Reza Kalhor

Genome architectures revealed by tethered chromosome conformation capture and population-based modeling

We describe tethered conformation capture (TCC), a method for genome-wide mapping of chromatin interactions. By performing ligations on solid substrates rather than in solution, TCC substantially enhances the signal-to-noise ratio, thereby facilitating a detailed analysis of interactions within and between chromosomes. We identified a group of regions in each chromosome in human cells that account for the majority of interchromosomal interactions. These regions are marked by high transcriptional activity, suggesting that their interactions are mediated by transcriptional machinery. Each of these regions interacts with numerous other such regions throughout the genome in an indiscriminate fashion, partly driven by the accessibility of the partners. As a different combination of interactions is likely present in different cells, we developed a computational method to translate the TCC data into physical chromatin contacts in a population of three-dimensional genome structures. Statistical analysis of the resulting population demonstrates that the indiscriminate properties of interchromosomal interactions are consistent with the well-known architectural features of the human genome.

Highly multiplexed subcellular RNA sequencing in situ

Transcripts Visualized in Situ Despite advances, current methods for single-cell sequencing are unable to resolve transcript location within the cell, so Lee et al. (p. 1360, published online 27 February) developed a method of fluorescent in situ RNA sequencing (FISSEQ) that works in vivo to show messenger RNA localization within cells. The method amplifies complementary DNA targets by rolling circle amplification, and then in situ cross-linking locks amplicons to produce ample, highly localized templates for three-dimensional sequencing. The technique was tested in fibroblasts to reveal the differences between individual cells during wound repair. Reads of cellular RNA transcripts demonstrate spatial expression differences during simulated wound healing. Understanding the spatial organization of gene expression with single-nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here, we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked complementary DNA (cDNA) amplicons are sequenced within a biological sample. Using 30-base reads from 8102 genes in situ, we examined RNA expression and localization in human primary fibroblasts with a simulated wound-healing assay. FISSEQ is compatible with tissue sections and whole-mount embryos and reduces the limitations of optical resolution and noisy signals on single-molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ.

Genome-wide Linkage Analysis of a Parkinsonian-Pyramidal Syndrome Pedigree by 500 K SNP Arrays

Robust SNP genotyping technologies and data analysis programs have encouraged researchers in recent years to use SNPs for linkage studies. Platforms used to date have been 10 K chip arrays, but the possible value of interrogating SNPs at higher densities has been considered. Here, we present a genome-wide linkage analysis by means of a 500 K SNP platform. The analysis was done on a large pedigree affected with Parkinsonian-pyramidal syndrome (PPS), and the results showed linkage to chromosome 22. Sequencing of candidate genes revealed a disease-associated homozygous variation (R378G) in FBXO7. FBXO7 codes for a member of the F-box family of proteins, all of which may have a role in the ubiquitin-proteosome protein-degradation pathway. This pathway has been implicated in various neurodegenerative diseases, and identification of FBXO7 as the causative gene of PPS is expected to shed new light on its role. The performance of the array was assessed and systematic analysis of effects of SNP density reduction was performed with the real experimental data. Our results suggest that linkage in our pedigree may have been missed had we used chips containing less than 100,000 SNPs across the genome.

Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

RNA-sequencing (RNA-seq) measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. In contrast, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq, our method enriches for context-specific transcripts over housekeeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d.

Rapidly evolving homing CRISPR barcodes

We present an approach for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9–hgRNA complex to the DNA locus of the hgRNA itself. We show that this homing CRISPR–Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show that they can be used to record lineage history and that the barcode RNA can be amplified in situ, a prerequisite for in situ sequencing. This integrated approach will have wide-ranging applications, such as in deep lineage tracing, cellular barcoding, molecular recording, dissecting cancer biology, and connectome mapping.

Population-based 3D genome structure analysis reveals driving forces in spatial genome organization

Significance We provide a method for population-based structure modeling of whole diploid genomes using Hi-C data. The method considers the stochastic nature of chromosome structures, which allows a detailed analysis of the dynamic landscape of genome organizations. We predict and experimentally validate the presence of chromosome-specific higher-order centromere clusters, which can play a key role in the spatial organization of the human genome, specifically influencing the overall chromosome positioning, as well as the preference of specific chromosome conformations. Our approach generate predictive structural models of diploid genomes from Hi-C data, which can provide insights into the guiding principles of 3D genome organizations. Conformation capture technologies (e.g., Hi-C) chart physical interactions between chromatin regions on a genome-wide scale. However, the structural variability of the genome between cells poses a great challenge to interpreting ensemble-averaged Hi-C data, particularly for long-range and interchromosomal interactions. Here, we present a probabilistic approach for deconvoluting Hi-C data into a model population of distinct diploid 3D genome structures, which facilitates the detection of chromatin interactions likely to co-occur in individual cells. Our approach incorporates the stochastic nature of chromosome conformations and allows a detailed analysis of alternative chromatin structure states. For example, we predict and experimentally confirm the presence of large centromere clusters with distinct chromosome compositions varying between individual cells. The stability of these clusters varies greatly with their chromosome identities. We show that these chromosome-specific clusters can play a key role in the overall chromosome positioning in the nucleus and stabilizing specific chromatin interactions. By explicitly considering genome structural variability, our population-based method provides an important tool for revealing novel insights into the key factors shaping the spatial genome organization.

Conserved forkhead dimerization motif controls DNA replication timing and spatial organization of chromosomes in S. cerevisiae

Significance The spatial organization of chromatin within the nucleus regulates genomic functions including DNA repair, transcription, and replication. For example, replication origins cluster prior to initiating replication, likely to aggregate the many necessary factors, but the mechanism is poorly understood. We recently discovered yeast “Forkhead Box” (Fox) DNA binding proteins, Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2), as required for this origin clustering and regulation of initiation timing. This study reveals that Fkh1 and Fkh2 share a structural motif that allows dimerization to bring distal DNA binding sites into close proximity. Mutation that disrupts dimerization ablates origin clustering and deregulates origin activation, suggesting causality between origin clustering and initiation control. We propose that Fkh1 and Fkh2 and related Fox proteins in metazoans establish chromatin architecture. Forkhead Box (Fox) proteins share the Forkhead domain, a winged-helix DNA binding module, which is conserved among eukaryotes from yeast to humans. These sequence-specific DNA binding proteins have been primarily characterized as transcription factors regulating diverse cellular processes from cell cycle control to developmental fate, deregulation of which contributes to developmental defects, cancer, and aging. We recently identified Saccharomyces cerevisiae Forkhead 1 (Fkh1) and Forkhead 2 (Fkh2) as required for the clustering of a subset of replication origins in G1 phase and for the early initiation of these origins in the ensuing S phase, suggesting a mechanistic role linking the spatial organization of the origins and their activity. Here, we show that Fkh1 and Fkh2 share a unique structural feature of human FoxP proteins that enables FoxP2 and FoxP3 to form domain-swapped dimers capable of bridging two DNA molecules in vitro. Accordingly, Fkh1 self-associates in vitro and in vivo in a manner dependent on the conserved domain-swapping region, strongly suggestive of homodimer formation. Fkh1- and Fkh2-domain-swap-minus (dsm) mutations are functional as transcription factors yet are defective in replication origin timing control. Fkh1-dsm binds replication origins in vivo but fails to cluster them, supporting the conclusion that Fkh1 and Fkh2 dimers perform a structural role in the spatial organization of chromosomal elements with functional importance.

Conneconomics: The Economics of Large-Scale Neural Connectomics

We analyze the scaling and cost-performance characteristics of current and projected connectomics approaches, with reference to the potential implications of recent advances in diverse contributing fields. This analysis suggests potential cost-effective strategies for dense connectivity mapping at the scale of whole mammalian brains.

Developmental barcoding of whole mouse via homing CRISPR

Lineage tracing in mouse using CRISPR A homing guide RNA (hgRNA) that directs CRISPR-Cas9 to its own DNA locus can diversify its sequence and act as an expressed genetic barcode. Kalhor et al. engineered a mouse line carrying 60 independent loci of hgRNAs, thus generating a large number of unique barcodes in various embryonic and extraembryonic tissues in fully developed mice. This method demonstrates lineage tracing from the very first branches of the development tree up to organogenesis events and was used to elucidate embryonic brain patterning. Science, this issue p. eaat9804 Multiple, independent barcoding elements enable lineage tracing in the whole mouse embryo during brain development and patterning. INTRODUCTION The remarkable development of a single cell, the zygote, into the full organism occurs through a complex series of division and differentiation events that resemble a tree, with the zygote at the base branching through lineages that end in the terminal cell types at the top. Characterizing this tree of development has long been a subject of interest, and the combination of modern genome engineering and sequencing technologies promises a powerful strategy in its service: in vivo barcoding. For in vivo barcoding, heritable random mutations are induced to accumulate during development and sequenced post hoc to reconstruct the lineage tree. Demonstrations thus far have largely focused on lower vertebrates and have used a barcoding element with a constrained window of activity for clonal tracing of individual cells or cell types. Implementation in mammalian model systems, such as the mouse, incurs unique challenges that require major enhancements. RATIONALE To address the complexity of mammalian development, we reasoned that multiple independent in vivo barcoding elements could be deployed in parallel to exponentially expand their recording power. Independence requires both an absence of cross-talk between the elements and an absence of interference between their mutation outcomes. A system with the potential to deliver on these requirements is homing CRISPR, a modified version of canonical CRISPR wherein the homing guide RNA (hgRNA) combines with CRISPR-Cas9 nuclease for repeated targeting of its own locus, leading to diverse mutational outcomes. Therefore, in mouse embryonic stem cells, we scattered multiple hgRNA loci with distinct spacers in the genome to serve as barcoding elements. With this arrangement, each hgRNA acts independently as a result of its unique spacer sequence, and undesirable deletion events between multiple adjacent cut sites are less likely. Using these cells, we generated a chimeric mouse with 60 hgRNAs as the founder of the MARC1 (Mouse for Actively Recording Cells 1) line that enables barcoding and recording of cell lineages. RESULTS In the absence of Cas9, hgRNAs are stable and dormant; to initiate barcoding, we crossed MARC1 mice with Cas9 knock-in mice. In the resulting offspring, hgRNAs were activated, creating diverse mutations such that an estimated 1023 distinct barcode combinations can be generated with only 10 hgRNAs. Furthermore, hgRNAs showed a range of activity profiles, with some mutating soon after conception while others exhibited lower activity through most of the gestation period. This range resulted in sustained barcoding throughout gestation and recording of developmental lineages: Each cell inherits a set of unique mutations that are passed on to its daughter cells, where further unique mutations can be added. Consequently, at any stage in such developmentally barcoded mice, closely related cells have a more similar mutation profile, or barcode, than the more distant ones. These recordings remain embedded in the genomes of the cells and can be extracted by sequencing. We used these recordings to carry out bottom-up reconstruction of the mouse lineage tree, starting with the first branches that emerged after the zygote, and observed robust reconstruction of the correct tree. We also investigated axis development in the brain by sequencing barcodes from the left and right side of the forebrain, midbrain, and hindbrain regions. We found that barcodes from the left and right sides of the same region were more closely related than those from different regions; this result suggests that in the precursor of the brain, commitment to the anterior-posterior axis is established prior to the lateral axis. CONCLUSION This system provides an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system. It can straightforwardly create developmentally barcoded mice in which lineage information is prerecorded in cell genomes. Combining multiple independently acting molecular recording devices greatly enhances their capacity and allows for reliable information recovery and reconstruction of deep lineage trees. Developmental barcoding and lineage reconstruction in mice. Crossing the MARC1 mouse line, which carries multiple hgRNAs, with a CRISPR-Cas9 mouse line results in developmentally barcoded offspring that record lineages in their cells. These recordings were extracted and used to reconstruct lineage trees. A combination of the trees extracted from different developmentally barcoded mice is shown. ICM, inner cell mass; E0, embryonic day 0. In vivo barcoding using nuclease-induced mutations is a powerful approach for recording biological information, including developmental lineages; however, its application in mammalian systems has been limited. We present in vivo barcoding in the mouse with multiple homing guide RNAs that each generate hundreds of mutant alleles and combine to produce an exponential diversity of barcodes. Activation upon conception and continued mutagenesis through gestation resulted in developmentally barcoded mice wherein information is recorded in lineage-specific mutations. We used these recordings for reliable post hoc reconstruction of the earliest lineages and investigation of axis development in the brain. Our results provide an enabling and versatile platform for in vivo barcoding and lineage tracing in a mammalian model system.

A homing CRISPR mouse resource for barcoding and lineage tracing

Cellular barcoding using nuclease-induced genetic mutations is an effective approach that is emerging for recording biological information, including developmental lineages. We have previously introduced the homing CRISPR system as a promising methodology for generating such barcodes with scalable diversity and without crosstalk. Here, we present a mouse line (MARC1) with multiple genomically-integrated and heritable homing guide RNAs (hgRNAs). We determine the genomic locations of these hgRNAs, their activity profiles during gestation, and the diversity of their mutants. We apply the line for unique barcoding of mouse embryos and differential barcoding of embryonic tissues. We conclude that this mouse line can address the unique challenges associated with in vivo barcoding in mammalian model organisms and is thus an enabling platform for recording and lineage tracing applications in a mammalian model system.