Spatial Single-cell Analysis Decodes Cortical Layer and Area Specification.

The human cerebral cortex, pivotal for advanced cognitive functions, is composed of six distinct layers and dozens of functionally specialized areas1,2. The layers and areas are distinguished both molecularly, by diverse neuronal and glial cell subtypes, and structurally, through intricate spatial organization3,4. While single-cell transcriptomics studies have advanced molecular characterization of human cortical development, a critical gap exists due to the loss of spatial context during cell dissociation5,6,7,8. Here, we utilized multiplexed error-robust fluorescence in situ hybridization (MERFISH)9, augmented with deep-learning-based cell segmentation, to examine the molecular, cellular, and cytoarchitectural development of human fetal cortex with spatially resolved single-cell resolution. Our extensive spatial atlas, encompassing 16 million single cells, spans eight cortical areas across four time points in the second and third trimesters. We uncovered an early establishment of the six-layer structure, identifiable in the laminar distribution of excitatory neuronal subtypes by mid-gestation, long before the emergence of cytoarchitectural layers. Notably, while anterior-posterior gradients of neuronal subtypes were generally observed in most cortical areas, a striking exception was the sharp molecular border between primary (V1) and secondary visual cortices (V2) at gestational week 20. Here we discovered an abrupt binary shift in neuronal subtype specification at the earliest stages, challenging the notion that continuous morphogen gradients dictate mid-gestation cortical arealization6,10. Moreover, integrating single-nuclei RNA-sequencing and in situ whole transcriptomics revealed an early upregulation of synaptogenesis in V1-specific Layer 4 neurons, suggesting a role of synaptogenesis in this discrete border formation. Collectively, our findings underscore the crucial role of spatial relationships in determining the molecular specification of cortical layers and areas. This work not only provides a valuable resource for the field, but also establishes a spatially resolved single-cell analysis paradigm that paves the way for a comprehensive developmental atlas of the human brain.

Jpx RNA regulates CTCF anchor site selection and formation of chromosome loops.

Chromosome loops shift dynamically during development, homeostasis, and disease. CCCTC-binding factor (CTCF) is known to anchor loops and construct 3D genomes, but how anchor sites are selected is not yet understood. Here, we unveil Jpx RNA as a determinant of anchor selectivity. Jpx RNA targets thousands of genomic sites, preferentially binding promoters of active genes. Depleting Jpx RNA causes ectopic CTCF binding, massive shifts in chromosome looping, and downregulation of >700 Jpx target genes. Without Jpx, thousands of lost loops are replaced by de novo loops anchored by ectopic CTCF sites. Although Jpx controls CTCF binding on a genome-wide basis, it acts selectively at the subset of developmentally sensitive CTCF sites. Specifically, Jpx targets low-affinity CTCF motifs and displaces CTCF protein through competitive inhibition. We conclude that Jpx acts as a CTCF release factor and shapes the 3D genome by regulating anchor site usage.

Four-dimensional chromosome reconstruction elucidates the spatiotemporal reorganization of the mammalian X chromosome.

Chromosomes are segmented into domains and compartments, but how these structures are spatially related in three dimensions (3D) is unclear. Here, we developed tools that directly extract 3D information from Hi-C experiments and integrate the data across time. With our "4DHiC" method, we use X chromosome inactivation (XCI) as a model to examine the time evolution of 3D chromosome architecture during large-scale changes in gene expression. Our modeling resulted in several insights. Both A/B and S1/S2 compartments divide the X chromosome into hemisphere-like structures suggestive of a spatial phase-separation. During the XCI, the X chromosome transits through A/B, S1/S2, and megadomain structures by undergoing only partial mixing to assume new structures. Interestingly, when an active X chromosome (Xa) is reorganized into an inactive X chromosome (Xi), original underlying compartment structures are not fully eliminated within the Xi superstructure. Our study affirms slow mixing dynamics in the inner chromosome core and faster dynamics near the surface where escapees reside. Once established, the Xa and Xi resemble glassy polymers where mixing no longer occurs. Finally, Xist RNA molecules initially reside within the A compartment but transition to the interface between the A and B hemispheres and then spread between hemispheres via both surface and core to establish the Xi.

Balancing cohesin eviction and retention prevents aberrant chromosomal interactions, Polycomb-mediated repression, and X-inactivation.

Depletion of architectural factors globally alters chromatin structure but only modestly affects gene expression. We revisit the structure-function relationship using the inactive X chromosome (Xi) as a model. We investigate cohesin imbalances by forcing its depletion or retention using degron-tagged RAD21 (cohesin subunit) or WAPL (cohesin release factor). Cohesin loss disrupts the Xi superstructure, unveiling superloops between escapee genes with minimal effect on gene repression. By contrast, forced cohesin retention markedly affects Xi superstructure, compromises spreading of Xist RNA-Polycomb complexes, and attenuates Xi silencing. Effects are greatest at distal chromosomal ends, where looping contacts with the Xist locus are weakened. Surprisingly, cohesin loss creates an Xi superloop, and cohesin retention creates Xi megadomains on the active X chromosome. Across the genome, a proper cohesin balance protects against aberrant inter-chromosomal interactions and tempers Polycomb-mediated repression. We conclude that a balance of cohesin eviction and retention regulates X inactivation and inter-chromosomal interactions across the genome.

Decapping enzyme 1A breaks X-chromosome symmetry by controlling Tsix elongation and RNA turnover.

How allelic asymmetry is generated remains a major unsolved problem in epigenetics. Here we model the problem using X-chromosome inactivation by developing "BioRBP", an enzymatic RNA-proteomic method that enables probing of low-abundance interactions and an allelic RNA-depletion and -tagging system. We identify messenger RNA-decapping enzyme 1A (DCP1A) as a key regulator of Tsix, a noncoding RNA implicated in allelic choice through X-chromosome pairing. DCP1A controls Tsix half-life and transcription elongation. Depleting DCP1A causes accumulation of X-X pairs and perturbs the transition to monoallelic Tsix expression required for Xist upregulation. While ablating DCP1A causes hyperpairing, forcing Tsix degradation resolves pairing and enables Xist upregulation. We link pairing to allelic partitioning of CCCTC-binding factor (CTCF) and show that tethering DCP1A to one Tsix allele is sufficient to drive monoallelic Xist expression. Thus, DCP1A flips a bistable switch for the mutually exclusive determination of active and inactive Xs.

Xist Deletional Analysis Reveals an Interdependency between Xist RNA and Polycomb Complexes for Spreading along the Inactive X.

During X-inactivation, Xist RNA spreads along an entire chromosome to establish silencing. However, the mechanism and functional RNA elements involved in spreading remain undefined. By performing a comprehensive endogenous Xist deletion screen, we identify Repeat B as crucial for spreading Xist and maintaining Polycomb repressive complexes 1 and 2 (PRC1/PRC2) along the inactive X (Xi). Unexpectedly, spreading of these three factors is inextricably linked. Deleting Repeat B or its direct binding partner, HNRNPK, compromises recruitment of PRC1 and PRC2. In turn, ablating PRC1 or PRC2 impairs Xist spreading. Therefore, Xist and Polycomb complexes require each other to propagate along the Xi, suggesting a positive feedback mechanism between RNA initiator and protein effectors. Perturbing Xist/Polycomb spreading causes failure of de novo Xi silencing, with partial compensatory downregulation of the active X, and also disrupts topological Xi reconfiguration. Thus, Repeat B is a multifunctional element that integrates interdependent Xist/Polycomb spreading, silencing, and changes in chromosome architecture.

Exploration of CTCF post-translation modifications uncovers Serine-224 phosphorylation by PLK1 at pericentric regions during the G2/M transition.

The zinc finger CCCTC-binding protein (CTCF) carries out many functions in the cell. Although previous studies sought to explain CTCF multivalency based on sequence composition of binding sites, few examined how CTCF post-translational modification (PTM) could contribute to function. Here, we performed CTCF mass spectrometry, identified a novel phosphorylation site at Serine 224 (Ser224-P), and demonstrate that phosphorylation is carried out by Polo-like kinase 1 (PLK1). CTCF Ser224-P is chromatin-associated, mapping to at least a subset of known CTCF sites. CTCF Ser224-P accumulates during the G2/M transition of the cell cycle and is enriched at pericentric regions. The phospho-obviation mutant, S224A, appeared normal. However, the phospho-mimic mutant, S224E, is detrimental to mouse embryonic stem cell colonies. While ploidy and chromatin architecture appear unaffected, S224E mutants differentially express hundreds of genes, including p53 and p21. We have thus identified a new CTCF PTM and provided evidence of biological function.

Megadomains and superloops form dynamically but are dispensable for X-chromosome inactivation and gene escape.

The mammalian inactive X-chromosome (Xi) is structurally distinct from all other chromosomes and serves as a model for how the 3D genome is organized. The Xi shows weakened topologically associated domains and is instead organized into megadomains and superloops directed by the noncoding loci, Dxz4 and Firre. Their functional significance is presently unclear, though one study suggests that they permit Xi genes to escape silencing. Here, we find that megadomains do not precede Xist expression or Xi gene silencing. Deleting Dxz4 disrupts the sharp megadomain border, whereas deleting Firre weakens intra-megadomain interactions. However, deleting Dxz4 and/or Firre has no impact on Xi silencing and gene escape. Nor does it affect Xi nuclear localization, stability, or H3K27 methylation. Additionally, ectopic integration of Dxz4 and Xist is not sufficient to form megadomains on autosomes. We conclude that Dxz4 and megadomains are dispensable for Xi silencing and escape from X-inactivation.

Transcriptional Pause Sites Delineate Stable Nucleosome-Associated Premature Polyadenylation Suppressed by U1 snRNP.

Regulation of RNA polymerase II (Pol II) elongation is a critical step in gene regulation. Here, we report that U1 snRNP recognition and transcription pausing at stable nucleosomes are linked through premature polyadenylation signal (PAS) termination. By generating RNA exosome conditional deletion mouse embryonic stem cells, we identified a large class of polyadenylated short transcripts in the sense direction destabilized by the RNA exosome. These PAS termination events are enriched at the first few stable nucleosomes flanking CpG islands and suppressed by U1 snRNP. Thus, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing, with PAS termination coinciding with the latter. While pausing factors NELF/DSIF only function in the former step, flavopiridol-sensitive mechanism(s) and Myc modulate both steps. We propose that premature PAS termination near the nucleosome-associated pause site represents a common transcriptional elongation checkpoint regulated by U1 snRNP recognition, nucleosome stability, and Myc activity.

In vivo genome editing using Staphylococcus aureus Cas9.

The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that use the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologues and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further assess the genome-wide targeting specificity of SaCas9 and SpCas9 using BLESS, and demonstrate that SaCas9-mediated in vivo genome editing has the potential to be efficient and specific.

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.

Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.

Target specificity of the CRISPR-Cas9 system.

The CRISPR-Cas9 system, naturally a defense mechanism in prokaryotes, has been repurposed as an RNA-guided DNA targeting platform. It has been widely used for genome editing and transcriptome modulation, and has shown great promise in correcting mutations in human genetic diseases. Off-target effects are a critical issue for all of these applications. Here we review the current status on the target specificity of the CRISPR-Cas9 system.

Promoter directionality is controlled by U1 snRNP and polyadenylation signals.

Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms. In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction. Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5' end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1-PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppressed by U1 snRNP. We propose that the U1-PAS axis limits pervasive transcription throughout the genome.