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Clinical RNA-seq has become an essential tool for resolving variants of uncertain significance (VUS), particularly those affecting gene expression and splicing. However, most reference data and diagnostic protocols employ relatively modest sequencing depths (∼50-150 million reads), which may fail to capture low-abundance transcripts and rare splicing events critical for accurate diagnoses. We evaluated the diagnostic and translational utility of ultra-high-depth (up to ∼one billion unique reads) RNA-seq in four clinically accessible tissues (blood, fibroblast, LCL, and iPSC) using Ultima sequencing platform. After validating the performance of Ultima RNA-seq, we investigated how increasing depth affects gene and isoform detection, splicing variant discovery, and clinical interpretation of VUS. Deep RNA-seq substantially improved sensitivity for detecting lowly-expressed genes and isoforms. At ∼1 billion reads, near-saturation was achieved for gene-level detection, although isoform-level coverage continued to benefit from even deeper sequencing. In two clinical cases with VUS, pathogenic splicing abnormalities were undetected at ∼50 million reads but emerged at 200 million reads, becoming even more pronounced at ∼one billion reads. Using deep RNA-seq data, we constructed a novel resource, MRSD-deep, to estimate the minimum required sequencing depth to achieve desired coverage thresholds. MRSD-deep provided gene- and junction-level guidelines, aiding labs in selecting suitable coverage targets for specific applications. Leveraging deep RNA-seq data on fibroblast, we also built an expanded splicing-variation reference that successfully identified rare splicing events missed by standard-depth data. Our findings underscore the diagnostic and research benefits of deep RNA-seq for Mendelian disease investigations. By capturing rare transcripts and splicing events, ultra-high-depth RNA-seq can facilitate more definitive variant interpretations and enrich splicing-reference databases. We anticipate that cost-effective deep sequencing technologies and robust reference cohorts will further advance RNA-based diagnostics in precision medicine.

Single-cell methods to assess DNA methylation have not achieved the same level of cell throughput per experiment compared to other modalities, with large-scale datasets requiring extensive automation, time, and other resources. Here, we describe sciMETv3, a combinatorial indexing-based technique that enables atlas-scale libraries to be produced in a single experiment. To reduce the sequencing burden, we demonstrate the compatibility of sciMETv3 with capture techniques to enrich regulatory regions, as well as the ability to leverage enzymatic conversion, which can yield higher library diversity. We showcase the throughput of sciMETv3 by producing a >140,000 cell library from human middle frontal gyrus split across four multiplexed individuals using both Illumina and Ultima sequencing instrumentation. Finally, we introduce sciMET+ATAC to enable high-throughput exploration of the interplay between chromatin accessibility and DNA methylation within the same cell., Competing Interests: Declaration of interests A.C.A. is an author of one or more patents that pertain to sciMET technology and an advisor to Scale Biosciences. This potential conflict is managed by the office of research integrity at OHSU. Z.S. and N.I. are employees of Ultima Genomics., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although three-dimensional (3D) genome architecture is crucial for gene regulation, its role in disease remains elusive. We traced the evolution and malignant transformation of colorectal cancer (CRC) by generating high-resolution chromatin conformation maps of 33 colon samples spanning different stages of early neoplastic growth in persons with familial adenomatous polyposis (FAP). Our analysis revealed a substantial progressive loss of genome-wide cis-regulatory connectivity at early malignancy stages, correlating with nonlinear gene regulation effects. Genes with high promoter-enhancer (P-E) connectivity in unaffected mucosa were not linked to elevated baseline expression but tended to be upregulated in advanced stages. Inhibiting highly connected promoters preferentially represses gene expression in CRC cells compared to normal colonic epithelial cells. Our results suggest a two-phase model whereby neoplastic transformation reduces P-E connectivity from a redundant state to a rate-limiting one for transcriptional levels, highlighting the intricate interplay between 3D genome architecture and gene regulation during early CRC progression., Competing Interests: Competing interests: G.K., A.J., D.L. and Z.S. are employees and shareholders of Ultima Genomics. M.P.S is a cofounder and scientific advisor of Personalis, Qbio, SensOmics, January AI, Mirvie, Protos, NiMo and Onza and is on the advisory board of Genapsys. E.D.E. is an employee and stockholder of Invitae and an advisor and stockholder of Taproot Health and Exir Bio. W.J.G. has affiliations with Guardant Health (consultant and scientific advisory board), Protillion Biosciences (Scientifica cofounder) and 10x and has licensed patents associated with ATAC-seq. All other authors declare no competing interests., (© 2024. The Author(s).)

The ability to analyze the transcriptomic and epigenomic states of individual single cells has in recent years transformed our ability to measure and understand biological processes. Recent advancements have focused on increasing sensitivity and throughput to provide richer and deeper biological insights at the cellular level. The next frontier is the development of multiomic methods capable of analyzing multiple features from the same cell, such as the simultaneous measurement of the transcriptome and the chromatin accessibility of candidate regulatory elements. In this chapter, we discuss and describe SHARE-seq (Simultaneous high-throughput ATAC, and RNA expression with sequencing) for carrying out simultaneous chromatin accessibility and transcriptome measurements in single cells, together with the experimental and analytical considerations for achieving optimal results., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Active cis-regulatory elements (cREs) in eukaryotes are characterized by nucleosomal depletion and, accordingly, higher accessibility. This property has turned out to be immensely useful for identifying cREs genome-wide and tracking their dynamics across different cellular states and is the basis of numerous methods taking advantage of the preferential enzymatic cleavage/labeling of accessible DNA. ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) has emerged as the most versatile and widely adaptable method and has been widely adopted as the standard tool for mapping open chromatin regions. Here, we discuss the current optimal practices and important considerations for carrying out ATAC-seq experiments, primarily in the context of mammalian systems., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aging is characterized by an increased vulnerability to infection and the development of inflammatory diseases, such as atherosclerosis, frailty, cancer and neurodegeneration. Here, we find that aging is associated with the loss of diurnally rhythmic innate immune responses, including monocyte trafficking from bone marrow to blood, response to lipopolysaccharide and phagocytosis. This decline in homeostatic immune responses was associated with a striking disappearance of circadian gene transcription in aged compared to young tissue macrophages. Chromatin accessibility was significantly greater in young macrophages than in aged macrophages; however, this difference did not explain the loss of rhythmic gene transcription in aged macrophages. Rather, diurnal expression of Kruppel-like factor 4 (Klf4), a transcription factor (TF) well established in regulating cell differentiation and reprogramming, was selectively diminished in aged macrophages. Ablation of Klf4 expression abolished diurnal rhythms in phagocytic activity, recapitulating the effect of aging on macrophage phagocytosis. Examination of individuals harboring genetic variants of KLF4 revealed an association with age-dependent susceptibility to death caused by bacterial infection. Our results indicate that loss of rhythmic Klf4 expression in aged macrophages is associated with disruption of circadian innate immune homeostasis, a mechanism that may underlie age-associated loss of protective immune responses., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

A hallmark feature of active cis-regulatory elements (CREs) in eukaryotes is their nucleosomal depletion and, accordingly, higher accessibility to enzymatic treatment. This property has been the basis of a number of sequencing-based assays for genome-wide identification and tracking the activity of CREs across different biological conditions, such as DNAse-seq, ATAC-seq , NOMeseq, and others. However, the fragmentation of DNA inherent to many of these assays and the limited read length of short-read sequencing platforms have so far not allowed the simultaneous measurement of the chromatin accessibility state of CREs located distally from each other. The combination of labeling accessible DNA with DNA modifications and nanopore sequencing has made it possible to develop such assays. Here, we provide a detailed protocol for carrying out the SMAC-seq assay (Single-Molecule long-read Accessible Chromatin mapping sequencing), in its m6A-SMAC-seq and m6A-CpG-GpC-SMAC-seq variants, together with methods for data processing and analysis, and discuss key experimental and analytical considerations for working with SMAC-seq datasets., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mammalian SWI/SNF (BAF) chromatin remodelers play dosage-sensitive roles in many human malignancies and neurologic disorders. The gene encoding the BAF subunit actin-like 6a (ACTL6A) is amplified early in the development of many squamous cell carcinomas (SCCs), but its oncogenic role remains unclear. Here we demonstrate that ACTL6A overexpression leads to its stoichiometric assembly into BAF complexes and drives their interaction and engagement with specific regulatory regions in the genome. In normal epithelial cells, ACTL6A was substoichiometric to other BAF subunits. However, increased ACTL6A levels by ectopic expression or in SCC cells led to near saturation of ACTL6A within BAF complexes. Increased ACTL6A occupancy enhanced polycomb opposition genome-wide to activate SCC genes and facilitated the co-dependent loading of BAF and TEAD-YAP complexes on chromatin. Both mechanisms appeared to be critical and function as a molecular AND gate for SCC initiation and maintenance, thereby explaining the specificity of the role of ACTL6A amplification in SCCs., Competing Interests: Declaration of interests G.R.C. is a founder and stockholder in Foghorn Therapeutics., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting autoreactive B cells. Dysfunction in these processes can lead to defective immune responses to infection or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type–specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC-associated cellular populations. These included gene loci linked with known roles in GC biology ( IL21 , IL21R , IL4R , and BCL6 ) and transcription factors regulating B cell differentiation ( POU2AF1 and HHEX ). Together, these analyses provide a powerful new cell type–resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.

ATAC-seq is a versatile, adaptable, and widely adopted technique for mapping open chromatin regions. However, some biological systems, such as primary neurons, present unique challenges to its application. Conventional ATAC-seq would require the dissociation of the primary neurons after plating but dissociating them leads to rapid cell death and major changes in cell state, affecting ATAC-seq results. We have developed this modified ATAC-seq protocol to address this challenge for primary neurons, providing a high-quality and high-resolution accessible chromatin profile. For complete details on the use and execution of this protocol, please refer to Maor-Nof et al. (2021)., Competing Interests: A.D.G. is a scientific founder of Maze Therapeutics. W.J.G. has affiliations with 10× Genomics (consultant), Guardant Health (consultant), and Protillion Biosciences (co-founder and consultant)., (© 2021 The Author(s).)

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration., Competing Interests: Declaration of interests A.D.G. has served as a consultant for Aquinnah Pharmaceuticals, Prevail Therapeutics, and Third Rock Ventures and is a scientific founder of Maze Therapeutics. W.J.G. has affiliations with 10x Genomics (consultant), Guardant Health (consultant), and Protillion Biosciences (co-founder and consultant). J.K.I. is a co-founder of Acurastem. D.H.G has served as a consultant for Acurastem, Axial Biosciences, and Roche, (Copyright © 2020 Elsevier Inc. All rights reserved.)

The ATAC-seq assay has emerged as the most useful, versatile, and widely adaptable method for profiling accessible chromatin regions and tracking the activity of cis-regulatory elements (cREs) in eukaryotes. Thanks to its great utility, it is now being applied to map active chromatin in the context of a very wide diversity of biological systems and questions. In the course of these studies, considerable experience working with ATAC-seq data has accumulated and a standard set of computational tasks that need to be carried for most ATAC-seq analyses has emerged. Here, we review and provide examples of common such analytical procedures (including data processing, quality control, peak calling, identifying differentially accessible open chromatin regions, and variable transcription factor (TF) motif accessibility) and discuss recommended optimal practices.

Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such "early activation" genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b ). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors ( Fos , Fosl2 , Fosb , and Junb ), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism., Competing Interests: Competing interest statement: G.R.C. is a founder of Foghorn Therapeutics., (Copyright © 2020 the Author(s). Published by PNAS.)

Mapping open chromatin regions has emerged as a widely used tool for identifying active regulatory elements in eukaryotes. However, existing approaches, limited by reliance on DNA fragmentation and short-read sequencing, cannot provide information about large-scale chromatin states or reveal coordination between the states of distal regulatory elements. We have developed a method for profiling the accessibility of individual chromatin fibers, a single-molecule long-read accessible chromatin mapping sequencing assay (SMAC-seq), enabling the simultaneous, high-resolution, single-molecule assessment of chromatin states at multikilobase length scales. Our strategy is based on combining the preferential methylation of open chromatin regions by DNA methyltransferases with low sequence specificity, in this case EcoGII, an N 6 -methyladenosine (m 6 A) methyltransferase, and the ability of nanopore sequencing to directly read DNA modifications. We demonstrate that aggregate SMAC-seq signals match bulk-level accessibility measurements, observe single-molecule nucleosome and transcription factor protection footprints, and quantify the correlation between chromatin states of distal genomic elements.

Patchy infiltration of tumors by cytotoxic T cells (CTLs) predicts poorer prognosis for cancer patients. The factors limiting intratumoral CTL dissemination, though, are poorly understood. To study CTL dissemination in tumors, we histologically examined human melanoma samples and used mice to image B16-OVA tumors infiltrated by OT-I CTLs using intravital two-photon microscopy. In patients, most CTLs concentrated around peripheral blood vessels, especially in poorly infiltrated tumors. In mice, OT-I CTLs had to cluster around tumor cells to efficiently kill them in a contact-and perforin-dependent manner and cytotoxicity was strictly antigen-specific. OT-I CTLs as well as non-specific CTLs concentrated around peripheral vessels, and cleared the tumor cells around them. This was also the case when CTLs were injected directly into the tumors. CTLs crawled rapidly only in areas within 50 µm of flowing blood vessels and transient occlusion of vessels immediately, though reversibly, stopped their migration. In vitro, oxygen depletion and blockade of oxidative phosphorylation also reduced CTL motility. Taken together, these results suggest that hypoxia limits CTL migration away from blood vessels, providing immune-privileged niches for tumor cells to survive. Normalizing intratumoral vasculature may thus synergize with tumor immunotherapy.

Physical access to DNA is a highly dynamic property of chromatin that plays an essential role in establishing and maintaining cellular identity. The organization of accessible chromatin across the genome reflects a network of permissible physical interactions through which enhancers, promoters, insulators and chromatin-binding factors cooperatively regulate gene expression. This landscape of accessibility changes dynamically in response to both external stimuli and developmental cues, and emerging evidence suggests that homeostatic maintenance of accessibility is itself dynamically regulated through a competitive interplay between chromatin-binding factors and nucleosomes. In this Review, we examine how the accessible genome is measured and explore the role of transcription factors in initiating accessibility remodelling; our goal is to illustrate how chromatin accessibility defines regulatory elements within the genome and how these epigenetic features are dynamically established to control gene expression.

DNA methylation is a key regulator of embryonic stem cell (ESC) biology, dynamically changing between naïve, primed, and differentiated states. The p53 tumor suppressor is a pivotal guardian of genomic stability, but its contributions to epigenetic regulation and stem cell biology are less explored. We report that, in naïve mouse ESCs (mESCs), p53 restricts the expression of the de novo DNA methyltransferases Dnmt3a and Dnmt3b while up-regulating Tet1 and Tet2, which promote DNA demethylation. The DNA methylation imbalance in p53-deficient (p53 -/- ) mESCs is the result of augmented overall DNA methylation as well as increased methylation landscape heterogeneity. In differentiating p53 -/- mESCs, elevated methylation persists, albeit more mildly. Importantly, concomitant with DNA methylation heterogeneity, p53 -/- mESCs display increased cellular heterogeneity both in the "naïve" state and upon induced differentiation. This impact of p53 loss on 5-methylcytosine (5mC) heterogeneity was also evident in human ESCs and mouse embryos in vivo. Hence, p53 helps maintain DNA methylation homeostasis and clonal homogeneity, a function that may contribute to its tumor suppressor activity., (© 2017 Tovy et al.; Published by Cold Spring Harbor Laboratory Press.)

Stable maintenance of gene regulatory programs is essential for normal function in multicellular organisms. Epigenetic mechanisms, and DNA methylation in particular, are hypothesized to facilitate such maintenance by creating cellular memory that can be written during embryonic development and then guide cell-type-specific gene expression. Here we develop new methods for quantitative inference of DNA methylation turnover rates, and show that human embryonic stem cells preserve their epigenetic state by balancing antagonistic processes that add and remove methylation marks rather than by copying epigenetic information from mother to daughter cells. In contrast, somatic cells transmit considerable epigenetic information to progenies. Paradoxically, the persistence of the somatic epigenome makes it more vulnerable to noise, since random epimutations can accumulate to massively perturb the epigenomic ground state. The rate of epigenetic perturbation depends on the genomic context, and, in particular, DNA methylation loss is coupled to late DNA replication dynamics. Epigenetic perturbation is not observed in the pluripotent state, because the rapid turnover-based equilibrium continuously reinforces the canonical state. This dynamic epigenetic equilibrium also explains how the epigenome can be reprogrammed quickly and to near perfection after induced pluripotency.

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.

Coordinate regulation of ribosomal protein (RP) genes is key for controlling cell growth. In yeast, it is unclear how this regulation achieves the required equimolar amounts of the different RP components, given that some RP genes exist in duplicate copies, while others have only one copy. Here, we tested whether the solution to this challenge is partly encoded within the DNA sequence of the RP promoters, by fusing 110 different RP promoters to a fluorescent gene reporter, allowing us to robustly detect differences in their promoter activities that are as small as ~10%. We found that single-copy RP promoters have significantly higher activities, suggesting that proper RP stoichiometry is indeed partly encoded within the RP promoters. Notably, we also partially uncovered how this regulation is encoded by finding that RP promoters with higher activity have more nucleosome-disfavoring sequences and characteristic spatial organizations of these sequences and of binding sites for key RP regulators. Mutations in these elements result in a significant decrease of RP promoter activity. Thus, our results suggest that intrinsic (DNA-dependent) nucleosome organization may be a key mechanism by which genomes encode biologically meaningful promoter activities. Our approach can readily be applied to uncover how transcriptional programs of other promoters are encoded.

Dendritic cells (DCs) must travel through lymphatics to carry skin antigens into lymph nodes. The processes controlling their mobilization and migration have not been completely delineated. We studied how DCs in live mice respond to skin inflammation, transmigrate through lymphatic endothelium, and propagate in initial lymphatics. At steady state, dermal DCs remain sessile along blood vessels. Inflammation mobilizes them, accelerating their interstitial motility 2.5-fold. CCR7-deficient BMDCs crawl as fast as wild-type DCs but less persistently. We observed discrete depositions of CCL21 complexed with collagen-IV on the basement membrane of initial lymphatics. Activated DCs move directionally toward lymphatics, contact CCL21 puncta, and migrate through portals into the lumen. CCR7-deficient DCs arrive at lymphatics through random migration but fail to dock and transmigrate. Once inside vessels, wild-type DCs use lamellipodia to crawl along lymphatic endothelium and, sensing lymph flow, proceed downstream. DCs start drifting freely only in collecting lymphatics. These results demonstrate in vivo that the CCL21-CCR7 axis plays a dual role in DC mobilization: promoting both chemotaxis and arrest of DCs on lymphatic endothelium. Intralymphatic crawling, in which DCs combine active adhesion-based migration and directional cues from lymph flow, represents a new step in DC mobilization which may be amenable to regulation.