High-resolution CTCF footprinting reveals impact of chromatin state on cohesin extrusion dynamics.

DNA looping is vital for establishing many enhancer-promoter interactions. While CTCF is known to anchor many cohesin-mediated loops, the looped chromatin fiber appears to predominantly exist in a poorly characterized actively extruding state. To better characterize extruding chromatin loop structures, we used CTCF MNase HiChIP data to determine both CTCF binding at high resolution and 3D contact information. Here we present FactorFinder, a tool that identifies CTCF binding sites at near base-pair resolution. We leverage this substantial advance in resolution to determine that the fully extruded (CTCF-CTCF) state is rare genome-wide with locus-specific variation from ~1-10%. We further investigate the impact of chromatin state on loop extrusion dynamics, and find that active enhancers and RNA Pol II impede cohesin extrusion, facilitating an enrichment of enhancer-promoter contacts in the partially extruded loop state. We propose a model of topological regulation whereby the transient, partially extruded states play active roles in transcription.

Tracing cancer evolution and heterogeneity using Hi-C.

Chromosomal rearrangements can initiate and drive cancer progression, yet it has been challenging to evaluate their impact, especially in genetically heterogeneous solid cancers. To address this problem we developed HiDENSEC, a new computational framework for analyzing chromatin conformation capture in heterogeneous samples that can infer somatic copy number alterations, characterize large-scale chromosomal rearrangements, and estimate cancer cell fractions. After validating HiDENSEC with in silico and in vitro controls, we used it to characterize chromosome-scale evolution during melanoma progression in formalin-fixed tumor samples from three patients. The resulting comprehensive annotation of the genomic events includes copy number neutral translocations that disrupt tumor suppressor genes such as NF1, whole chromosome arm exchanges that result in loss of CDKN2A, and whole-arm copy-number neutral loss of homozygosity involving PTEN. These findings show that large-scale chromosomal rearrangements occur throughout cancer evolution and that characterizing these events yields insights into drivers of melanoma progression.

A non-genetic switch triggers alternative telomere lengthening and cellular immortalization in ATRX deficient cells.

Alternative Lengthening of Telomeres (ALT) is an aberrant DNA recombination pathway which grants replicative immortality to approximately 10% of all cancers. Despite this high prevalence of ALT in cancer, the mechanism and genetics by which cells activate this pathway remain incompletely understood. A major challenge in dissecting the events that initiate ALT is the extremely low frequency of ALT induction in human cell systems. Guided by the genetic lesions that have been associated with ALT from cancer sequencing studies, we genetically engineered primary human pluripotent stem cells to deterministically induce ALT upon differentiation. Using this genetically defined system, we demonstrate that disruption of the p53 and Rb pathways in combination with ATRX loss-of-function is sufficient to induce all hallmarks of ALT and results in functional immortalization in a cell type-specific manner. We further demonstrate that ALT can be induced in the presence of telomerase, is neither dependent on telomere shortening nor crisis, but is rather driven by continuous telomere instability triggered by the induction of differentiation in ATRX-deficient stem cells.

Loss of epigenetic information as a cause of mammalian aging.

All living things experience an increase in entropy, manifested as a loss of genetic and epigenetic information. In yeast, epigenetic information is lost over time due to the relocalization of chromatin-modifying proteins to DNA breaks, causing cells to lose their identity, a hallmark of yeast aging. Using a system called "ICE" (inducible changes to the epigenome), we find that the act of faithful DNA repair advances aging at physiological, cognitive, and molecular levels, including erosion of the epigenetic landscape, cellular exdifferentiation, senescence, and advancement of the DNA methylation clock, which can be reversed by OSK-mediated rejuvenation. These data are consistent with the information theory of aging, which states that a loss of epigenetic information is a reversible cause of aging.

Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer.

The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodelling1 and is altered in over 20% of cancers2,3. Here we developed a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supra-physiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumour growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers.

Defining the expression of piRNA and transposable elements in Drosophila ovarian germline stem cells and somatic support cells.

Piwi-interacting RNAs (piRNAs) are important for repressing transposable elements (TEs) and modulating gene expression in germ cells, thereby maintaining genome stability and germ cell function. Although they are also important for maintaining germline stem cells (GSCs) in the Drosophila ovary by repressing TEs and preventing DNA damage, piRNA expression has not been investigated in GSCs or their early progeny. Here, we show that the canonical piRNA clusters are more active in GSCs and their early progeny than late germ cells and also identify more than 3,000 new piRNA clusters from deep sequencing data. The increase in piRNAs in GSCs and early progeny can be attributed to both canonical and newly identified piRNA clusters. As expected, piRNA clusters in GSCs, but not those in somatic support cells (SCs), exhibit ping-pong signatures. Surprisingly, GSCs and early progeny express more TE transcripts than late germ cells, suggesting that the increase in piRNA levels may be related to the higher levels of TE transcripts in GSCs and early progeny. GSCs also have higher piRNA levels and lower TE levels than SCs. Furthermore, the 3' UTRs of 171 mRNA transcripts may produce sense, antisense, or dual-stranded piRNAs. Finally, we show that alternative promoter usage and splicing are frequently used to modulate gene function in GSCs and SCs. Overall, this study has provided important insight into piRNA production and TE repression in GSCs and SCs. The rich information provided by this study will be a beneficial resource to the fields of piRNA biology and germ cell development.

A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing.

FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.

Structural Variation Detection by Proximity Ligation from Formalin-Fixed, Paraffin-Embedded Tumor Tissue.

The clinical management and therapy of many solid tumor malignancies depends on detection of medically actionable or diagnostically relevant genetic variation. However, a principal challenge for genetic assays from tumors is the fragmented and chemically damaged state of DNA in formalin-fixed, paraffin-embedded (FFPE) samples. From highly fragmented DNA and RNA there is no current technology for generating long-range DNA sequence data as is required to detect genomic structural variation or long-range genotype phasing. We have developed a high-throughput chromosome conformation capture approach for FFPE samples that we call Fix-C, which is similar in concept to Hi-C. Fix-C enables structural variation detection from archival FFPE samples. This method was applied to 15 clinical adenocarcinoma- and sarcoma-positive control specimens spanning a broad range of tumor purities. In this panel, Fix-C analysis achieves a 90% concordance rate with fluorescence in situ hybridization assays, the current clinical gold standard. In addition, novel structural variation undetected by other methods could be identified, and long-range chromatin configuration information recovered from these FFPE samples harboring highly degraded DNA. This powerful approach will enable detailed resolution of global genome rearrangement events during cancer progression from FFPE material and will inform the development of targeted molecular diagnostic assays for patient care.

Regulated Intron Removal Integrates Motivational State and Experience.

Myriad experiences produce transient memory, yet, contingent on the internal state of the organism and the saliency of the experience, only some memories persist over time. How experience and internal state influence the duration of memory at the molecular level remains unknown. A self-assembled aggregated state of Drosophila Orb2A protein is required specifically for long-lasting memory. We report that in the adult fly brain the mRNA encoding Orb2A protein exists in an unspliced non-protein-coding form. The convergence of experience and internal drive transiently increases the spliced protein-coding Orb2A mRNA. A screen identified pasilla, the fly ortholog of mammalian Nova-1/2, as a mediator of Orb2A mRNA processing. A single-nucleotide substitution in the intronic region that reduces Pasilla binding and intron removal selectively impairs long-term memory. We posit that pasilla-mediated processing of unspliced Orb2A mRNA integrates experience and internal state to control Orb2A protein abundance and long-term memory formation.

Aubergine Controls Germline Stem Cell Self-Renewal and Progeny Differentiation via Distinct Mechanisms.

Piwi family protein Aubergine (Aub) maintains genome integrity in late germ cells of the Drosophila ovary through Piwi-associated RNA-mediated repression of transposon activities. Although it is highly expressed in germline stem cells (GSCs) and early progeny, it remains unclear whether it plays any roles in early GSC lineage development. Here we report that Aub promotes GSC self-renewal and GSC progeny differentiation. RNA-iCLIP results show that Aub binds the mRNAs encoding self-renewal and differentiation factors in cultured GSCs. Aub controls GSC self-renewal by preventing DNA-damage-induced Chk2 activation and by translationally controlling the expression of self-renewal factors. It promotes GSC progeny differentiation by translationally controlling the expression of differentiation factors, including Bam. Therefore, this study reveals a function of Aub in GSCs and their progeny, which promotes translation of self-renewal and differentiation factors by directly binding to its target mRNAs and interacting with translational initiation factors.

Sex-Specific Transcript Diversity in the Fly Head Is Established during Pupal Stages and Adulthood and Is Largely Independent of the Mating Process and the Germline.

Alternative splicing (AS), the process which generates multiple RNA and protein isoforms from a single pre-mRNA, greatly contributes to transcript diversity and compensates for the fact that the gene number does not scale with organismal complexity. A number of genomic approaches have established that the extent of AS is much higher than previously expected, raising questions on its spatio-temporal regulation and function. In the present study, we address AS in the context of sex-specific neuronal development in the model Drosophila melanogaster. We report that at least 47 genes display sex-specific AS in the adult fly head. Unlike targets of the classical Sex lethal-dependent sex determination cascade, sex-specific isoforms of the vast majority of these genes are not present during larval development but start accumulating during metamorphosis or later, indicating the existence of novel mechanisms in the induction of sex-specific AS. We also established that sex-specific AS in the adult fly head is largely independent of the germline or the mating process. Finally, we investigated the role of sex-specific AS of the sulfotransferase Tango13 pre-mRNA and provide first evidence that differential expression of certain isoforms of this protein significantly affects courtship and mating behavior in male flies.

Modulation of the splicing regulatory function of SRSF10 by a novel compound that impairs HIV-1 replication.

We recently identified the 4-pyridinone-benzisothiazole carboxamide compound 1C8 as displaying strong anti-HIV-1 potency against a variety of clinical strains in vitro. Here we show that 1C8 decreases the expression of HIV-1 and alters splicing events involved in the production of HIV-1 mRNAs. Although 1C8 was designed to be a structural mimic of the fused tetracyclic indole compound IDC16 that targets SRSF1, it did not affect the splice site shifting activity of SRSF1. Instead, 1C8 altered splicing regulation mediated by SRSF10. Depleting SRSF10 by RNA interference affected viral splicing and, like 1C8, decreased expression of Tat, Gag and Env. Incubating cells with 1C8 promoted the dephosphorylation of SRSF10 and increased its interaction with hTra2ß, a protein previously implicated in the control of HIV-1 RNA splicing. While 1C8 affects the alternative splicing of cellular transcripts controlled by SRSF10 and hTra2ß, concentrations greater than those needed to inhibit HIV-1 replication were required to elicit significant alterations. Thus, the ability of 1C8 to alter the SRSF10-dependent splicing of HIV-1 transcripts, with minor effects on cellular splicing, supports the view that SRSF10 may be used as a target for the development of new anti-viral agents.

Exon junction complex proteins bind nascent transcripts independently of pre-mRNA splicing in Drosophila melanogaster.

Although it is currently understood that the exon junction complex (EJC) is recruited on spliced mRNA by a specific interaction between its central protein, eIF4AIII, and splicing factor CWC22, we found that eIF4AIII and the other EJC core proteins Y14 and MAGO bind the nascent transcripts of not only intron-containing but also intronless genes on Drosophila polytene chromosomes. Additionally, Y14 ChIP-seq demonstrates that association with transcribed genes is also splicing-independent in Drosophila S2 cells. The association of the EJC proteins with nascent transcripts does not require CWC22 and that of Y14 and MAGO is independent of eIF4AIII. We also show that eIF4AIII associates with both polysomal and monosomal RNA in S2 cell extracts, whereas Y14 and MAGO fractionate separately. Cumulatively, our data indicate a global role of eIF4AIII in gene expression, which would be independent of Y14 and MAGO, splicing, and of the EJC, as currently understood.

Chromosome-scale shotgun assembly using an in vitro method for long-range linkage.

Long-range and highly accurate de novo assembly from short-read data is one of the most pressing challenges in genomics. Recently, it has been shown that read pairs generated by proximity ligation of DNA in chromatin of living tissue can address this problem, dramatically increasing the scaffold contiguity of assemblies. Here, we describe a simpler approach ("Chicago") based on in vitro reconstituted chromatin. We generated two Chicago data sets with human DNA and developed a statistical model and a new software pipeline ("HiRise") that can identify poor quality joins and produce accurate, long-range sequence scaffolds. We used these to construct a highly accurate de novo assembly and scaffolding of a human genome with scaffold N50 of 20 Mbp. We also demonstrated the utility of Chicago for improving existing assemblies by reassembling and scaffolding the genome of the American alligator. With a single library and one lane of Illumina HiSeq sequencing, we increased the scaffold N50 of the American alligator from 508 kbp to 10 Mbp.

The SMC Loader Scc2 Promotes ncRNA Biogenesis and Translational Fidelity.

The Scc2-Scc4 complex is essential for loading the cohesin complex onto DNA. Cohesin has important roles in chromosome segregation, DSB repair, and chromosome condensation. Here we report that Scc2 is important for gene expression in budding yeast. Scc2 and the transcriptional regulator Paf1 collaborate to promote the production of Box H/ACA snoRNAs which guide pseudouridylation of RNAs including ribosomal RNA. Mutation of SCC2 was associated with defects in the production of ribosomal RNA, ribosome assembly, and splicing. While the scc2 mutant does not have a general defect in protein synthesis, it shows increased frameshifting and reduced cap-independent translation. These findings suggest Scc2 normally promotes a gene expression program that supports translational fidelity. We hypothesize that translational dysfunction may contribute to the human disorder Cornelia de Lange syndrome, which is caused by mutations in NIPBL, the human ortholog of SCC2.

A Function for the hnRNP A1/A2 Proteins in Transcription Elongation.

The hnRNP A1 and A2 proteins regulate processes such as alternative pre-mRNA splicing and mRNA stability. Here, we report that a reduction in the levels of hnRNP A1 and A2 by RNA interference or their cytoplasmic retention by osmotic stress drastically increases the transcription of a reporter gene. Based on previous work, we propose that this effect may be linked to a decrease in the activity of the transcription elongation factor P-TEFb. Consistent with this hypothesis, the transcription of the reporter gene was stimulated when the catalytic component of P-TEFb, CDK9, was inhibited with DRB. While low levels of A1/A2 stimulated the association of RNA polymerase II with the reporter gene, they also increased the association of CDK9 with the repressor 7SK RNA, and compromised the recovery of promoter-distal transcription on the Kitlg gene after the release of pausing. Transcriptome analysis revealed that more than 50% of the genes whose expression was affected by the siRNA-mediated depletion of A1/A2 were also affected by DRB. RNA polymerase II-chromatin immunoprecipitation assays on DRB-treated and A1/A2-depleted cells identified a common set of repressed genes displaying increased occupancy of polymerases at promoter-proximal locations, consistent with pausing. Overall, our results suggest that lowering the levels of hnRNP A1/A2 elicits defective transcription elongation on a fraction of P-TEFb-dependent genes, hence favoring the transcription of P-TEFb-independent genes.

SR proteins control a complex network of RNA-processing events.

SR proteins are a well-conserved class of RNA-binding proteins that are essential for regulation of splice-site selection, and have also been implicated as key regulators during other stages of RNA metabolism. For many SR proteins, the complexity of the RNA targets and specificity of RNA-binding location are poorly understood. It is also unclear if general rules governing SR protein alternative pre-mRNA splicing (AS) regulation uncovered for individual SR proteins on few model genes, apply to the activity of all SR proteins on endogenous targets. Using RNA-seq, we characterize the global AS regulation of the eight Drosophila SR protein family members. We find that a majority of AS events are regulated by multiple SR proteins, and that all SR proteins can promote exon inclusion, but also exon skipping. Most coregulated targets exhibit cooperative regulation, but some AS events are antagonistically regulated. Additionally, we found that SR protein levels can affect alternative promoter choices and polyadenylation site selection, as well as overall transcript levels. Cross-linking and immunoprecipitation coupled with high-throughput sequencing (iCLIP-seq), reveals that SR proteins bind a distinct and functionally diverse class of RNAs, which includes several classes of noncoding RNAs, uncovering possible novel functions of the SR protein family. Finally, we find that SR proteins exhibit positional RNA binding around regulated AS events. Therefore, regulation of AS by the SR proteins is the result of combinatorial regulation by multiple SR protein family members on most endogenous targets, and SR proteins have a broader role in integrating multiple layers of gene expression regulation.

Two new and distinct roles for Drosophila Argonaute-2 in the nucleus: alternative pre-mRNA splicing and transcriptional repression.

Transcription and pre-mRNA alternative splicing are highly regulated processes that play major roles in modulating eukaryotic gene expression. It is increasingly apparent that other pathways of RNA metabolism, including small RNA biogenesis, can regulate these processes. However, a direct link between alternative pre-mRNA splicing and small RNA pathways has remained elusive. Here we show that the small RNA pathway protein Argonaute-2 (Ago-2) regulates alternative pre-mRNA splicing patterns of specific transcripts in the Drosophila nucleus using genome-wide methods in conjunction with RNAi in cell culture and Ago-2 deletion or catalytic site mutations in Drosophila adults. Moreover, we show that nuclear Argonaute-2 binds to specific chromatin sites near gene promoters and negatively regulates the transcription of the Ago-2-associated target genes. These transcriptional target genes are also bound by Polycomb group (PcG) transcriptional repressor proteins and change during development, implying that Ago-2 may regulate Drosophila development. Importantly, both of these activities were independent of the catalytic activity of Ago-2, suggesting new roles for Ago-2 in the nucleus. Finally, we determined the nuclear RNA-binding profile of Ago-2, found it bound to several splicing target transcripts, and identified a G-rich RNA-binding site for Ago-2 that was enriched in these transcripts. These results suggest two new nuclear roles for Ago-2: one in pre-mRNA splicing and one in transcriptional repression.