CCCTC-binding factor (CTCF) is essential for chromatin organization. CTCF interacts with endogenous RNAs, and deletion of its ZF1 RNA binding region (∆ZF1) disrupts chromatin loops in mouse embryonic stem cells (ESCs). However, the functional significance of CTCF-ZF1 RNA interactions during cell differentiation is unknown. Using an ESC-to-neural progenitor cell (NPC) differentiation model, we show that CTCF-ZF1 is crucial for maintaining cell type-specific chromatin loops. Expression of CTCF-∆ZF1 leads to disrupted loops and dysregulation of genes within these loops, particularly those involved in neuronal development and function. We identified NPC-specific, CTCF-ZF1 interacting RNAs. Truncation of two such coding RNAs, Podxl and Grb10, disrupted chromatin loops in cis, similar to the disruption seen in CTCF-∆ZF1-expressing NPCs. These findings underscore the inherent importance of CTCF-ZF1 RNA interactions in preserving cell-specific genome structure and cellular identity.

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.

Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development.

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.

AbstractCCCTC-binding factor (CTCF) and MAZ are recognized insulators required for shielding repressed posterior genes from active anterior genes within theHoxclusters 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.Patz1KO mESCs exhibited altered global gene expression. While the absence of MAZ impacts anterior CTCF-boundaries as shown previously1,Patz1KO led to derepression of posteriorHoxgenes, 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.

Abstract 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.

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.

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.

Amyotrophic Lateral Sclerosis (ALS) is a fatal adult-onset neurodegenerative disease without cure or understanding of its molecular basis. It is characterized by progressive loss of spinal and upper motor neurons, but surprising resistance of cranial motor neurons until the late stages of the disease. There is no known single-gene cause of ALS, suggesting a failure of the entire cellular system to maintain proteostasis. To generate new hypotheses on the molecular differences between cranial and spinal motor neurons that underlie ALS-sensitivity, we exploited a unique platform that generates highly pure populations of induced cranial and spinal motor neurons (iCrMN and iSpMN). Exposing both iCrMNs and iSpMNs to proteotoxic stress, we monitored transcriptome and proteome changes over 36 hours and identified significant hundreds of statistically significant differences at either or both the RNA and protein level from a core set of 8,206 genes with complete data. While we observed a response to misfolding stress coordinated between transcript and protein changes, differences between the cell type were specific to RNA and protein expression. iCrMNs and iSpMNs differed in expression of many membrane proteins, including synaptic proteins, solute carriers, channels, and signaling molecules, as well as genes involved in ribosome biogenesis and subunits of the core proteasome. Differences exclusive to the transcriptome involved chaperones and the tricarboxylic acid cycle. Further, we found that the superior proteostasis capability of iCrMNs might, in part, be caused due to higher activity and abundance of the fully assembled 26S/30S proteasome in the nucleus, pointing to an increased role of ubiquitin-mediated, targeted protein degradation. The formation of the 26S/30S proteasome was highly dynamic under stress, likely mediated by a surplus of regulatory particles that attached and detached based on the action of proteasome regulators. Several lines of evidence supported this notion and described an intricate network of differentially expressed proteasome regulators and ubiquitination enzymes that defined the iCrMNand iSpMN-specific ability to maintain proteostasis.

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. ZBTB11 and ZFP131 maintain promoter-proximally paused Polymerase II at pro-differentiation genes in ESCs. ZBTB11 or ZFP131 loss leads to NELF pausing factor release, an increase in H3K4me3, and transcriptional upregulation of genes associated with all three germ layers. 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. One-sentence summary A Transcription Factor-wide CRISPR screen identifies ZBTB11 and ZFP131 maintaining pluripotency by pausing POL II at pro-differentiation genes

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.

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.

Despite clear therapeutic potential, the mechanisms that confer differential neuronal sensitivity are not well understood. During Amyotrophic Lateral Sclerosis (ALS), sensitive spinal motor neurons (SpMN) die while a subset of rostral cranial motor neurons (CrMN) survive. In this work, we optimized a protocol to differentiate CrMNs and SpMNs from human induced pluripotent stem cells (iPSCs) by direct programming and positional patterning. Human iCrMNs are more resistant than iSpMNs to proteotoxic stress and rely on the proteasome to maintain proteostasis. iCrMNs better prevent mislocalization of TDP43 from the nucleus, a hallmark of ALS progression. iSpMNs contain more splicing defects than iCrMNs in response to ALS-related stress with genes involved in splicing and proteostasis maintenance. Therefore, iCrMNs resist ALS at two levels, preventing protein accumulation and reducing splicing defects in response to TDP43 nuclear depletion. Thus, ALS-sensitive iSpMNs appear to enter a downward spiral compromising their ability to maintain proteostasis and splicing.

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.

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.

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.

Amyotrophic Lateral Sclerosis (ALS) is a fatal adult neurodegenerative disease characterized by proteostasis dysregulation, resulting in progressive loss of spinal and upper motor neurons. A subset of cranial motor neurons resistant to ALS-stress survive until late stages of the disease. To investigate these differences, we exploited a unique platform of induced cranial and spinal motor neurons (iCrMNs and iSpMNs, respectively). Exposing both cell types to proteotoxic stress, we quantified transcriptome and proteome changes over 36 hours for a core set of >8,200 genes. While mRNA and protein changes under stress were congruent for many genes, cell-type specific differences manifested at either the RNA or protein level, but less at both. At the protein level, iCrMNs and iSpMNs differed significantly with respect to abundance of many membrane proteins, including synaptic proteins, solute carriers, adhesion molecules, and signaling molecules suggesting that the superior stress survival of iCrMNs involve diverse pathways supporting neuronal function. Other differences included genes involved in ribosome biogenesis and subunits of the core proteasome. We investigated the role of proteasomal degradation in more detail. Our data showed that although stress reduces proteasome activity in both neuronal types, iCrMNs had significantly more abundant and active 26S proteasome than iSpMNs, which indicate a higher capacity for the degradation of ubiquitinated proteins. We identified a new regulator of this better performance, i.e. the nuclear proteasome activator Ublcp1, whose inhibition sensitized iCrMNs, but not iSpMNs, to stress and abolished their higher survival rates. The results suggest that the two neuronal cell types regulate and use the degradation machinery differently under normal and stress conditions. Overall, this work demonstrates the value of unbiased system-wide analyses in generating hypotheses on differential proteostasis regulation in cranial and spinal motor neurons.

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 101kb 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.

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.

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.