Shank3/PROSAP2 gene mutations are associated with cognitive impairment ranging from mental retardation to autism. Shank3 is a large scaffold postsynaptic density protein implicated in dendritic spines and synapse formation; however, its specific functions have not been clearly demonstrated. We have used RNAi to knockdown Shank3 expression in neuronal cultures and showed that this treatment specifically reduced the synaptic expression of the metabotropic glutamate receptor 5 (mGluR5), but did not affect the expression of other major synaptic proteins. The functional consequence of Shank3 RNAi knockdown was impaired signaling via mGluR5, as shown by reduction in ERK1/2 and CREB phosphorylation induced by stimulation with (S)-3,5-dihydroxyphenylglycine (DHPG) as the agonist of mGluR5 receptors, impaired mGluR5-dependent synaptic plasticity (DHPG-induced long-term depression), and impaired mGluR5-dependent modulation of neural network activity. We also found morphological abnormalities in the structure of synapses (spine number, width, and length) and impaired glutamatergic synaptic transmission, as shown by reduction in the frequency of miniature excitatory postsynaptic currents (mEPSC). Notably, pharmacological augmentation of mGluR5 activity using 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide as the positive allosteric modulator of these receptors restored mGluR5-dependent signaling (DHPG-induced phosphorylation of ERK1/2) and normalized the frequency of mEPSCs in Shank3-knocked down neurons. These data demonstrate that a deficit in mGluR5-mediated intracellular signaling in Shank3 knockdown neurons can be compensated by 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)-benzamide; this raises the possibility that pharmacological augmentation of mGluR5 activity represents a possible new therapeutic approach for patients with Shank3 mutations.

Brief expression of pluripotency-associated factors such as Oct4, Klf4, Sox2 and c-Myc (OKSM), in combination with differentiation-inducing signals, has been reported to trigger transdifferentiation of fibroblasts into other cell types. Here we show that OKSM expression in mouse fibroblasts gives rise to both induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) under conditions previously shown to induce only iNSCs. Fibroblast-derived iNSC colonies silenced retroviral transgenes and reactivated silenced X chromosomes, both hallmarks of pluripotent stem cells. Moreover, lineage tracing with an Oct4-CreER labeling system demonstrated that virtually all iNSC colonies originated from cells transiently expressing Oct4, whereas ablation of Oct4(+) cells prevented iNSC formation. Lastly, an alternative transdifferentiation cocktail that lacks Oct4 and was reportedly unable to support induced pluripotency yielded iPSCs and iNSCs carrying the Oct4-CreER-derived lineage label. Together, these data suggest that iNSC generation from fibroblasts using OKSM and other pluripotency-related reprogramming factors requires passage through a transient iPSC state.

The molecular mechanisms underlying specification from embryonic stem cells (ESCs) and maintenance of neural progenitor cells (NPCs) are largely unknown. Recently, we reported that the Zuotin-related factor 1 (Zrf1) is necessary for chromatin displacement of the Polycomb-repressive complex 1 (PRC1). We found that Zrf1 is required for NPC specification from ESCs and that it promotes the expression of NPC markers, including the key regulator Pax6. Moreover, Zrf1 is essential to establish and maintain Wnt ligand expression levels, which are necessary for NPC self-renewal. Reactivation of proper Wnt signaling in Zrf1-depleted NPCs restores Pax6 expression and the self-renewal capacity. ESC-derived NPCs in vitro resemble most of the characteristics of the self-renewing NPCs located in the developing embryonic cortex, which are termed radial glial cells (RGCs). Depletion of Zrf1 in vivo impairs the expression of key self-renewal regulators and Wnt ligand genes in RGCs. Thus, we demonstrate that Zrf1 plays an essential role in NPC generation and maintenance.

Here, we report DNA methylation and hydroxymethylation dynamics at nucleotide resolution using C/EBPα-enhanced reprogramming of B cells into induced pluripotent cells (iPSCs). We observed successive waves of hydroxymethylation at enhancers, concomitant with a decrease in DNA methylation, suggesting active demethylation. Consistent with this finding, ablation of the DNA demethylase Tet2 almost completely abolishes reprogramming. C/EBPα, Klf4, and Tfcp2l1 each interact with Tet2 and recruit the enzyme to specific DNA sites. During reprogramming, some of these sites maintain high levels of 5hmC, and enhancers and promoters of key pluripotency factors become demethylated as early as 1 day after Yamanaka factor induction. Surprisingly, methylation changes precede chromatin opening in distinct chromatin regions, including Klf4 bound sites, revealing a pioneer factor activity associated with alternation in DNA methylation. Rapid changes in hydroxymethylation similar to those in B cells were also observed during compound-accelerated reprogramming of fibroblasts into iPSCs, highlighting the generality of our observations.

Somatic cell reprogramming is the process that allows differentiated cells to revert to a pluripotent state. In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we study the dynamics of alternative splicing changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells and compare them to those occurring during reprogramming of mouse embryonic fibroblasts.

Here, we describe how the speed of C/EBPα-induced B cell to macrophage transdifferentiation (BMT) can be regulated, using both mouse and human models. The identification of a mutant of C/EBPα (C/EBPαR35A) that greatly accelerates BMT helped to illuminate the mechanism. Thus, incoming C/EBPα binds to PU.1, an obligate partner expressed in B cells, leading to the release of PU.1 from B cell enhancers, chromatin closing and silencing of the B cell program. Released PU.1 redistributes to macrophage enhancers newly occupied by C/EBPα, causing chromatin opening and activation of macrophage genes. All these steps are accelerated by C/EBPαR35A, initiated by its increased affinity for PU.1. Wild-type C/EBPα is methylated by Carm1 at arginine 35 and the enzyme's perturbations modulate BMT velocity as predicted from the observations with the mutant. Increasing the proportion of unmethylated C/EBPα in granulocyte/macrophage progenitors by inhibiting Carm1 biases the cell's differentiation toward macrophages, suggesting that cell fate decision velocity and lineage directionality are closely linked processes.

Forced transcription factor expression can transdifferentiate somatic cells into other specialised cell types or reprogram them into induced pluripotent stem cells (iPSCs) with variable efficiency. To better understand the heterogeneity of these processes, we used single-cell RNA sequencing to follow the transdifferentation of murine pre-B cells into macrophages as well as their reprogramming into iPSCs. Even in these highly efficient systems, there was substantial variation in the speed and path of fate conversion. We predicted and validated that these differences are inversely coupled and arise in the starting cell population, with Mychigh large pre-BII cells transdifferentiating slowly but reprogramming efficiently and Myclow small pre-BII cells transdifferentiating rapidly but failing to reprogram. Strikingly, differences in Myc activity predict the efficiency of reprogramming across a wide range of somatic cell types. These results illustrate how single cell expression and computational analyses can identify the origins of heterogeneity in cell fate conversion processes.

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.

Forced expression of reprogramming factors can convert somatic cells into induced pluripotent stem cells (iPSCs). Here we studied genome topology dynamics during reprogramming of different somatic cell types with highly distinct genome conformations. We find large-scale topologically associated domain (TAD) repositioning and alterations of tissue-restricted genomic neighborhoods and chromatin loops, effectively erasing the somatic-cell-specific genome structures while establishing an embryonic stem-cell-like 3D genome. Yet, early passage iPSCs carry topological hallmarks that enable recognition of their cell of origin. These hallmarks are not remnants of somatic chromosome topologies. Instead, the distinguishing topological features are acquired during reprogramming, as we also find for cell-of-origin-dependent gene expression patterns.

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.

In conformational disorders, it is not evident which amyloid aggregates affect specific molecular mechanisms or cellular pathways, which cause disease because of their quantity and mechanical features and which states in aggregate formation are pathogenic. Due to the increasing consensus that prefibrillar oligomers play a major role in conformational diseases, there is a growing interest in understanding the characteristics of metastable polypeptide associations.

B cell progenitor acute lymphoblastic leukemia (B-ALL) treatment has been revolutionized by T cell-based immunotherapies-including chimeric antigen receptor T cell therapy (CAR-T) and the bispecific T cell engager therapeutic, blinatumomab-targeting surface glycoprotein CD19. Unfortunately, many patients with B-ALL will fail immunotherapy due to 'antigen escape'-the loss or absence of leukemic CD19 targeted by anti-leukemic T cells. In the present study, we utilized a genome-wide CRISPR-Cas9 screening approach to identify modulators of CD19 abundance on human B-ALL blasts. These studies identified a critical role for the transcriptional activator ZNF143 in CD19 promoter activation. Conversely, the RNA-binding protein, NUDT21, limited expression of CD19 by regulating CD19 messenger RNA polyadenylation and stability. NUDT21 deletion in B-ALL cells increased the expression of CD19 and the sensitivity to CD19-specific CAR-T and blinatumomab. In human B-ALL patients treated with CAR-T and blinatumomab, upregulation of NUDT21 mRNA coincided with CD19 loss at disease relapse. Together, these studies identify new CD19 modulators in human B-ALL.

The generation of myotubes from fibroblasts upon forced MyoD expression is a classic example of transcription factor-induced reprogramming. We recently discovered that additional modulation of signaling pathways with small molecules facilitates reprogramming to more primitive induced myogenic progenitor cells (iMPCs). Here, we dissected the transcriptional and epigenetic dynamics of mouse fibroblasts undergoing reprogramming to either myotubes or iMPCs using a MyoD-inducible transgenic model. Induction of MyoD in fibroblasts combined with small molecules generated Pax7+ iMPCs with high similarity to primary muscle stem cells. Analysis of intermediate stages of iMPC induction revealed that extinction of the fibroblast program preceded induction of the stem cell program. Moreover, key stem cell genes gained chromatin accessibility prior to their transcriptional activation, and these regions exhibited a marked loss of DNA methylation dependent on the Tet enzymes. In contrast, myotube generation was associated with few methylation changes, incomplete and unstable reprogramming, and an insensitivity to Tet depletion. Finally, we showed that MyoD's ability to bind to unique bHLH targets was crucial for generating iMPCs but dispensable for generating myotubes. Collectively, our analyses elucidate the role of MyoD in myogenic reprogramming and derive general principles by which transcription factors and signaling pathways cooperate to rewire cell identity.

Cell fate transitions involve rapid gene expression changes and global chromatin remodeling, yet the underlying regulatory pathways remain incompletely understood. Here, we identified the RNA-processing factor Nudt21 as a novel regulator of cell fate change using transcription-factor-induced reprogramming as a screening assay. Suppression of Nudt21 enhanced the generation of induced pluripotent stem cells, facilitated transdifferentiation into trophoblast stem cells, and impaired differentiation of myeloid precursors and embryonic stem cells, suggesting a broader role for Nudt21 in cell fate change. We show that Nudt21 directs differential polyadenylation of over 1,500 transcripts in cells acquiring pluripotency, although only a fraction changed protein levels. Remarkably, these proteins were strongly enriched for chromatin regulators, and their suppression neutralized the effect of Nudt21 during reprogramming. Collectively, our data uncover Nudt21 as a novel post-transcriptional regulator of cell fate and establish a direct, previously unappreciated link between alternative polyadenylation and chromatin signaling.

Chromosomal architecture is known to influence gene expression, yet its role in controlling cell fate remains poorly understood. Reprogramming of somatic cells into pluripotent stem cells (PSCs) by the transcription factors (TFs) OCT4, SOX2, KLF4 and MYC offers an opportunity to address this question but is severely limited by the low proportion of responding cells. We have recently developed a highly efficient reprogramming protocol that synchronously converts somatic into pluripotent stem cells. Here, we used this system to integrate time-resolved changes in genome topology with gene expression, TF binding and chromatin-state dynamics. The results showed that TFs drive topological genome reorganization at multiple architectural levels, often before changes in gene expression. Removal of locus-specific topological barriers can explain why pluripotency genes are activated sequentially, instead of simultaneously, during reprogramming. Together, our results implicate genome topology as an instructive force for implementing transcriptional programs and cell fate in mammals.

Post-transcriptional mechanisms have the potential to influence complex changes in gene expression, yet their role in cell fate transitions remains largely unexplored. Here, we show that suppression of the RNA helicase DDX6 endows human and mouse primed embryonic stem cells (ESCs) with a differentiation-resistant, "hyper-pluripotent" state, which readily reprograms to a naive state resembling the preimplantation embryo. We further demonstrate that DDX6 plays a key role in adult progenitors where it controls the balance between self-renewal and differentiation in a context-dependent manner. Mechanistically, DDX6 mediates the translational suppression of target mRNAs in P-bodies. Upon loss of DDX6 activity, P-bodies dissolve and release mRNAs encoding fate-instructive transcription and chromatin factors that re-enter the ribosome pool. Increased translation of these targets impacts cell fate by rewiring the enhancer, heterochromatin, and DNA methylation landscapes of undifferentiated cell types. Collectively, our data establish a link between P-body homeostasis, chromatin organization, and stem cell potency.

The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is lengthy and inefficient. The development of a reprogramming system that allows rapid and synchronous reprogramming to pluripotency is imperative for understanding the mechanism of iPSC formation and for future therapeutic applications. We have recently reported that a short expression in mouse primary B cells of the transcription factor C/EBPα before the induction of pluripotency factors increases the iPSC reprogramming efficiency >100-fold, involving 95% of the cells within a week. Here we present a dataset containing the time course of gene expression during this process as determined by microarray and RNA-seq techniques.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) upon overexpression of OCT4, KLF4, SOX2 and c-MYC (OKSM) provides a powerful system to interrogate basic mechanisms of cell fate change. However, iPSC formation with standard methods is typically protracted and inefficient, resulting in heterogeneous cell populations. We show that exposure of OKSM-expressing cells to both ascorbic acid and a GSK3-β inhibitor (AGi) facilitates more synchronous and rapid iPSC formation from several mouse cell types. AGi treatment restored the ability of refractory cell populations to yield iPSC colonies, and it attenuated the activation of developmental regulators commonly observed during the reprogramming process. Moreover, AGi supplementation gave rise to chimera-competent iPSCs after as little as 48 h of OKSM expression. Our results offer a simple modification to the reprogramming protocol, facilitating iPSC induction at unparalleled efficiencies and enabling dissection of the underlying mechanisms in more homogeneous cell populations.

Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display naïve ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. Characterization of the key molecular signalling pathways revealed FGF-iPSCs to depend on the Activin/Nodal and FGF pathways, while signalling through the JAK-STAT pathway is not required for FGF-iPS cell maintenance. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions.

Despite significant advances in the identification of specific genes and pathways important in the onset and progression of colorectal cancer (CRC), mechanistic insight into the relationship between driver and susceptibility genes is needed. In this paper, we systematically explore physical interactions between causative and putative CRC susceptibility genes to reveal the molecular mechanisms involved in tumor biology. In total, we identify 622 high-confidence protein-protein interactions between 42 CRC causative and 65 candidate susceptibility genes. Among the latter, 28 are located in the CRCS9 loci, related to the etiology of CRC, and 17 are co-expressed with well-established CRC drivers, which makes them excellent candidates for further functional studies. Moreover, we find a high degree of functional coherence between connected driver and susceptibility genes, which indicates that our network-based strategy is useful to gain insight into the underlying mechanisms of those proteins with unknown roles in CRC.