles and discuss their utility in our understanding of RBP function, regulation of biological processes and contribution to human disease

Canonical and alternate functions of the microRNA biogenesis machinery

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