To elucidate mechanisms of cancer progression, we generated inducible human neoplasia in three-dimensionally intact epithelial tissue. Gene expression profiling of both epithelia and stroma at specific time points during tumor progression revealed sequential enrichment of genes mediating discrete biologic functions in each tissue compartment. A core cancer progression signature was distilled using the increased signaling specificity of downstream oncogene effectors and subjected to network modeling. Network topology predicted that tumor development depends on specific extracellular matrix-interacting network hubs. Blockade of one such hub, the beta1 integrin subunit, disrupted network gene expression and attenuated tumorigenesis in vivo. Thus, integrating network modeling and temporal gene expression analysis of inducible human neoplasia provides an approach to prioritize and characterize genes functioning in cancer progression.

Sustained angiogenesis, through either local sprouting (angiogenesis) or the recruitment of bone marrow-derived cells (BMDCs) (vasculogenesis), is essential to the development of a tumor. How BMDCs are recruited to the tumor and their contribution to the tumor vasculature is poorly understood. Here, we demonstrate that both IL-8 and angiogenin contribute to the complementary pathways of angiogenesis and BMDC mobilization to increase tumor growth. These two factors are regulated by PHD2 in a HIF-independent but NF-kappaB-dependent manner. PHD2 levels are decreased in human cancers, compared with corresponding normal tissue, and correlate with an increase in mature blood vessels. Thus, PHD2 plays a critical role in regulating tumor angiogenesis.

The sirtuin Sirt6 is a NAD-dependent histone deacetylase that is implicated in gene regulation and lifespan control. Sirt6 can interact with the stress-responsive transcription factor NF-κB and regulate some NF-κB target genes, but the full scope of Sirt6 target genes as well as dynamics of Sirt6 occupancy on chromatin are not known. Here we map Sirt6 occupancy on mouse promoters genome-wide and show that Sirt6 occupancy is highly dynamic in response to TNF-α. More than half of Sirt6 target genes are only revealed upon stress-signaling. The majority of genes bound by NF-κB subunit RelA recruit Sirt6, and dynamic Sirt6 relocalization is largely driven in a RelA-dependent manner. Integrative analysis with global gene expression patterns in wild-type, Sirt6-/-, and double Sirt6-/- RelA-/- cells reveals the epistatic relationships between Sirt6 and RelA in shaping diverse temporal patterns of gene expression. Genes under the direct joint control of Sirt6 and RelA include several with prominent roles in cell senescence and organismal aging. These data suggest dynamic chromatin relocalization of Sirt6 as a key output of NF-κB signaling in stress response and aging.

We present an integrative approach, SeqFold, that combines high-throughput RNA structure profiling data with computational prediction for genome-scale reconstruction of RNA secondary structures. SeqFold transforms experimental RNA structure information into a structure preference profile (SPP) and uses it to select stable RNA structure candidates representing the structure ensemble. Under a high-dimensional classification framework, SeqFold efficiently matches a given SPP to the most likely cluster of structures sampled from the Boltzmann-weighted ensemble. SeqFold is able to incorporate diverse types of RNA structure profiling data, including parallel analysis of RNA structure (PARS), selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), fragmentation sequencing (FragSeq) data generated by deep sequencing, and conventional SHAPE data. Using the known structures of a wide range of mRNAs and noncoding RNAs as benchmarks, we demonstrate that SeqFold outperforms or matches existing approaches in accuracy and is more robust to noise in experimental data. Application of SeqFold to reconstruct the secondary structures of the yeast transcriptome reveals the diverse impact of RNA secondary structure on gene regulation, including translation efficiency, transcription initiation, and protein-RNA interactions. SeqFold can be easily adapted to incorporate any new types of high-throughput RNA structure profiling data and is widely applicable to analyze RNA structures in any transcriptome.

Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types.

Complex biomedical analyses require the use of multiple software tools in concert and remain challenging for much of the biomedical research community. We introduce GenomeSpace (http://www.genomespace.org), a cloud-based, cooperative community resource that currently supports the streamlined interaction of 20 bioinformatics tools and data resources. To facilitate integrative analysis by non-programmers, it offers a growing set of 'recipes', short workflows to guide investigators through high-utility analysis tasks.

Here we survey variation and dynamics of active regulatory elements genome-wide using longitudinal samples from human individuals. We applied Assay of Transposase Accessible Chromatin with sequencing (ATAC-seq) to map chromatin accessibility in primary CD4+ T cells isolated from standard blood draws of 12 healthy volunteers over time, from cancer patients, and during T cell activation. Over 4,000 predicted regulatory elements (7.2%) showed reproducible variation in accessibility between individuals. Gender was the most significant attributable source of variation. ATAC-seq revealed previously undescribed elements that escape X chromosome inactivation and predicted gender-specific gene regulatory networks across autosomes, which coordinately affect genes with immune function. Noisy regulatory elements with personal variation in accessibility are significantly enriched for autoimmune disease loci. Over one third of regulome variation lacked genetic variation in cis, suggesting contributions from environmental or epigenetic factors. These results refine concepts of human individuality and provide a foundational reference for comparing disease-associated regulomes.

Recurrent mutations in cohesin complex proteins have been identified in pre-leukemic hematopoietic stem cells and during the early development of acute myeloid leukemia and other myeloid malignancies. Although cohesins are involved in chromosome separation and DNA damage repair, cohesin complex functions during hematopoiesis and leukemic development are unclear. Here, we show that mutant cohesin proteins block differentiation of human hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo and enforce stem cell programs. These effects are restricted to immature HSPC populations, where cohesin mutants show increased chromatin accessibility and likelihood of transcription factor binding site occupancy by HSPC regulators including ERG, GATA2, and RUNX1, as measured by ATAC-seq and ChIP-seq. Epistasis experiments show that silencing these transcription factors rescues the differentiation block caused by cohesin mutants. Together, these results show that mutant cohesins impair HSPC differentiation by controlling chromatin accessibility and transcription factor activity, possibly contributing to leukemic disease.

DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3's interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5'UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3's role in this process. Arsenite-induced stress shifts DDX3 binding from the 5'UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.

Studies in model organisms suggest that aged cells can be functionally rejuvenated, but whether this concept applies to human skin is unclear. Here we apply 3'-end sequencing for expression quantification ("3-seq") to discover the gene expression program associated with human photoaging and intrinsic skin aging (collectively termed "skin aging"), and the impact of broadband light (BBL) treatment. We find that skin aging was associated with a significantly altered expression level of 2,265 coding and noncoding RNAs, of which 1,293 became "rejuvenated" after BBL treatment; i.e., they became more similar to their expression level in youthful skin. Rejuvenated genes (RGs) included several known key regulators of organismal longevity and their proximal long noncoding RNAs. Skin aging is not associated with systematic changes in 3'-end mRNA processing. Hence, BBL treatment can restore gene expression pattern of photoaged and intrinsically aged human skin to resemble young skin. In addition, our data reveal, to our knowledge, a previously unreported set of targets that may lead to new insights into the human skin aging process.

When different types of functional genomics data are generated on single cells from different samples of cells from the same heterogeneous population, the clustering of cells in the different samples should be coupled. We formulate this "coupled clustering" problem as an optimization problem and propose the method of coupled nonnegative matrix factorizations (coupled NMF) for its solution. The method is illustrated by the integrative analysis of single-cell RNA-sequencing (RNA-seq) and single-cell ATAC-sequencing (ATAC-seq) data.

Over 90% of genetic variants associated with complex human traits map to non-coding regions, but little is understood about how they modulate gene regulation in health and disease. One possible mechanism is that genetic variants affect the activity of one or more cis-regulatory elements leading to gene expression variation in specific cell types. To identify such cases, we analyzed ATAC-seq and RNA-seq profiles from stimulated primary CD4+ T cells in up to 105 healthy donors. We found that regions of accessible chromatin (ATAC-peaks) are co-accessible at kilobase and megabase resolution, consistent with the three-dimensional chromatin organization measured by in situ Hi-C in T cells. Fifteen percent of genetic variants located within ATAC-peaks affected the accessibility of the corresponding peak (local-ATAC-QTLs). Local-ATAC-QTLs have the largest effects on co-accessible peaks, are associated with gene expression and are enriched for autoimmune disease variants. Our results provide insights into how natural genetic variants modulate cis-regulatory elements, in isolation or in concert, to influence gene expression.

Stem cell regulation and hierarchical organization of human skeletal progenitors remain largely unexplored. Here, we report the isolation of a self-renewing and multipotent human skeletal stem cell (hSSC) that generates progenitors of bone, cartilage, and stroma, but not fat. Self-renewing and multipotent hSSCs are present in fetal and adult bones and can also be derived from BMP2-treated human adipose stroma (B-HAS) and induced pluripotent stem cells (iPSCs). Gene expression analysis of individual hSSCs reveals overall similarity between hSSCs obtained from different sources and partially explains skewed differentiation toward cartilage in fetal and iPSC-derived hSSCs. hSSCs undergo local expansion in response to acute skeletal injury. In addition, hSSC-derived stroma can maintain human hematopoietic stem cells (hHSCs) in serum-free culture conditions. Finally, we combine gene expression and epigenetic data of mouse skeletal stem cells (mSSCs) and hSSCs to identify evolutionarily conserved and divergent pathways driving SSC-mediated skeletogenesis. VIDEO ABSTRACT.

Direct reprogramming of fibroblasts to neurons induces widespread cellular and transcriptional reconfiguration. Here, we characterized global epigenomic changes during the direct reprogramming of mouse fibroblasts to neurons using whole-genome base-resolution DNA methylation (mC) sequencing. We found that the pioneer transcription factor Ascl1 alone is sufficient for inducing the uniquely neuronal feature of non-CG methylation (mCH), but co-expression of Brn2 and Mytl1 was required to establish a global mCH pattern reminiscent of mature cortical neurons. Ascl1 alone induced promoter CG methylation (mCG) of fibroblast specific genes, while BAM overexpression additionally targets a competing myogenic program and directs a more faithful conversion to neuronal cells. Ascl1 induces local demethylation at its binding sites. Surprisingly, co-expression with Brn2 and Mytl1 inhibited the ability of Ascl1 to induce demethylation, suggesting a contextual regulation of transcription factor - epigenome interaction. Finally, we found that de novo methylation by DNMT3A is required for efficient neuronal reprogramming.

Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.

RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, called 'RNA structuromes'. However, previous whole-cell analyses lacked the resolution to unravel the landscape and also the regulatory mechanisms of RNA structural changes across subcellular compartments. Here we reveal the RNA structuromes in three compartments, chromatin, nucleoplasm and cytoplasm, in human and mouse cells. The cytotopic structuromes substantially expand RNA structural information and enable detailed investigation of the central role of RNA structure in linking transcription, translation and RNA decay. We develop a resource with which to visualize the interplay of RNA-protein interactions, RNA modifications and RNA structure and predict both direct and indirect reader proteins of RNA modifications. We also validate a novel role for the RNA-binding protein LIN28A as an N6-methyladenosine modification 'anti-reader'. Our results highlight the dynamic nature of RNA structures and its functional importance in gene regulation.

Transposable elements (TEs) comprise nearly half of the human genome and are often transcribed or exhibit cis-regulatory properties with unknown function in specific processes such as heart development. In the case of endogenous retroviruses (ERVs), a TE subclass, experimental interrogation is constrained as many are primate-specific or human-specific. Here, we use primate pluripotent stem-cell-derived cardiomyocytes that mimic fetal cardiomyocytes in vitro to discover hundreds of ERV transcripts from the primate-specific MER41 family, some of which are regulated by the cardiogenic transcription factor TBX5. The most significant of these are located within BANCR, a long non-coding RNA (lncRNA) exclusively expressed in primate fetal cardiomyocytes. Functional studies reveal that BANCR promotes cardiomyocyte migration in vitro and ventricular enlargement in vivo. We conclude that recently evolved TE loci such as BANCR may represent potent de novo developmental regulatory elements that can be interrogated with species-matching pluripotent stem cell models.

Understanding the genomic logic that underlies cellular diversity and developmental potential in the human pancreas will accelerate the growth of cell replacement therapies and reveal genetic risk mechanisms in diabetes. Here, we identified and characterized thousands of chromatin regions governing cell-specific gene regulation in human pancreatic endocrine and exocrine lineages, including islet β cells, α cells, duct, and acinar cells. Our findings have captured cellular ontogenies at the chromatin level, identified lineage-specific regulators potentially acting on these sites, and uncovered hallmarks of regulatory plasticity between cell types that suggest mechanisms to regenerate β cells from pancreatic endocrine or exocrine cells. Our work shows that disease risk variants related to pancreas are significantly enriched in these regulatory regions and reveals previously unrecognized links between endocrine and exocrine pancreas in diabetes risk.

Here, we present Perturb-ATAC, a method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning and identified a hierarchy of TFs that govern B cell state, variation, and disease-associated cis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules of cis-elements that specify keratinocyte fate. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful strategy to dissect gene regulatory networks in development and disease.

The SARS-CoV-2 coronavirus is driving a global pandemic, but its biological mechanisms are less well understood. SARS-CoV-2 is an RNA virus whose multiple genomic and subgenomic RNA (sgRNA) transcripts hijack the host cell's machinery, located across distinct cytotopic locations. Subcellular localization of its viral RNA could play important roles in viral replication and host antiviral immune response. Here we perform computational modeling of SARS-CoV-2 viral RNA localization across eight subcellular neighborhoods. We compare hundreds of SARS-CoV-2 genomes to the human transcriptome and other coronaviruses and perform systematic sub-sequence analyses to identify the responsible signals. Using state-of-the-art machine learning models, we predict that the SARS-CoV-2 RNA genome and all sgRNAs are enriched in the host mitochondrial matrix and nucleolus. The 5' and 3' viral untranslated regions possess the strongest and most distinct localization signals. We discuss the mitochondrial localization signal in relation to the formation of double-membrane vesicles, a critical stage in the coronavirus life cycle. Our computational analysis serves as a hypothesis generation tool to suggest models for SARS-CoV-2 biology and inform experimental efforts to combat the virus.