An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

The alx3 gene shapes the zebrafish neurocranium by regulating frontonasal neural crest cell differentiation timing.

During craniofacial development, different populations of cartilage- and bone-forming cells develop in precise locations in the head. Most of these cells are derived from pluripotent cranial neural crest cells and differentiate with distinct developmental timing and cellular morphologies. The mechanisms that divide neural crest cells into discrete populations are not fully understood. Here, we use single-cell RNA sequencing to transcriptomically define different populations of cranial neural crest cells. We discovered that the gene family encoding the Alx transcription factors is enriched in the frontonasal population of neural crest cells. Genetic mutant analyses indicate that alx3 functions to regulate the distinct differentiation timing and cellular morphologies among frontonasal neural crest cell subpopulations. This study furthers our understanding of how genes controlling developmental timing shape craniofacial skeletal elements.

Adrenal gene expression dynamics support hibernation in 13-lined ground squirrels.

Hibernation is a natural model of extreme physiology in a mammal. Throughout winter, small hibernators repeatedly undergo rapid, dramatic swings in body temperature, perfusion, and oxygen delivery. To gain insight into the molecular mechanisms that support homeostasis despite the numerous challenges posed by this dynamic physiology, we collected 13-lined ground squirrel adrenal glands from at least five individuals representing six key timepoints across the year using body temperature telemetry. Differentially expressed genes were identified using RNA-seq, revealing both strong seasonal and torpor-arousal cycle effects on gene expression. Two novel findings emerge from this study. First, transcripts encoding multiple genes involved in steroidogenesis decreased seasonally. Taken together with morphometric analyses, the data are consistent with preservation of mineralocorticoids but suppression of glucocorticoid and androgen output throughout winter hibernation. Second, a temporally orchestrated, serial gene expression program unfolds across the brief arousal periods. This program initiates during early rewarming with the transient activation of a set of immediate early response (IER) genes, comprised of both transcription factors and the RNA degradation proteins that assure their rapid turnover. This pulse in turn activates a cellular stress response program to restore proteostasis comprised of protein turnover, synthesis, and folding machinery. These and other data support a general model for gene expression across the torpor-arousal cycle that is facilitated in synchrony with whole body temperature shifts; induction of the immediate early response upon rewarming activates a proteostasis program followed by a restored tissue-specific gene expression profile enabling renewal, repair, and survival of the torpid state.NEW & NOTEWORTHY This pioneer study of adrenal gland gene expression dynamics in hibernating ground squirrels leverages the power of RNA-seq on multiple precisely timed samples to demonstrate: 1) steroidogenesis is seasonally reorganized to preserve aldosterone at the expense of glucocorticoids and androgens throughout winter hibernation; 2) a serial gene expression program unfolds during each short arousal whereby immediate early response genes induce the gene expression machinery that restores proteostasis and the cell-specific expression profile before torpor reentry.

Single-cell RNA sequencing and binary hierarchical clustering define lung interstitial macrophage heterogeneity in response to hypoxia.

Few studies have examined lung interstitial macrophage (IM) molecular phenotypes after being exposed to hypoxia in vivo at the single-cell level, even though macrophages contribute to hypoxic pulmonary hypertension (PH). We aimed to determine IM diversity and its association with hypoxia-induced PH. We hypothesized that integrating single-cell RNA sequencing (scRNAseq) and binary hierarchal clustering (BHC) could resolve IM heterogeneity under normal homeostatic conditions and changes induced by hypoxia exposure. Cx3cr1GFP/+ reporter mice were exposed to normoxic conditions (∼21% [Formula: see text]) or exposed to 1 day (D1) or 7 days (D7) of hypoxia (∼10% [Formula: see text]). We used flow cytometry to isolate Cx3cr1+ IMs and the 10X Genomics platform for scRNAseq, Cell Ranger, Seurat, ClusterMap, monocle, ingenuity pathway analysis, and Fisher's exact test (q value < 0.05) for functional investigations. n = 374 (normoxia), n = 2,526 (D1), and n = 1,211 (D7) IMs were included in the analyses. We identified three normoxia-related cell types, five hypoxia-associated cell types that emerged at D1, and three that appeared at D7. We describe the existence of a putative resident trained innate IM, which is present in normoxia, transiently depleted at D1, and recovered after 7 days of sustained hypoxia. We also define a rare putative pathogenic population associated with transcripts implicated in PH development that emerges at D7. In closing, we describe the successful integration of BHC with scRNAseq to determine IM heterogeneity and its association with PH. These results shed light on how resident-trained innate IMs become more heterogeneous but ultimately accustomed to hypoxia.

LABRAT reveals association of alternative polyadenylation with transcript localization, RNA binding protein expression, transcription speed, and cancer survival.

BACKGROUND: The sequence content of the 3' UTRs of many mRNA transcripts is regulated through alternative polyadenylation (APA). The study of this process using RNAseq data, though, has been historically challenging. RESULTS: To combat this problem, we developed LABRAT, an APA isoform quantification method. LABRAT takes advantage of newly developed transcriptome quantification techniques to accurately determine relative APA site usage and how it varies across conditions. Using LABRAT, we found consistent relationships between gene-distal APA and subcellular RNA localization in multiple cell types. We also observed connections between transcription speed and APA site choice as well as tumor-specific transcriptome-wide shifts in APA isoform abundance in hundreds of patient-derived tumor samples that were associated with patient prognosis. We investigated the effects of APA on transcript expression and found a weak overall relationship, although many individual genes showed strong correlations between relative APA isoform abundance and overall gene expression. We interrogated the roles of 191 RNA-binding proteins in the regulation of APA isoforms, finding that dozens promote broad, directional shifts in relative APA isoform abundance both in vitro and in patient-derived samples. Finally, we find that APA site shifts in the two classes of APA, tandem UTRs and alternative last exons, are strongly correlated across many contexts, suggesting that they are coregulated. CONCLUSIONS: We conclude that LABRAT has the ability to accurately quantify APA isoform ratios from RNAseq data across a variety of sample types. Further, LABRAT is able to derive biologically meaningful insights that connect APA isoform regulation to cellular and molecular phenotypes.

Recovery and analysis of transcriptome subsets from pooled single-cell RNA-seq libraries.

Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries often prevents full characterization of transcriptomes from individual cells. To extract more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in cell-centric and gene-centric modes to isolate cDNA fragments from scRNA-seq libraries. First, we resampled the transcriptomes of rare, single megakaryocytes from a complex mixture of lymphocytes and analyzed them in a second round of DNA sequencing, yielding up to 20-fold greater sequencing depth per cell and increasing the number of genes detected per cell from a median of 1313 to 2002. We similarly isolated mRNAs from targeted T cells to improve the reconstruction of their VDJ-rearranged immune receptor mRNAs. Second, we isolated CD3D mRNA fragments expressed across cells in a scRNA-seq library prepared from a clonal T cell line, increasing the number of cells with detected CD3D expression from 59.7% to 100%. Transcriptome resampling is a general approach to recover targeted gene expression information from single-cell RNA sequencing libraries that enhances the utility of these costly experiments, and may be applicable to the targeted recovery of molecules from other single-cell assays.

Analysis of circulating breast cancer cell heterogeneity and interactions with peripheral blood mononuclear cells.

For solid tumors, extravasation of cancer cells and their survival in circulation represents a critical stage of the metastatic process that lacks complete understanding. Gaining insight into interactions between circulating tumor cells (CTCs) and other peripheral blood mononuclear cells (PBMCs) may provide valuable prognostic information. The purpose of this study was to use single-cell RNA-sequencing (scRNA-seq) of liquid biopsies from breast cancer patients to begin defining intravascular interactions. We captured CTCs from the peripheral blood of breast cancer patients using size-exclusion membranes followed by scRNA-seq of enriched CTCs and carry-over PBMCs. Transcriptome analysis identified two populations of CTCs: one enriched for transcripts indicative of estrogen responsiveness and increased proliferation and another enriched for transcripts characteristic of reduced proliferation and epithelial-mesenchymal transition (EMT). We applied interactome and pathway analysis to determine interactions between CTCs and other captured cells. Our analysis predicted for enhanced immune evasion in the CTC population with EMT characteristics. In addition, PD-1/PD-L1 pathway activation and T cell exhaustion were predicted in T cells isolated from breast cancer patients compared with normal T cells. We conclude that scRNA-seq of breast cancer CTCs generally stratifies them into two types based on their proliferative and epithelial state and differential potential to interact with PBMCs. Better understanding of CTC subtypes and their intravascular interactions may help design treatments directed against CTCs with high metastatic and immune-evasive competence.

Dynamic temperature-sensitive A-to-I RNA editing in the brain of a heterothermic mammal during hibernation.

RNA editing diversifies genomically encoded information to expand the complexity of the transcriptome. In ectothermic organisms, including Drosophila and Cephalopoda, where body temperature mirrors ambient temperature, decreases in environmental temperature lead to increases in A-to-I RNA editing and cause amino acid recoding events that are thought to be adaptive responses to temperature fluctuations. In contrast, endothermic mammals, including humans and mice, typically maintain a constant body temperature despite environmental changes. Here, A-to-I editing primarily targets repeat elements, rarely results in the recoding of amino acids, and plays a critical role in innate immune tolerance. Hibernating ground squirrels provide a unique opportunity to examine RNA editing in a heterothermic mammal whose body temperature varies over 30°C and can be maintained at 5°C for many days during torpor. We profiled the transcriptome in three brain regions at six physiological states to quantify RNA editing and determine whether cold-induced RNA editing modifies the transcriptome as a potential mechanism for neuroprotection at low temperature during hibernation. We identified 5165 A-to-I editing sites in 1205 genes with dynamically increased editing after prolonged cold exposure. The majority (99.6%) of the cold-increased editing sites are outside of previously annotated coding regions, 82.7% lie in SINE-derived repeats, and 12 sites are predicted to recode amino acids. Additionally, A-to-I editing frequencies increase with increasing cold-exposure, demonstrating that ADAR remains active during torpor. Our findings suggest that dynamic A-to-I editing at low body temperature may provide a neuroprotective mechanism to limit aberrant dsRNA accumulation during torpor in the mammalian hibernator.

Improvements to the HITS-CLIP protocol eliminate widespread mispriming artifacts.

BACKGROUND: High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) allows for high resolution, genome-wide mapping of RNA-binding proteins. This methodology is frequently used to validate predicted targets of microRNA binding, as well as direct targets of other RNA-binding proteins. Hence, the accuracy and sensitivity of binding site identification is critical. RESULTS: We found that substantial mispriming during reverse transcription results in the overrepresentation of sequences complementary to the primer used for reverse transcription. Up to 45 % of peaks in publicly available HITS-CLIP libraries are attributable to this mispriming artifact, and the majority of libraries have detectable levels of mispriming. We also found that standard techniques for validating microRNA-target interactions fail to differentiate between artifactual peaks and physiologically relevant peaks. CONCLUSIONS: Here, we present a modification to the HITS-CLIP protocol that effectively eliminates this artifact and improves the sensitivity and complexity of resulting libraries.

Characterization of a large human transgene following invasin-mediated delivery in a bacterial artificial chromosome.

Bacterial artificial chromosomes (BACs) are widely used in transgenesis, particularly for the humanization of animal models. Moreover, due to their extensive capacity, BACs provide attractive tools to study distal regulatory elements associated with large gene loci. However, despite their widespread use, little is known about the integration dynamics of these large transgenes in mammalian cells. Here, we investigate the post-integration structure of a ~260 kb BAC carrying the cystic fibrosis transmembrane conductance regulator (CFTR) locus following delivery by bacterial invasion and compare this to the outcome of a more routine lipid-based delivery method. We find substantial variability in integrated copy number and expression levels of the BAC CFTR transgene after bacterial invasion-mediated delivery. Furthermore, we frequently observed variation in the representation of different regions of the CFTR transgene within individual cell clones, indicative of BAC fragmentation. Finally, using fluorescence in situ hybridization, we observed that the integrated BAC forms extended megabase-scale structures in some clones that are apparently stably maintained at cell division. These data demonstrate that the utility of large BACs to investigate cis-regulatory elements in the genomic context may be limited by recombination events that complicate their use.

HITS-CLIP reveals key regulators of nuclear receptor signaling in breast cancer.

miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA-target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA-mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.

Nucleosome occupancy reveals regulatory elements of the CFTR promoter.

Access to regulatory elements of the genome can be inhibited by nucleosome core particles arranged along the DNA strand. Hence, sites that are accessible by transcription factors may be located by using nuclease digestion to identify the relative nucleosome occupancy of a genomic region. In order to define novel cis regulatory elements in the ∼2.7-kb promoter region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, we define its nucleosome occupancy. This profile reveals the precise positions of nucleosome-free regions (NFRs), both cell-type specific and others apparently unrelated to CFTR-expression level and offer the first high-resolution map of the chromatin structure of the entire CFTR promoter in relevant cell types. Several of these NFRs are strongly bound by nuclear factors in a sequence-specific manner, and directly influence CFTR promoter activity. Sequences within the NFR1 and NFR4 elements are highly conserved in many human gene promoters. Moreover, NFR1 contributes to promoter activity of another gene, angiopoietin-like 3 (ANGPTL3), while NFR4 is constitutively nucleosome-free in promoters genome wide. Conserved motifs within NFRs of the CFTR promoter also show a high level of protection from DNase I digestion genome-wide, and likely have important roles in the positioning of nucleosome core particles more generally.

Antigen-driven CD8 + T cell clonal expansion is a prominent feature of MASH in humans and mice.

Background and Aims: Chronic liver disease due to metabolic dysfunction-associated steatohepatitis (MASH) is a rapidly increasing global epidemic. MASH progression is a consequence of the complex interplay between inflammatory insults and dysregulated hepatic immune responses. T lymphocytes have been shown to accumulate in the liver during MASH, but the cause and consequence of T cell accumulation in the liver remain unclear. Our study aimed to define the phenotype and T cell receptor diversity of T cells from human cirrhotic livers and an animal model of MASH to begin resolving their function in disease. Approach and Results: In these studies, we evaluated differences in T cell phenotype in the context of liver disease we isolated liver resident T cell populations from individuals with cirrhosis and a murine model of MASH. Using both 5' single cell sequencing and flow cytometry we defined the phenotype and T cell receptor repertoire of liver resident T cells during health and disease. Conclusions: MASH-induced cirrhosis and diet-induced MASH in mice resulted in the accumulation of activated and clonally expanded T cells in the liver. The clonally expanded T cells in the liver expressed markers of chronic antigenic stimulation, including PD1 , TIGIT and TOX . Overall, this study establishes for the first time that T cells undergo antigen-dependent clonal expansion and functional differentiation during the progression of MASH. These studies could lead to the identification of potential antigenic targets that drive T cell activation, clonal expansion, and recruitment to the liver during MASH.

Targeting Acute Myeloid Leukemia Stem Cells Through Perturbation of Mitochondrial Calcium.

Acute myeloid leukemia stem cells (LSCs) are uniquely reliant on oxidative phosphorylation (OXPHOS) for survival. Moreover, maintenance of OXPHOS is dependent on BCL-2, creating a therapeutic opportunity to target LSCs using the BCL-2 inhibitor venetoclax. While venetoclax-based regimens have shown promising clinical activity, the emergence of drug resistance is prevalent. Thus, in the present study, we investigated how mitochondrial properties may influence venetoclax responsiveness. Our data show that utilization of mitochondrial calcium is fundamentally different between drug-responsive and non-responsive LSCs. By comparison, venetoclax-resistant LSCs demonstrate a more active metabolic (i.e. OXPHOS) status with relatively high levels of calcium. Consequently, we tested genetic and pharmacological approaches to target the mitochondrial calcium uniporter, MCU. We demonstrate that inhibition of calcium uptake reduces OXPHOS and leads to eradication of venetoclax-resistant LSCs. These findings demonstrate a central role for calcium signaling in LSCs and provide an avenue for clinical management of venetoclax resistance.

A genome-wide analysis of open chromatin in human epididymis epithelial cells reveals candidate regulatory elements for genes coordinating epididymal function.

The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters, and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells, we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis, including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were overrepresented in epididymis-selective DHS identified epithelial transcription factors, including ELF5 and ELF3, the androgen receptor, Pax2, and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium.

Compromised nonsense-mediated RNA decay results in truncated RNA-binding protein production upon DUX4 expression.

Nonsense-mediated RNA decay (NMD) degrades transcripts carrying premature termination codons. NMD is thought to prevent the synthesis of toxic truncated proteins. However, whether loss of NMD results in widespread production of truncated proteins is unclear. A human genetic disease, facioscapulohumeral muscular dystrophy (FSHD), features acute inhibition of NMD upon expression of the disease-causing transcription factor, DUX4. Using a cell-based model of FSHD, we show production of truncated proteins from physiological NMD targets and find that RNA-binding proteins are enriched for aberrant truncations. The NMD isoform of one RNA-binding protein, SRSF3, is translated to produce a stable truncated protein, which is detected in FSHD patient-derived myotubes. Ectopic expression of truncated SRSF3 confers toxicity, and its downregulation is cytoprotective. Our results delineate the genome-scale impact of NMD loss. This widespread production of potentially deleterious truncated proteins has implications for FSHD biology as well as other genetic diseases where NMD is therapeutically modulated.

A Novel Type of Monocytic Leukemia Stem Cell Revealed by the Clinical Use of Venetoclax-Based Therapy.

The BCL2 inhibitor venetoclax has recently emerged as an important component of acute myeloid leukemia (AML) therapy. Notably, use of this agent has revealed a previously unrecognized form of pathogenesis characterized by monocytic disease progression. We demonstrate that this form of disease arises from a fundamentally different type of leukemia stem cell (LSC), which we designate as monocytic LSC (m-LSC), that is developmentally and clinically distinct from the more well-described primitive LSC (p-LSC). The m-LSC is distinguished by a unique immunophenotype (CD34-, CD4+, CD11b-, CD14-, CD36-), unique transcriptional state, reliance on purine metabolism, and selective sensitivity to cladribine. Critically, in some instances, m-LSC and p-LSC subtypes can co-reside in the same patient with AML and simultaneously contribute to overall tumor biology. Thus, our findings demonstrate that LSC heterogeneity has direct clinical significance and highlight the need to distinguish and target m-LSCs as a means to improve clinical outcomes with venetoclax-based regimens. SIGNIFICANCE: These studies identify and characterize a new type of human acute myeloid LSC that is responsible for monocytic disease progression in patients with AML treated with venetoclax-based regimens. Our studies describe the phenotype, molecular properties, and drug sensitivities of this unique LSC subclass. This article is featured in Selected Articles from This Issue, p. 1949.

MicroRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene.

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex temporal and spatial pattern of expression that is controlled by multiple cis-acting elements interacting with the basal promoter. Although significant progress has been made towards understanding these genomic elements, there have been no reports of post-transcriptional regulation of CFTR by miRNAs (microRNAs). In the present study, we identify two miRNAs, hsa-miR-145 and hsa-miR-494, which regulate CFTR expression by directly targeting discrete sites in the CFTR 3' UTR (untranslated region). We show that at least 12 miRNAs are capable of repressing endogenous CFTR mRNA expression in the Caco-2 cell line. Ten of these also inhibit expression of a reporter construct containing the CFTR 3' UTR in one or more cell lines, and five repress endogenous CFTR protein expression in Caco-2 cells. Moreover, at least three are expressed in primary human airway epithelial cells, where CFTR expression is maintained at low levels in comparison with intestinal cell lines. Three of the miRNAs that target CFTR, hsa-miR-384, hsa-miR-494 and hsa-miR-1246, also inhibit expression of a reporter carrying the Na(+)-K(+)-Cl(-) co-transporter SLC12A2 [solute carrier family 12 (Na(+)-K(+)-Cl(-) transporters), member 2] 3' UTR, suggesting that these miRNAs may play a more general role in regulating chloride transport in epithelial cells.

Diverse cell-specific patterns of alternative polyadenylation in Drosophila.

Most genes in higher eukaryotes express isoforms with distinct 3' untranslated regions (3' UTRs), generated by alternative polyadenylation (APA). Since 3' UTRs are predominant locations of post-transcriptional regulation, APA can render such programs conditional, and can also alter protein sequences via alternative last exon (ALE) isoforms. We previously used 3'-sequencing from diverse Drosophila samples to define multiple tissue-specific APA landscapes. Here, we exploit comprehensive single nucleus RNA-sequencing data (Fly Cell Atlas) to elucidate cell-type expression of 3' UTRs across >250 adult Drosophila cell types. We reveal the cellular bases of multiple tissue-specific APA/ALE programs, such as 3' UTR lengthening in differentiated neurons and 3' UTR shortening in spermatocytes and spermatids. We trace dynamic 3' UTR patterns across cell lineages, including in the male germline, and discover new APA patterns in the intestinal stem cell lineage. Finally, we correlate expression of RNA binding proteins (RBPs), miRNAs and global levels of cleavage and polyadenylation (CPA) factors in several cell types that exhibit characteristic APA landscapes, yielding candidate regulators of transcriptome complexity. These analyses provide a comprehensive foundation for future investigations of mechanisms and biological impacts of alternative 3' isoforms across the major cell types of this widely-studied model organism.

The role of CTCF in coordinating the expression of single gene loci.

The CCCTC-binding factor (CTCF), which binds insulator elements in vertebrates, also facilitates coordinated gene expression at several gene clusters, including the ß-globin, Igf2/H19 (insulin like growth factor 2/H19 noncoding RNA), and major histocompatibility complex (MHC) class II loci. CTCF controls expression of these genes both by enabling insulator function and facilitating higher order chromatin interactions. While the role of CTCF in gene regulation is best studied at these multi-gene loci, there is also evidence that CTCF contributes to the regulated expression of single genes. Here, we discuss how CTCF participates in coordinating gene expression at the CFTR (cystic fibrosis transmembrane conductance regulator) and IFNG (interferon-gamma) loci. We consider the structural similarities between the loci with regard to CTCF-binding elements, the possible interaction between nuclear receptors and CTCF, and the role of CTCF in chromatin looping at these genes. These comparisons reveal a functional model that may be applicable to other single-gene loci that require CTCF for coordinated gene expression.