The selfish yeast plasmid exploits a SWI/SNF-type chromatin remodeling complex for hitchhiking on chromosomes and ensuring high-fidelity propagation.

Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF-type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and interaction analyses. We describe a Rep-STB bypass system in which a plasmid engineered to non-covalently associate with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.

MicroRNAs reinforce repression of PRC2 transcriptional targets independently and through a feed-forward regulatory network.

Gene expression can be regulated at multiple levels, but it is not known if and how there is broad coordination between regulation at the transcriptional and post-transcriptional levels. Transcription factors and chromatin regulate gene expression transcriptionally, whereas microRNAs (miRNAs) are small regulatory RNAs that function post-transcriptionally. Systematically identifying the post-transcriptional targets of miRNAs and the mechanism of transcriptional regulation of the same targets can shed light on regulatory networks connecting transcriptional and post-transcriptional control. We used individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) for the RNA-induced silencing complex (RISC) component AGO2 and global miRNA depletion to identify genes directly targeted by miRNAs. We found that Polycomb repressive complex 2 (PRC2) and its associated histone mark, H3K27me3, is enriched at hundreds of miRNA-repressed genes. We show that these genes are directly repressed by PRC2 and constitute a significant proportion of direct PRC2 targets. For just over half of the genes corepressed by PRC2 and miRNAs, PRC2 promotes their miRNA-mediated repression by increasing expression of the miRNAs that are likely to target them. miRNAs also repress the remainder of the PRC2 target genes, but independently of PRC2. Thus, miRNAs post-transcriptionally reinforce silencing of PRC2-repressed genes that are inefficiently repressed at the level of chromatin, by either forming a feed-forward regulatory network with PRC2 or repressing them independently of PRC2.

The histone variant H2A.Z in yeast is almost exclusively incorporated into the +1 nucleosome in the direction of transcription.

Transcription start sites (TSS) in eukaryotes are characterized by a nucleosome-depleted region (NDR), which appears to be flanked upstream and downstream by strongly positioned nucleosomes incorporating the histone variant H2A.Z. H2A.Z associates with both active and repressed TSS and is important for priming genes for rapid transcriptional activation. However, the determinants of H2A.Z occupancy at specific nucleosomes and its relationship to transcription initiation remain unclear. To further elucidate the specificity of H2A.Z, we determined its genomic localization at single nucleosome resolution, as well as the localization of its chromatin remodelers Swr1 and Ino80. By analyzing H2A.Z occupancy in conjunction with RNA expression data that captures promoter-derived antisense initiation, we find that H2A.Z's bimodal incorporation on either side of the NDR is not a general feature of TSS, but is specifically a marker for bidirectional transcription, such that the upstream flanking -1 H2A.Z-containing nucleosome is more appropriately considered as a +1 H2A.Z nucleosome for antisense transcription. The localization of H2A.Z almost exclusively at the +1 nucleosome suggests that a transcription-initiation dependent process could contribute to its specific incorporation.

The specificity of H2A.Z occupancy in the yeast genome and its relationship to transcription.

The incorporation of histone variants into nucleosomes has important functional consequences in all aspects of eukaryotic chromatin biology. H2A.Z is a conserved histone variant found in all eukaryotes from yeast to mammals. Recent studies in yeast have shed light on the questions of where and how nucleosomes containing this variant are situated at promoters and in relation to genes, and what its specificity implies with regard to transcription. In yeast, H2A.Z appears to be primarily incorporated into the first nucleosome in the direction of transcription initiation, either of an mRNA transcript or a divergently transcribed upstream antisense non-coding RNA. This specificity of H2A.Z is due in part to the localization at promoters of SWR1, the ATP-dependent chromatin remodeler that incorporates H2A.Z into nucleosomes. Replacement of H2A.Z with canonical H2A is dependent on the function of the transcription pre-initiation complex. The recent studies summarized in this review reveal that the directionality of H2A.Z occupancy in relation to transcription thus reflects a balance of incorporation and eviction activities, which likely have varying contributions at distinct sets of genes across the genome.

Identification and removal of sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies.

The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Nonspecific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published data sets. We show that mispriming can occur with as little as two bases of complementarity at the 3' end of the primer followed by intermittent regions of complementarity. We also provide a computational pipeline that identifies cDNA reads produced from RT mispriming, allowing users to filter them out from any aligned data set. Using this analysis pipeline, we identify thousands of mispriming events in a dozen published data sets from diverse technologies including short RNA-seq, total/mRNA-seq, HITS-CLIP, and GRO-seq. We further show how RT mispriming can lead to misinterpretation of data. In addition to providing a solution to computationally remove RT-misprimed reads, we also propose an experimental solution to completely avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq).

The Determinants of Directionality in Transcriptional Initiation.

A new paradigm has emerged in recent years characterizing transcription initiation as a bidirectional process encompassing a larger proportion of the genome than previously thought. Past concepts of coding genes thinly scattered among a vast background of transcriptionally inert noncoding DNA have been abandoned. A richer picture has taken shape, integrating transcription of coding genes, enhancer RNAs (eRNAs), and various other noncoding transcriptional events. In this review we give an overview of recent studies detailing the mechanisms of RNA polymerase II (RNA Pol II)-based transcriptional initiation and discuss the ways in which transcriptional direction is established as well as its functional implications.

The ATP-dependent chromatin remodeler Chd1 is recruited by transcription elongation factors and maintains H3K4me3/H3K36me3 domains at actively transcribed and spliced genes.

Chd1 (Chromodomain Helicase DNA Binding Protein 1) is a conserved ATP-dependent chromatin remodeler that maintains the nucleosomal structure of chromatin, but the determinants of its specificity and its impact on gene expression are not well defined. To identify the determinants of Chd1 binding specificity in the yeast genome, we investigated Chd1 occupancy in mutants of several candidate factors. We found that several components of the PAF1 transcription elongation complex contribute to Chd1 recruitment to highly transcribed genes and identified Spt4 as a factor that appears to negatively modulate Chd1 binding to chromatin. We discovered that CHD1 loss alters H3K4me3 and H3K36me3 patterns throughout the yeast genome. Interestingly, the aberrant histone H3 methylation patterns were predominantly observed within 1 kb from the transcription start site, where both histone H3 methylation marks co-occur. A reciprocal change between the two marks was obvious in the absence of Chd1, suggesting a role for CHD1 in establishing or maintaining the boundaries of these largely mutually exclusive histone marks. Strikingly, intron-containing genes were most susceptible to CHD1 loss and exhibited a high degree of histone H3 methylation changes. Intron retention was significantly lower in the absence of CHD1, suggesting that CHD1 function as a chromatin remodeler could indirectly affect RNA splicing.

Subtype-specific addiction of the activated B-cell subset of diffuse large B-cell lymphoma to FOXP1.

High expression of the forkhead box P1 (FOXP1) transcription factor distinguishes the aggressive activated B cell (ABC) diffuse large B-cell lymphoma (DLBCL) subtype from the better prognosis germinal center B-cell (GCB)-DLBCL subtype and is highly correlated with poor outcomes. A genetic or functional role for FOXP1 in lymphomagenesis, however, remains unknown. Here, we report that sustained FOXP1 expression is vital for ABC-DLBCL cell-line survival. Genome-wide analyses revealed direct and indirect FOXP1 transcriptional enforcement of ABC-DLBCL hallmarks, including the classical NF-κB and MYD88 (myeloid differentiation primary response gene 88) pathways. FOXP1 promoted gene expression underlying transition of the GCB cell to the plasmablast--the transient B-cell stage targeted in ABC-DLBCL transformation--by antagonizing pathways distinctive of GCB-DLBCL, including that of the GCB "master regulator," BCL6 (B-cell lymphoma 6). Cell-line derived FOXP1 target genes that were highly correlated with FOXP1 expression in primary DLBCL accurately segregated the corresponding clinical subtypes of a large cohort of primary DLBCL isolates and identified conserved pathways associated with ABC-DLBCL pathology.

Correction: Quantitative genetics of CTCF binding reveal local sequence effects and different modes of X-chromosome association.

[This corrects the article DOI: 10.1371/journal.pgen.1004798.].

Ethylene induces combinatorial effects of histone H3 acetylation in gene expression in Arabidopsis.

BACKGROUND: Histone acetylation and deacetylation are essential for gene regulation and have been implicated in the regulation of plant hormone responses. Many studies have indicated the role of histone acetylation in ethylene signaling; however, few studies have investigated how ethylene signaling regulates the genomic landscape of chromatin states. Recently, we found that ethylene can specifically elevate histone H3K14 acetylation and the non-canonical histone H3K23 acetylation in etiolated seedlings and the gene activation is positively associated with the elevation of H3K14Ac and H3K23Ac in response to ethylene. To assess the role of H3K9, H3K14, and H3K23 histone modifications in the ethylene response, we examined how ethylene regulates histone acetylation and the transcriptome at global level and in ethylene regulated genes both in wild type (Col-0) and ein2-5 seedlings. RESULTS: Our results revealed that H3K9Ac, H3K14Ac, and H3K23Ac are preferentially enriched around the transcription start sites and are positively correlated with gene expression levels in Col-0 and ein2-5 seedlings both with and without ethylene treatment. In the absence of ethylene, no combinatorial effect of H3K9Ac, H3K14Ac, and H3K23Ac on gene expression was detected. In the presence of ethylene, however, combined enrichment of the three histone acetylation marks was associated with high gene expression levels, and this ethylene-induced change was EIN2 dependent. In addition, we found that ethylene-regulated genes are expressed at medium or high levels, and a group of ethylene regulated genes are marked by either one of H3K9Ac, H3K14Ac or H3K23Ac. In this group of genes, the levels of H3K9Ac were altered by ethylene, but in the absence of ethylene the levels of H3K9Ac and peak breadths are distinguished in up- and down- regulated genes. In the presence of ethylene, the changes in the peak breadths and levels of H3K14Ac and H3K23Ac are required for the alteration of gene expressions. CONCLUSIONS: Our study reveals that the plant hormone ethylene induces combinatorial effects of H3K9Ac, K14Ac and K23Ac histone acetylation in gene expression genome widely. Further, for a group of ethylene regulated genes, in the absence of ethylene the levels and the covered breadths of H3K9Ac are the preexist markers for distinguishing up- and down- regulated genes, the change in the peak breadths and levels of H3K14Ac and H3K23Ac are required for the alteration of gene expression in the presence of ethylene.

Quantitative genetics of CTCF binding reveal local sequence effects and different modes of X-chromosome association.

Associating genetic variation with quantitative measures of gene regulation offers a way to bridge the gap between genotype and complex phenotypes. In order to identify quantitative trait loci (QTLs) that influence the binding of a transcription factor in humans, we measured binding of the multifunctional transcription and chromatin factor CTCF in 51 HapMap cell lines. We identified thousands of QTLs in which genotype differences were associated with differences in CTCF binding strength, hundreds of them confirmed by directly observable allele-specific binding bias. The majority of QTLs were either within 1 kb of the CTCF binding motif, or in linkage disequilibrium with a variant within 1 kb of the motif. On the X chromosome we observed three classes of binding sites: a minority class bound only to the active copy of the X chromosome, the majority class bound to both the active and inactive X, and a small set of female-specific CTCF sites associated with two non-coding RNA genes. In sum, our data reveal extensive genetic effects on CTCF binding, both direct and indirect, and identify a diversity of patterns of CTCF binding on the X chromosome.

Dendritic cell fate is determined by BCL11A.

The plasmacytoid dendritic cell (pDC) is vital to the coordinated action of innate and adaptive immunity. pDC development has not been unequivocally traced, nor has its transcriptional regulatory network been fully clarified. Here we confirm an essential requirement for the BCL11A transcription factor in fetal pDC development, and demonstrate this lineage-specific requirement in the adult organism. Furthermore, we identify BCL11A gene targets and provide a molecular mechanism for its action in pDC commitment. Embryonic germ-line deletion of Bcl11a revealed an absolute cellular, molecular, and functional absence of pDCs in fetal mice. In adults, deletion of Bcl11a in hematopoietic stem cells resulted in perturbed yet continued generation of progenitors, loss of downstream pDC and B-cell lineages, and persisting myeloid, conventional dendritic, and T-cell lineages. Challenge with virus resulted in a marked reduction of antiviral response in conditionally deleted adults. Genome-wide analyses of BCL11A DNA binding and expression revealed that BCL11A regulates transcription of E2-2 and other pDC differentiation modulators, including ID2 and MTG16. Our results identify BCL11A as an essential, lineage-specific factor that regulates pDC development, supporting a model wherein differentiation into pDCs represents a primed "default" pathway for common dendritic cell progenitors.

Simultaneous mapping of transcript ends at single-nucleotide resolution and identification of widespread promoter-associated non-coding RNA governed by TATA elements.

Understanding the relationships between regulatory factor binding, chromatin structure, cis-regulatory elements and RNA-regulation mechanisms relies on accurate information about transcription start sites (TSS) and polyadenylation sites (PAS). Although several approaches have identified transcript ends in yeast, limitations of resolution and coverage have remained, and definitive identification of TSS and PAS with single-nucleotide resolution has not yet been achieved. We developed SMORE-seq (simultaneous mapping of RNA ends by sequencing) and used it to simultaneously identify the strongest TSS for 5207 (90%) genes and PAS for 5277 (91%) genes. The new transcript annotations identified by SMORE-seq showed improved distance relationships with TATA-like regulatory elements, nucleosome positions and active RNA polymerase. We found 150 genes whose TSS were downstream of the annotated start codon, and additional analysis of evolutionary conservation and ribosome footprinting suggests that these protein-coding sequences are likely to be mis-annotated. SMORE-seq detected short non-coding RNAs transcribed divergently from more than a thousand promoters in wild-type cells under normal conditions. These divergent non-coding RNAs were less evident at promoters containing canonical TATA boxes, suggesting a model where transcription initiation at promoters by RNAPII is bidirectional, with TATA elements serving to constrain the directionality of initiation.

Genetic reconstruction of a functional transcriptional regulatory network.

Although global analyses of transcription factor binding provide one view of potential transcriptional regulatory networks, regulation also occurs at levels distinct from transcription factor binding. Here, we use a genetic approach to identify targets of transcription factors in yeast and reconstruct a functional regulatory network. First, we profiled transcriptional responses in S. cerevisiae strains with individual deletions of 263 transcription factors. Then we used directed-weighted graph modeling and regulatory epistasis analysis to identify indirect regulatory relationships between these transcription factors, and from this we reconstructed a functional transcriptional regulatory network. The enrichment of promoter motifs and Gene Ontology annotations provide insight into the biological functions of the transcription factors.

miR-503 represses human cell proliferation and directly targets the oncogene DDHD2 by non-canonical target pairing.

BACKGROUND: The pathways regulating the transition of mammalian cells from quiescence to proliferation are mediated by multiple miRNAs. Despite significant improvements in our understanding of miRNA targeting, the majority of miRNA regulatory networks are still largely unknown and require experimental validation. RESULTS: Here we identified miR-503, miR-103, and miR-494 as negative regulators of proliferation in primary human cells. We experimentally determined their genome wide target profiles using RNA-induced silencing complex (RISC) immunoprecipitations and gene expression profiling. Analysis of the genome wide target profiles revealed evidence of extensive regulation of gene expression through non-canonical target pairing by miR-503. We identified the proto-oncogene DDHD2 as a target of miR-503 that requires pairing outside of the canonical 5' seed region of miR-503, representing a novel mode of miRNA-target pairing. Further bioinformatics analysis implicated miR-503 and DDHD2 in breast cancer tumorigenesis. CONCLUSIONS: Our results provide an extensive genome wide set of targets for miR-503, miR-103, and miR-494, and suggest that miR-503 may act as a tumor suppressor in breast cancer by its direct non-canonical targeting of DDHD2.

Cell-type specific and combinatorial usage of diverse transcription factors revealed by genome-wide binding studies in multiple human cells.

Cell-type diversity is governed in part by differential gene expression programs mediated by transcription factor (TF) binding. However, there are few systematic studies of the genomic binding of different types of TFs across a wide range of human cell types, especially in relation to gene expression. In the ENCODE Project, we have identified the genomic binding locations across 11 different human cell types of CTCF, RNA Pol II (RNAPII), and MYC, three TFs with diverse roles. Our data and analysis revealed how these factors bind in relation to genomic features and shape gene expression and cell-type specificity. CTCF bound predominantly in intergenic regions while RNAPII and MYC preferentially bound to core promoter regions. CTCF sites were relatively invariant across diverse cell types, while MYC showed the greatest cell-type specificity. MYC and RNAPII co-localized at many of their binding sites and putative target genes. Cell-type specific binding sites, in particular for MYC and RNAPII, were associated with cell-type specific functions. Patterns of binding in relation to gene features were generally conserved across different cell types. RNAPII occupancy was higher over exons than adjacent introns, likely reflecting a link between transcriptional elongation and splicing. TF binding was positively correlated with the expression levels of their putative target genes, but combinatorial binding, in particular of MYC and RNAPII, was even more strongly associated with higher gene expression. These data illuminate how combinatorial binding of transcription factors in diverse cell types is associated with gene expression and cell-type specific biology.

ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals.

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility ("retrohoming") by a process that requires reverse transcription of a highly structured, 2-2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3' ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

A Myc-microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes.

The transition of mammalian cells from quiescence to proliferation is accompanied by the differential expression of several microRNAs (miRNAs) and transcription factors. However, the interplay between transcription factors and miRNAs in modulating gene regulatory networks involved in human cell proliferation is largely unknown. Here we show that the miRNA miR-22 promotes proliferation in primary human cells, and through a combination of Argonaute-2 immunoprecipitation and reporter assays, we identified multiple novel targets of miR-22, including several cell-cycle arrest genes that mediate the effects of the tumor-suppressor p53. In addition, we found that miR-22 suppresses interferon gene expression by directly targeting high mobility group box-1 and interferon regulatory factor (IRF)-5, preventing activation of IRF3 and NF-κB, which are activators of interferon genes. The expression of interferon genes is elevated in quiescent cells and their expression is inhibitory for cell proliferation. In addition, we find that miR-22 is activated by the transcription factor Myc when quiescent cells enter proliferation and that miR-22 inhibits the Myc transcriptional repressor MXD4, mediating a feed-forward loop to elevate Myc expression levels. Our results implicate miR-22 in downregulating the anti-proliferative p53 and interferon pathways and reveal a new transcription factor-miRNA network that regulates the transition of primary human cells from quiescence to proliferation.

Extensive evolutionary changes in regulatory element activity during human origins are associated with altered gene expression and positive selection.

Understanding the molecular basis for phenotypic differences between humans and other primates remains an outstanding challenge. Mutations in non-coding regulatory DNA that alter gene expression have been hypothesized as a key driver of these phenotypic differences. This has been supported by differential gene expression analyses in general, but not by the identification of specific regulatory elements responsible for changes in transcription and phenotype. To identify the genetic source of regulatory differences, we mapped DNaseI hypersensitive (DHS) sites, which mark all types of active gene regulatory elements, genome-wide in the same cell type isolated from human, chimpanzee, and macaque. Most DHS sites were conserved among all three species, as expected based on their central role in regulating transcription. However, we found evidence that several hundred DHS sites were gained or lost on the lineages leading to modern human and chimpanzee. Species-specific DHS site gains are enriched near differentially expressed genes, are positively correlated with increased transcription, show evidence of branch-specific positive selection, and overlap with active chromatin marks. Species-specific sequence differences in transcription factor motifs found within these DHS sites are linked with species-specific changes in chromatin accessibility. Together, these indicate that the regulatory elements identified here are genetic contributors to transcriptional and phenotypic differences among primate species.