Genome-wide screening identifies Trim33 as an essential regulator of dendritic cell differentiation.

The development of dendritic cells (DCs), including antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs), is controlled by the growth factor Flt3 ligand (Flt3L) and its receptor Flt3. We genetically dissected Flt3L-driven DC differentiation using CRISPR-Cas9-based screening. Genome-wide screening identified multiple regulators of DC differentiation including subunits of TSC and GATOR1 complexes, which restricted progenitor growth but enabled DC differentiation by inhibiting mTOR signaling. An orthogonal screen identified the transcriptional repressor Trim33 (TIF-1γ) as a regulator of DC differentiation. Conditional targeting in vivo revealed an essential role of Trim33 in the development of all DCs, but not of monocytes or granulocytes. In particular, deletion of Trim33 caused rapid loss of DC progenitors, pDCs, and the cross-presenting cDC1 subset. Trim33-deficient Flt3+ progenitors up-regulated pro-inflammatory and macrophage-specific genes but failed to induce the DC differentiation program. Collectively, these data elucidate mechanisms that control Flt3L-driven differentiation of the entire DC lineage and identify Trim33 as its essential regulator.

Identification of molecular signatures defines the differential proteostasis response in induced spinal and cranial motor neurons.

Amyotrophic lateral sclerosis damages proteostasis, affecting spinal and upper motor neurons earlier than a subset of cranial motor neurons. To aid disease understanding, we exposed induced cranial and spinal motor neurons (iCrMNs and iSpMNs) to proteotoxic stress, under which iCrMNs showed superior survival, quantifying the transcriptome and proteome for >8,200 genes at 0, 12, and 36 h. Two-thirds of the proteome showed cell-type differences. iSpMN-enriched proteins related to DNA/RNA metabolism, and iCrMN-enriched proteins acted in the endoplasmic reticulum (ER)/ER chaperone complex, tRNA aminoacylation, mitochondria, and the plasma/synaptic membrane, suggesting that iCrMNs expressed higher levels of proteins supporting proteostasis and neuronal function. When investigating the increased proteasome levels in iCrMNs, we showed that the activity of the 26S proteasome, but not of the 20S proteasome, was higher in iCrMNs than in iSpMNs, even after a stress-induced decrease. We identified Ublcp1 as an iCrMN-specific regulator of the nuclear 26S activity.

Essential transcription factors for induced neuron differentiation.

Neurogenins are proneural transcription factors required to specify neuronal identity. Their overexpression in human pluripotent stem cells rapidly produces cortical-like neurons with spiking activity and, because of this, they have been widely adopted for human neuron disease models. However, we do not fully understand the key downstream regulatory effectors responsible for driving neural differentiation. Here, using inducible expression of NEUROG1 and NEUROG2, we identify transcription factors (TFs) required for directed neuronal differentiation by combining expression and chromatin accessibility analyses with a pooled in vitro CRISPR-Cas9 screen targeting all ~1900 TFs in the human genome. The loss of one of these essential TFs (ZBTB18) yields few MAP2-positive neurons. Differentiated ZBTB18-null cells have radically altered gene expression, leading to cytoskeletal defects and stunted neurites and spines. In addition to identifying key downstream TFs for neuronal differentiation, our work develops an integrative multi-omics and TFome-wide perturbation platform to rapidly characterize essential TFs for the differentiation of any human cell type.

Novel Chromatin Insulating Activities Uncovered upon Eliminating Known Insulators in vivo.

CCCTC-binding factor (CTCF) and MAZ are recognized insulators required for shielding repressed posterior genes from active anterior genes within the Hox clusters during motor neuron (MN) differentiation. CTCF and MAZ interact independently with cohesin and regulate three-dimensional genome organization. Here, we followed cohesin re-location upon CTCF and MAZ depletion in mouse embryonic stem cells (mESCs) to identify novel insulators. Cohesin relocated to DNA motifs for various transcription factors, including PATZ1 and other zinc finger proteins (ZNFs). Moreover, PATZ1 and ZNFs co-localized with CTCF, MAZ, and cohesin with apparent overlapping specificity as dictated by the site to be insulated. Similar to CTCF and MAZ, PATZ1 interacted with RAD21. Patz1 KO mESCs exhibited altered global gene expression. While the absence of MAZ impacts anterior CTCF-boundaries as shown previously 1 , Patz1 KO led to derepression of posterior Hox genes, resulting in cervicothoracic transformation of motor neuron (MN) fate during differentiation. These findings point to a varied, combinatorial binding of known and newly defined accessory factors as being critical for positional identity and cellular fate determination during differentiation.

ISSCR Education Committee syllabus and learning guide for enhancing stem cell literacy.

The rapidly evolving stem cell field puts much stress on developing educational resources. The ISSCR Education Committee has created a flexible stem cell syllabus rooted in core concepts to facilitate stem cell literacy. The free syllabus will be updated regularly to maintain accuracy and relevance.

Foxa2 and Pet1 Direct and Indirect Synergy Drive Serotonergic Neuronal Differentiation.

Neuronal programming by forced expression of transcription factors (TFs) holds promise for clinical applications of regenerative medicine. However, the mechanisms by which TFs coordinate their activities on the genome and control distinct neuronal fates remain obscure. Using direct neuronal programming of embryonic stem cells, we dissected the contribution of a series of TFs to specific neuronal regulatory programs. We deconstructed the Ascl1-Lmx1b-Foxa2-Pet1 TF combination that has been shown to generate serotonergic neurons and found that stepwise addition of TFs to Ascl1 canalizes the neuronal fate into a diffuse monoaminergic fate. The addition of pioneer factor Foxa2 represses Phox2b to induce serotonergic fate, similar to in vivo regulatory networks. Foxa2 and Pet1 appear to act synergistically to upregulate serotonergic fate. Foxa2 and Pet1 co-bind to a small fraction of genomic regions but mostly bind to different regulatory sites. In contrast to the combinatorial binding activities of other programming TFs, Pet1 does not strictly follow the Foxa2 pioneer. These findings highlight the challenges in formulating generalizable rules for describing the behavior of TF combinations that program distinct neuronal subtypes.

Synthetic regulatory reconstitution reveals principles of mammalian Hox cluster regulation.

Precise Hox gene expression is crucial for embryonic patterning. Intra-Hox transcription factor binding and distal enhancer elements have emerged as the major regulatory modules controlling Hox gene expression. However, quantifying their relative contributions has remained elusive. Here, we introduce "synthetic regulatory reconstitution," a conceptual framework for studying gene regulation, and apply it to the HoxA cluster. We synthesized and delivered variant rat HoxA clusters (130 to 170 kilobases) to an ectopic location in the mouse genome. We found that a minimal HoxA cluster recapitulated correct patterns of chromatin remodeling and transcription in response to patterning signals, whereas the addition of distal enhancers was needed for full transcriptional output. Synthetic regulatory reconstitution could provide a generalizable strategy for deciphering the regulatory logic of gene expression in complex genomes.

Capybara: A computational tool to measure cell identity and fate transitions.

Measuring cell identity in development, disease, and reprogramming is challenging as cell types and states are in continual transition. Here, we present Capybara, a computational tool to classify discrete cell identity and intermediate "hybrid" cell states, supporting a metric to quantify cell fate transition dynamics. We validate hybrid cells using experimental lineage tracing data to demonstrate the multi-lineage potential of these intermediate cell states. We apply Capybara to diagnose shortcomings in several cell engineering protocols, identifying hybrid states in cardiac reprogramming and off-target identities in motor neuron programming, which we alleviate by adding exogenous signaling factors. Further, we establish a putative in vivo correlate for induced endoderm progenitors. Together, these results showcase the utility of Capybara to dissect cell identity and fate transitions, prioritizing interventions to enhance the efficiency and fidelity of stem cell engineering.

The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by repressing pro-differentiation genes.

In pluripotent cells, a delicate activation-repression balance maintains pro-differentiation genes ready for rapid activation. The identity of transcription factors (TFs) that specifically repress pro-differentiation genes remains obscure. By targeting ∼1,700 TFs with CRISPR loss-of-function screen, we found that ZBTB11 and ZFP131 are required for embryonic stem cell (ESC) pluripotency. ESCs without ZBTB11 or ZFP131 lose colony morphology, reduce proliferation rate, and upregulate transcription of genes associated with three germ layers. ZBTB11 and ZFP131 bind proximally to pro-differentiation genes. ZBTB11 or ZFP131 loss leads to an increase in H3K4me3, negative elongation factor (NELF) complex release, and concomitant transcription at associated genes. Together, our results suggest that ZBTB11 and ZFP131 maintain pluripotency by preventing premature expression of pro-differentiation genes and present a generalizable framework to maintain cellular potency.

CRISPR and biochemical screens identify MAZ as a cofactor in CTCF-mediated insulation at Hox clusters.

CCCTC-binding factor (CTCF) is critical to three-dimensional genome organization. Upon differentiation, CTCF insulates active and repressed genes within Hox gene clusters. We conducted a genome-wide CRISPR knockout (KO) screen to identify genes required for CTCF-boundary activity at the HoxA cluster, complemented by biochemical approaches. Among the candidates, we identified Myc-associated zinc-finger protein (MAZ) as a cofactor in CTCF insulation. MAZ colocalizes with CTCF at chromatin borders and, similar to CTCF, interacts with the cohesin subunit RAD21. MAZ KO disrupts gene expression and local contacts within topologically associating domains. Similar to CTCF motif deletions, MAZ motif deletions lead to derepression of posterior Hox genes immediately after CTCF boundaries upon differentiation, giving rise to homeotic transformations in mouse. Thus, MAZ is a factor contributing to appropriate insulation, gene expression and genomic architecture during development.

PRC1 sustains the integrity of neural fate in the absence of PRC2 function.

Polycomb repressive complexes (PRCs) 1 and 2 maintain stable cellular memories of early fate decisions by establishing heritable patterns of gene repression. PRCs repress transcription through histone modifications and chromatin compaction, but their roles in neuronal subtype diversification are poorly defined. We found that PRC1 is essential for the specification of segmentally restricted spinal motor neuron (MN) subtypes, while PRC2 activity is dispensable to maintain MN positional identities during terminal differentiation. Mutation of the core PRC1 component Ring1 in mice leads to increased chromatin accessibility and ectopic expression of a broad variety of fates determinants, including Hox transcription factors, while neuronal class-specific features are maintained. Loss of MN subtype identities in Ring1 mutants is due to the suppression of Hox-dependent specification programs by derepressed Hox13 paralogs (Hoxa13, Hoxb13, Hoxc13, Hoxd13). These results indicate that PRC1 can function in the absence of de novo PRC2-dependent histone methylation to maintain chromatin topology and postmitotic neuronal fate.

An expansion of the non-coding genome and its regulatory potential underlies vertebrate neuronal diversity.

Proper assembly and function of the nervous system requires the generation of a uniquely diverse population of neurons expressing a cell-type-specific combination of effector genes that collectively define neuronal morphology, connectivity, and function. How countless partially overlapping but cell-type-specific patterns of gene expression are controlled at the genomic level remains poorly understood. Here we show that neuronal genes are associated with highly complex gene regulatory systems composed of independent cell-type- and cell-stage-specific regulatory elements that reside in expanded non-coding genomic domains. Mapping enhancer-promoter interactions revealed that motor neuron enhancers are broadly distributed across the large chromatin domains. This distributed regulatory architecture is not a unique property of motor neurons but is employed throughout the nervous system. The number of regulatory elements increased dramatically during the transition from invertebrates to vertebrates, suggesting that acquisition of new enhancers might be a fundamental process underlying the evolutionary increase in cellular complexity.

De novo assembly and delivery to mouse cells of a 101 kb functional human gene.

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.

An interpretable bimodal neural network characterizes the sequence and preexisting chromatin predictors of induced transcription factor binding.

BACKGROUND: Transcription factor (TF) binding specificity is determined via a complex interplay between the transcription factor's DNA binding preference and cell type-specific chromatin environments. The chromatin features that correlate with transcription factor binding in a given cell type have been well characterized. For instance, the binding sites for a majority of transcription factors display concurrent chromatin accessibility. However, concurrent chromatin features reflect the binding activities of the transcription factor itself and thus provide limited insight into how genome-wide TF-DNA binding patterns became established in the first place. To understand the determinants of transcription factor binding specificity, we therefore need to examine how newly activated transcription factors interact with sequence and preexisting chromatin landscapes. RESULTS: Here, we investigate the sequence and preexisting chromatin predictors of TF-DNA binding by examining the genome-wide occupancy of transcription factors that have been induced in well-characterized chromatin environments. We develop Bichrom, a bimodal neural network that jointly models sequence and preexisting chromatin data to interpret the genome-wide binding patterns of induced transcription factors. We find that the preexisting chromatin landscape is a differential global predictor of TF-DNA binding; incorporating preexisting chromatin features improves our ability to explain the binding specificity of some transcription factors substantially, but not others. Furthermore, by analyzing site-level predictors, we show that transcription factor binding in previously inaccessible chromatin tends to correspond to the presence of more favorable cognate DNA sequences. CONCLUSIONS: Bichrom thus provides a framework for modeling, interpreting, and visualizing the joint sequence and chromatin landscapes that determine TF-DNA binding dynamics.

Differential abilities to engage inaccessible chromatin diversify vertebrate Hox binding patterns.

Although Hox genes encode for conserved transcription factors (TFs), they are further divided into anterior, central and posterior groups based on their DNA-binding domain similarity. The posterior Hox group expanded in the deuterostome clade and patterns caudal and distal structures. We aimed to address how similar Hox TFs diverge to induce different positional identities. We studied Hox TF DNA-binding and regulatory activity during an in vitro motor neuron differentiation system that recapitulates embryonic development. We found diversity in the genomic binding profiles of different Hox TFs, even among the posterior group paralogs that share similar DNA-binding domains. These differences in genomic binding were explained by differing abilities to bind to previously inaccessible sites. For example, the posterior group HOXC9 had a greater ability to bind occluded sites than the posterior HOXC10, producing different binding patterns and driving differential gene expression programs. From these results, we propose that the differential abilities of posterior Hox TFs to bind to previously inaccessible chromatin drive patterning diversification.This article has an associated 'The people behind the papers' interview.

Simple Method to Quantify Protein Abundances from 1000 Cells.

The rise of single-cell transcriptomics has created an urgent need for similar approaches that use a minimal number of cells to quantify expression levels of proteins. We integrated and optimized multiple recent developments to establish a proteomics workflow to quantify proteins from as few as 1000 mammalian stem cells. The method uses chemical peptide labeling, does not require specific equipment other than cell lysis tools, and quantifies >2500 proteins with high reproducibility. We validated the method by comparing mouse embryonic stem cells and in vitro differentiated motor neurons. We identify differentially expressed proteins with small fold changes and a dynamic range in abundance similar to that of standard methods. Protein abundance measurements obtained with our protocol compared well to corresponding transcript abundance and to measurements using standard inputs. The protocol is also applicable to other systems, such as fluorescence-activated cell sorting (FACS)-purified cells from the tunicate Ciona. Therefore, we offer a straightforward and accurate method to acquire proteomics data from minimal input samples.

Cell Reprogramming: The Many Roads to Success.

Cellular reprogramming experiments from somatic cell types have demonstrated the plasticity of terminally differentiated cell states. Recent efforts in understanding the mechanisms of cellular reprogramming have begun to elucidate the differentiation trajectories along the reprogramming processes. In this review, we focus mainly on direct reprogramming strategies by transcription factors and highlight the variables that contribute to cell fate conversion outcomes. We review key studies that shed light on the cellular and molecular mechanisms by investigating differentiation trajectories and alternative cell states as well as transcription factor regulatory activities during cell fate reprogramming. Finally, we highlight a few concepts that we believe require attention, particularly when measuring the success of cell reprogramming experiments.

Stem cell-derived cranial and spinal motor neurons reveal proteostatic differences between ALS resistant and sensitive motor neurons.

In amyotrophic lateral sclerosis (ALS) spinal motor neurons (SpMN) progressively degenerate while a subset of cranial motor neurons (CrMN) are spared until late stages of the disease. Using a rapid and efficient protocol to differentiate mouse embryonic stem cells (ESC) to SpMNs and CrMNs, we now report that ESC-derived CrMNs accumulate less human (h)SOD1 and insoluble p62 than SpMNs over time. ESC-derived CrMNs have higher proteasome activity to degrade misfolded proteins and are intrinsically more resistant to chemically-induced proteostatic stress than SpMNs. Chemical and genetic activation of the proteasome rescues SpMN sensitivity to proteostatic stress. In agreement, the hSOD1 G93A mouse model reveals that ALS-resistant CrMNs accumulate less insoluble hSOD1 and p62-containing inclusions than SpMNs. Primary-derived ALS-resistant CrMNs are also more resistant than SpMNs to proteostatic stress. Thus, an ESC-based platform has identified a superior capacity to maintain a healthy proteome as a possible mechanism to resist ALS-induced neurodegeneration.

Proneural factors Ascl1 and Neurog2 contribute to neuronal subtype identities by establishing distinct chromatin landscapes.

Developmental programs that generate the astonishing neuronal diversity of the nervous system are not completely understood and thus present a major challenge for clinical applications of guided cell differentiation strategies. Using direct neuronal programming of embryonic stem cells, we found that two main vertebrate proneural factors, Ascl1 and neurogenin 2 (Neurog2), induce different neuronal fates by binding to largely different sets of genomic sites. Their divergent binding patterns are not determined by the previous chromatin state, but are distinguished by enrichment of specific E-box sequences that reflect the binding preferences of the DNA-binding domains. The divergent Ascl1 and Neurog2 binding patterns result in distinct chromatin accessibility and enhancer activity profiles that differentially shape the binding of downstream transcription factors during neuronal differentiation. This study provides a mechanistic understanding of how transcription factors constrain terminal cell fates, and it delineates the importance of choosing the right proneural factor in neuronal reprogramming strategies.

cAMPr: A single-wavelength fluorescent sensor for cyclic AMP.

Genetically encoded fluorescent sensors enable cell-specific measurements of ions and small molecules in real time. Cyclic adenosine monophosphate (cAMP) is one of the most important signaling molecules in virtually all cell types and organisms. We describe cAMPr, a new single-wavelength cAMP sensor. We developed cAMPr in bacteria and embryonic stem cells and validated the sensor in mammalian neurons in vitro and in Drosophila circadian pacemaker neurons in intact brains. Comparison with other single-wavelength cAMP sensors showed that cAMPr improved the quantitative detection of cAMP abundance. In addition, cAMPr is compatible with both single-photon and two-photon imaging. This enabled us to use cAMPr together with the red fluorescent Ca2+ sensor RCaMP1h to simultaneously monitor Ca2+ and cAMP in Drosophila brains. Thus, cAMPr is a new and versatile genetically encoded cAMP sensor.