The ETS transcription factor ERF controls the exit from the naïve pluripotent state in a MAPK-dependent manner.

The naïve epiblast transitions to a pluripotent primed state during embryo implantation. Despite the relevance of the FGF pathway during this period, little is known about the downstream effectors regulating this signaling. Here, we examined the molecular mechanisms coordinating the naïve to primed transition by using inducible ESC to genetically eliminate all RAS proteins. We show that differentiated RASKO ESC remain trapped in an intermediate state of pluripotency with naïve-associated features. Elimination of the transcription factor ERF overcomes the developmental blockage of RAS-deficient cells by naïve enhancer decommissioning. Mechanistically, ERF regulates NANOG expression and ensures naïve pluripotency by strengthening naïve transcription factor binding at ESC enhancers. Moreover, ERF negatively regulates the expression of the methyltransferase DNMT3B, which participates in the extinction of the naïve transcriptional program. Collectively, we demonstrated an essential role for ERF controlling the exit from naïve pluripotency in a MAPK-dependent manner during the progression to primed pluripotency.

CTCF is a barrier for 2C-like reprogramming.

Totipotent cells have the ability to generate embryonic and extra-embryonic tissues. Interestingly, a rare population of cells with totipotent-like potential, known as 2 cell (2C)-like cells, has been identified within ESC cultures. They arise from ESC and display similar features to those found in the 2C embryo. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that the CCCTC-binding factor (CTCF) is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by the transcription factor DUX is associated with DNA damage at a subset of CTCF binding sites. Depletion of CTCF in ESC efficiently promotes spontaneous and asynchronous conversion to a 2C-like state and is reversible upon restoration of CTCF levels. This phenotypic reprogramming is specific to pluripotent cells as neural progenitor cells do not show 2C-like conversion upon CTCF-depletion. Furthermore, we show that transcriptional activation of the ZSCAN4 cluster is necessary for successful 2C-like reprogramming. In summary, we reveal an unexpected relationship between CTCF and 2C-like reprogramming.

Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria.

Promoter-proximal pausing regulates eukaryotic gene expression and serves as checkpoints to assemble elongation/splicing machinery. Little is known how broadly this type of pausing regulates transcription in bacteria. We apply nascent elongating transcript sequencing combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Retention of σ70 induces strong backtracked pauses at a 10-20-bp distance from many promoters. The pauses in the 10-15-bp register of the promoter are dictated by the canonical -10 element, 6-7 nt spacer and "YR+1Y" motif centered at the transcription start site. The promoters for the pauses in the 16-20-bp register contain an additional -10-like sequence recognized by σ70. Our in vitro analysis reveals that DNA scrunching is involved in these pauses relieved by Gre cleavage factors. The genes coding for transcription factors are enriched in these pauses, suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

NusG controls transcription pausing and RNA polymerase translocation throughout the Bacillus subtilis genome.

Transcription is punctuated by RNA polymerase (RNAP) pausing. These pauses provide time for diverse regulatory events that can modulate gene expression. Transcription elongation factors dramatically affect RNAP pausing in vitro, but the genome-wide role of such factors on pausing has not been examined. Using native elongating transcript sequencing followed by RNase digestion (RNET-seq), we analyzed RNAP pausing in Bacillus subtilis genome-wide and identified an extensive role of NusG in pausing. This universally conserved transcription elongation factor is known as Spt5 in archaeal and eukaryotic organisms. B. subtilis NusG shifts RNAP to the posttranslocation register and induces pausing at 1,600 sites containing a consensus TTNTTT motif in the nontemplate DNA strand within the paused transcription bubble. The TTNTTT motif is necessary but not sufficient for NusG-dependent pausing. Approximately one-fourth of these pause sites were localized to untranslated regions and could participate in posttranscription initiation control of gene expression as was previously shown for tlrB and the trpEDCFBA operon. Most of the remaining pause sites were identified in protein-coding sequences. NusG-dependent pausing was confirmed for all 10 pause sites that we tested in vitro. Putative pause hairpins were identified for 225 of the 342 strongest NusG-dependent pause sites, and some of these hairpins were shown to function in vitro. NusG-dependent pausing in the ribD riboswitch provides time for cotranscriptional binding of flavin mononucleotide, which decreases the concentration required for termination upstream of the ribD coding sequence. Our phylogenetic analysis implicates NusG-dependent pausing as a widespread mechanism in bacteria.

GABPα and CREB1 Binding to Double Nucleotide Polymorphisms of Their Consensus Motifs and Cooperative Binding to the Composite ETS ⇔ CRE Motif (ACCGGAAGTGACGTCA).

Previously, cooperative binding of the bZIP domain of CREB1 and the ETS domain of GABPα was observed for the composite DNA ETS ⇔ CRE motif (A 0 C 1 C 2 G 3 G 4 A 5 A 6 G 7 T 8 G 9 A 10 C 11 G 12 T 13 C 14 A 15 ). Single nucleotide polymorphisms (SNPs) at the beginning and end of the ETS motif (ACCGGAAGT) increased cooperative binding. Here, we use an Agilent microarray of 60-mers containing all double nucleotide polymorphisms (DNPs) of the ETS ⇔ CRE motif to explore GABPα and CREB1 binding to their individual motifs and their cooperative binding. For GABPα, all DNPs were bound as if each SNP acted independently. In contrast, CREB1 binding to some DNPs was stronger or weaker than expected, depending on the locations of each SNP. CREB1 binding to DNPs where both SNPs were in the same half site, T 8 G 9 A 10 or T 13 C 14 A 15 , was greater than expected, indicating that an additional SNP cannot destroy binding as much as expected, suggesting that an individual SNP is enough to abolish sequence-specific DNA binding of a single bZIP monomer. If a DNP contains SNPs in each half site, binding is weaker than expected. Similar results were observed for additional ETS and bZIP family members. Cooperative binding between GABPα and CREB1 to the ETS ⇔ CRE motif was weaker than expected except for DNPs containing A 7 and SNPs at the beginning of the ETS motif.

Metagenomic Discovery of 83 New Human Papillomavirus Types in Patients with Immunodeficiency.

Several immunodeficiencies are associated with high susceptibility to persistent and progressive human papillomavirus (HPV) infection leading to a wide range of cutaneous and mucosal lesions. However, the HPV types most commonly associated with such clinical manifestations in these patients have not been systematically defined. Here, we used virion enrichment, rolling circle amplification, and deep sequencing to identify circular DNA viruses present in skin swabs and/or wart biopsy samples from 48 patients with rare genetic immunodeficiencies, including patients with warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, or epidermodysplasia verruciformis (EV). Their profiles were compared with the profiles of swabs from 14 healthy adults and warts from 6 immunologically normal children. Individual patients were typically infected with multiple HPV types; up to 26 different types were isolated from a single patient (multiple anatomical sites, one time point). Among these, we identified the complete genomes of 83 previously unknown HPV types and 35 incomplete genomes representing possible additional new types. HPV types in the genus Gammapapillomavirus were common in WHIM patients, whereas EV patients mainly shed HPVs from the genus Betapapillomavirus. Preliminary evidence based on three WHIM patients treated with plerixafor, a leukocyte mobilizing agent, suggest that longer-term therapy may correlate with decreased HPV diversity and increased predominance of HPV types associated with childhood skin warts.IMPORTANCE Although some members of the viral family Papillomaviridae cause benign skin warts (papillomas), many human papillomavirus (HPV) infections are not associated with visible symptoms. For example, most healthy adults chronically shed Gammapapillomavirus (Gamma) virions from apparently healthy skin surfaces. To further explore the diversity of papillomaviruses, we performed viromic surveys on immunodeficient individuals suffering from florid skin warts. Our results nearly double the number of known Gamma HPV types and suggest that WHIM syndrome patients are uniquely susceptible to Gamma HPV-associated skin warts. Preliminary results suggest that treatment with the drug plerixafor may promote resolution of the unusual Gamma HPV skin warts observed in WHIM patients.

Characterization of BK Polyomaviruses from Kidney Transplant Recipients Suggests a Role for APOBEC3 in Driving In-Host Virus Evolution.

BK polyomavirus (BKV) frequently causes nephropathy (BKVN) in kidney transplant recipients (KTRs). BKV has also been implicated in the etiology of bladder and kidney cancers. We characterized BKV variants from two KTRs who developed BKVN followed by renal carcinoma. Both patients showed a swarm of BKV sequence variants encoding non-silent mutations in surface loops of the viral major capsid protein. The temporal appearance and disappearance of these mutations highlights the intra-patient evolution of BKV. Some of the observed mutations conferred resistance to antibody-mediated neutralization. The mutations also modified the spectrum of receptor glycans engaged by BKV during host cell entry. Intriguingly, all observed mutations were consistent with DNA damage caused by antiviral APOBEC3 cytosine deaminases. Moreover, APOBEC3 expression was evident upon immunohistochemical analysis of renal biopsies from KTRs. These results provide a snapshot of in-host BKV evolution and suggest that APOBEC3 may drive BKV mutagenesis in vivo.

An Efficient Single-Cell RNA-Seq Approach to Identify Neoantigen-Specific T Cell Receptors.

The adoptive transfer of neoantigen-reactive tumor-infiltrating lymphocytes (TILs) can result in tumor regression in patients with metastatic cancer. To improve the efficacy of adoptive T cell therapy targeting these tumor-specific mutations, we have proposed a new therapeutic strategy, which involves the genetic modification of autologous T cells with neoantigen-specific T cell receptors (TCRs) and the transfer of these modified T cells back to cancer patients. However, the current techniques to isolate neoantigen-specific TCRs are labor intensive, time consuming, and technically challenging, not suitable for clinical applications. To facilitate this process, a new approach was developed, which included the co-culture of TILs with tandem minigene (TMG)-transfected or peptide-pulsed autologous antigen-presenting cells (APCs) and the single-cell RNA sequencing (RNA-seq) analysis of T cells to identify paired TCR sequences associated with cells expressing high levels of interferon-γ (IFN-γ) and interleukin-2 (IL-2). Following this new approach, multiple TCRs were identified, synthesized, cloned into a retroviral vector, and then transduced into donor T cells. These transduced T cells were shown to specifically recognize the neoantigens presented by autologous APCs. In conclusion, this approach provides an efficient procedure to isolate neoantigen-specific TCRs for clinical applications, as well as for basic and translational research.

Molecular basis of CENP-C association with the CENP-A nucleosome at yeast centromeres.

Histone CENP-A-containing nucleosomes play an important role in nucleating kinetochores at centromeres for chromosome segregation. However, the molecular mechanisms by which CENP-A nucleosomes engage with kinetochore proteins are not well understood. Here, we report the finding of a new function for the budding yeast Cse4/CENP-A histone-fold domain interacting with inner kinetochore protein Mif2/CENP-C. Strikingly, we also discovered that AT-rich centromere DNA has an important role for Mif2 recruitment. Mif2 contacts one side of the nucleosome dyad, engaging with both Cse4 residues and AT-rich nucleosomal DNA. Both interactions are directed by a contiguous DNA- and histone-binding domain (DHBD) harboring the conserved CENP-C motif, an AT hook, and RK clusters (clusters enriched for arginine-lysine residues). Human CENP-C has two related DHBDs that bind preferentially to DNA sequences of higher AT content. Our findings suggest that a DNA composition-based mechanism together with residues characteristic for the CENP-A histone variant contribute to the specification of centromere identity.

Methylated Cytosines Mutate to Transcription Factor Binding Sites that Drive Tetrapod Evolution.

In mammals, the cytosine in CG dinucleotides is typically methylated producing 5-methylcytosine (5mC), a chemically less stable form of cytosine that can spontaneously deaminate to thymidine resulting in a T•G mismatched base pair. Unlike other eukaryotes that efficiently repair this mismatched base pair back to C•G, in mammals, 5mCG deamination is mutagenic, sometimes producing TG dinucleotides, explaining the depletion of CG dinucleotides in mammalian genomes. It was suggested that new TG dinucleotides generate genetic diversity that may be critical for evolutionary change. We tested this conjecture by examining the DNA sequence properties of regulatory sequences identified by DNase I hypersensitive sites (DHSs) in human and mouse genomes. We hypothesized that the new TG dinucleotides generate transcription factor binding sites (TFBS) that become tissue-specific DHSs (TS-DHSs). We find that 8-mers containing the CG dinucleotide are enriched in DHSs in both species. However, 8-mers containing a TG and no CG dinucleotide are preferentially enriched in TS-DHSs when compared with 8-mers with neither a TG nor a CG dinucleotide. The most enriched 8-mer with a TG and no CG dinucleotide in tissue-specific regulatory regions in both genomes is the AP-1 motif ( TG: A(C)/GT CA: N), and we find evidence that TG dinucleotides in the AP-1 motif arose from CG dinucleotides. Additional TS-DHS-enriched TFBS containing the TG/CA dinucleotide are the E-Box motif (G CA: GC TG: C), the NF-1 motif (GG CATG: CC), and the GR (glucocorticoid receptor) motif (G-A CATG: T-C). Our results support the suggestion that cytosine methylation is mutagenic in tetrapods producing TG dinucleotides that create TFBS that drive evolution.

Genome Sequence of a Fish-Associated Polyomavirus, Black Sea Bass (Centropristis striata) Polyomavirus 1.

All known polyomaviruses are associated with mammals or birds. Using virion enrichment, random-primed rolling circle amplification, and deep sequencing, we identified a polyomavirus associated with black sea bass (Centropristis striata). The virus has a variety of novel genetic features, suggesting a long evolutionary separation from polyomaviruses of terrestrial animals.

Hamburger polyomaviruses.

Epidemiological studies have suggested that consumption of beef may correlate with an increased risk of colorectal cancer. One hypothesis to explain this proposed link might be the presence of a carcinogenic infectious agent capable of withstanding cooking. Polyomaviruses are a ubiquitous family of thermostable non-enveloped DNA viruses that are known to be carcinogenic. Using virion enrichment, rolling circle amplification (RCA) and next-generation sequencing, we searched for polyomaviruses in meat samples purchased from several supermarkets. Ground beef samples were found to contain three polyomavirus species. One species, bovine polyomavirus 1 (BoPyV1), was originally discovered as a contaminant in laboratory FCS. A previously unknown species, BoPyV2, occupies the same clade as human Merkel cell polyomavirus and raccoon polyomavirus, both of which are carcinogenic in their native hosts. A third species, BoPyV3, is related to human polyomaviruses 6 and 7. Examples of additional DNA virus families, including herpesviruses, adenoviruses, circoviruses and gyroviruses were also detected either in ground beef samples or in comparison samples of ground pork and ground chicken. The results suggest that the virion enrichment/RCA approach is suitable for random detection of essentially any DNA virus with a detergent-stable capsid. It will be important for future studies to address the possibility that animal viruses commonly found in food might be associated with disease.

Comparative validation of the D. melanogaster modENCODE transcriptome annotation.

Accurate gene model annotation of reference genomes is critical for making them useful. The modENCODE project has improved the D. melanogaster genome annotation by using deep and diverse high-throughput data. Since transcriptional activity that has been evolutionarily conserved is likely to have an advantageous function, we have performed large-scale interspecific comparisons to increase confidence in predicted annotations. To support comparative genomics, we filled in divergence gaps in the Drosophila phylogeny by generating draft genomes for eight new species. For comparative transcriptome analysis, we generated mRNA expression profiles on 81 samples from multiple tissues and developmental stages of 15 Drosophila species, and we performed cap analysis of gene expression in D. melanogaster and D. pseudoobscura. We also describe conservation of four distinct core promoter structures composed of combinations of elements at three positions. Overall, each type of genomic feature shows a characteristic divergence rate relative to neutral models, highlighting the value of multispecies alignment in annotating a target genome that should prove useful in the annotation of other high priority genomes, especially human and other mammalian genomes that are rich in noncoding sequences. We report that the vast majority of elements in the annotation are evolutionarily conserved, indicating that the annotation will be an important springboard for functional genetic testing by the Drosophila community.

A divergent variant of the eleventh human polyomavirus species, saint louis polyomavirus.

Saint Louis polyomavirus (STLPyV) was recently discovered in human feces. Using random-primed rolling circle amplification combined with deep sequencing, we have found a divergent variant of STLPyV in a sanitized human skin wart specimen. The result strongly suggests that STLPyV directly infects humans and is not simply a dietary contaminant.

Nucleosome-free region dominates histone acetylation in targeting SWR1 to promoters for H2A.Z replacement.

The histone variant H2A.Z is a genome-wide signature of nucleosomes proximal to eukaryotic regulatory DNA. Whereas the multisubunit chromatin remodeler SWR1 is known to catalyze ATP-dependent deposition of H2A.Z, the mechanism of SWR1 recruitment to S. cerevisiae promoters has been unclear. A sensitive assay for competitive binding of dinucleosome substrates revealed that SWR1 preferentially binds long nucleosome-free DNA and the adjoining nucleosome core particle, allowing discrimination of gene promoters over gene bodies. Analysis of mutants indicates that the conserved Swc2/YL1 subunit and the adenosine triphosphatase domain of Swr1 are mainly responsible for binding to substrate. SWR1 binding is enhanced on nucleosomes acetylated by the NuA4 histone acetyltransferase, but recognition of nucleosome-free and nucleosomal DNA is dominant over interaction with acetylated histones. Such hierarchical cooperation between DNA and histone signals expands the dynamic range of genetic switches, unifying classical gene regulation by DNA-binding factors with ATP-dependent nucleosome remodeling and posttranslational histone modifications.

Integration profiling of gene function with dense maps of transposon integration.

Understanding how complex networks of genes integrate to produce dividing cells is an important goal that is limited by the difficulty in defining the function of individual genes. Current resources for the systematic identification of gene function such as siRNA libraries and collections of deletion strains are costly and organism specific. We describe here integration profiling, a novel approach to identify the function of eukaryotic genes based upon dense maps of transposon integration. As a proof of concept, we used the transposon Hermes to generate a library of 360,513 insertions in the genome of Schizosaccharomyces pombe. On average, we obtained one insertion for every 29 bp of the genome. Hermes integrated more often into nucleosome free sites and 33% of the insertions occurred in ORFs. We found that ORFs with low integration densities successfully identified the genes that are essential for cell division. Importantly, the nonessential ORFs with intermediate levels of insertion correlated with the nonessential genes that have functions required for colonies to reach full size. This finding indicates that integration profiles can measure the contribution of nonessential genes to cell division. While integration profiling succeeded in identifying genes necessary for propagation, it also has the potential to identify genes important for many other functions such as DNA repair, stress response, and meiosis.

Evidence for two different regulatory mechanisms linking replication and segregation of vibrio cholerae chromosome II.

Understanding the mechanisms that coordinate replication initiation with subsequent segregation of chromosomes is an important biological problem. Here we report two replication-control mechanisms mediated by a chromosome segregation protein, ParB2, encoded by chromosome II of the model multichromosome bacterium, Vibrio cholerae. We find by the ChIP-chip assay that ParB2, a centromere binding protein, spreads beyond the centromere and covers a replication inhibitory site (a 39-mer). Unexpectedly, without nucleation at the centromere, ParB2 could also bind directly to a related 39-mer. The 39-mers are the strongest inhibitors of chromosome II replication and they mediate inhibition by binding the replication initiator protein. ParB2 thus appears to promote replication by out-competing initiator binding to the 39-mers using two mechanisms: spreading into one and direct binding to the other. We suggest that both these are novel mechanisms to coordinate replication initiation with segregation of chromosomes.

Contribution of nucleosome binding preferences and co-occurring DNA sequences to transcription factor binding.

BACKGROUND: Chromatin plays a critical role in regulating transcription factors (TFs) binding to their canonical transcription factor binding sites (TFBS). Recent studies in vertebrates show that many TFs preferentially bind to genomic regions that are well bound by nucleosomes in vitro. Co-occurring secondary motifs sometimes correlated with functional TFBS. RESULTS: We used a logistic regression to evaluate how well the propensity for nucleosome binding and co-occurrence of a secondary motif identify which canonical motifs are bound in vivo. We used ChIP-seq data for three transcription factors binding to their canonical motifs: c-Jun binding the AP-1 motif (TGA(C)/(G)TCA), GR (glucocorticoid receptor) binding the GR motif (G-ACA---(T)/(C)GT-C), and Hoxa2 (homeobox a2) binding the Pbx (Pre-B-cell leukemia homeobox) motif (TGATTGAT). For all canonical TFBS in the mouse genome, we calculated intrinsic nucleosome occupancy scores (INOS) for its surrounding 150-bps DNA and examined the relationship with in vivo TF binding. In mouse mammary 3134 cells, c-Jun and GR proteins preferentially bound regions calculated to be well-bound by nucleosomes in vitro with the canonical AP-1 and GR motifs themselves contributing to the high INOS. Functional GR motifs are enriched for AP-1 motifs if they are within a nucleosome-sized 150-bps region. GR and Hoxa2 also bind motifs with low INOS, perhaps indicating a different mechanism of action. CONCLUSION: Our analysis quantified the contribution of INOS and co-occurring sequence to the identification of functional canonical motifs in the genome. This analysis revealed an inherent competition between some TFs and nucleosomes for binding canonical TFBS. GR and c-Jun cooperate if they are within 150-bps. Binding of Hoxa2 and a fraction of GR to motifs with low INOS values suggesting they are not in competition with nucleosomes and may function using different mechanisms.

Overlapping ETS and CRE Motifs ((G/C)CGGAAGTGACGTCA) preferentially bound by GABPα and CREB proteins.

Previously, we identified 8-bps long DNA sequences (8-mers) that localize in human proximal promoters and grouped them into known transcription factor binding sites (TFBS). We now examine split 8-mers consisting of two 4-mers separated by 1-bp to 30-bps (X(4)-N(1-30)-X(4)) to identify pairs of TFBS that localize in proximal promoters at a precise distance. These include two overlapping TFBS: the ETS⇔ETS motif ((C/G)CCGGAAGCGGAA) and the ETS⇔CRE motif ((C/G)CGGAAGTGACGTCAC). The nucleotides in bold are part of both TFBS. Molecular modeling shows that the ETS⇔CRE motif can be bound simultaneously by both the ETS and the B-ZIP domains without protein-protein clashes. The electrophoretic mobility shift assay (EMSA) shows that the ETS protein GABPα and the B-ZIP protein CREB preferentially bind to the ETS⇔CRE motif only when the two TFBS overlap precisely. In contrast, the ETS domain of ETV5 and CREB interfere with each other for binding the ETS⇔CRE. The 11-mer (CGGAAGTGACG), the conserved part of the ETS⇔CRE motif, occurs 226 times in the human genome and 83% are in known regulatory regions. In vivo GABPα and CREB ChIP-seq peaks identified the ETS⇔CRE as the most enriched motif occurring in promoters of genes involved in mRNA processing, cellular catabolic processes, and stress response, suggesting that a specific class of genes is regulated by this composite motif.

Stepwise histone replacement by SWR1 requires dual activation with histone H2A.Z and canonical nucleosome.

Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or zero H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction.