Rate of elongation by RNA polymerase II is associated with specific gene features and epigenetic modifications.

The rate of transcription elongation plays an important role in the timing of expression of full-length transcripts as well as in the regulation of alternative splicing. In this study, we coupled Bru-seq technology with 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside (DRB) to estimate the elongation rates of over 2000 individual genes in human cells. This technique, BruDRB-seq, revealed gene-specific differences in elongation rates with a median rate of around 1.5 kb/min. We found that genes with rapid elongation rates showed higher densities of H3K79me2 and H4K20me1 histone marks compared to slower elongating genes. Furthermore, high elongation rates had a positive correlation with gene length, low complexity DNA sequence, and distance from the nearest active transcription unit. Features that negatively correlated with elongation rate included the density of exons, long terminal repeats, GC content of the gene, and DNA methylation density in the bodies of genes. Our results suggest that some static gene features influence transcription elongation rates and that cells may alter elongation rates by epigenetic regulation. The BruDRB-seq technique offers new opportunities to interrogate mechanisms of regulation of transcription elongation.

A bacteriophage-based platform for rapid trace detection of proteases.

Sensitive, inexpensive, and rapid protease activity assays are of great merit for clinical diagnostics. Detection of protease-based toxins produced by Clostridium botulinum and Bacillus anthracis represents a particularly challenging task, as exceptional sensitivity is a prerequisite because of the extreme potency of the toxins. Here we present an inexpensive and sensitive assay platform for activity-based protease quantification utilizing filamentous bacteriophage as an exponentially amplifiable reporter and its application to the detection of these bacterial toxins. The assay is based on specific cleavage of bacteriophage from a solid support and its subsequent quantification by means of infectivity or quantitative PCR. Detection of botulinum neurotoxin (BoNT) serotypes A and B and anthrax lethal factor in the picomolar range was demonstrated with a limit of detection of 2 pM for BoNT/A under optimized conditions.

3' Branch ligation: a novel method to ligate non-complementary DNA to recessed or internal 3'OH ends in DNA or RNA.

Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various genomic tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to insert 5' phosphorylated blunt-end double-stranded DNA to DNA breaks at 3'-recessive ends, gaps, or nicks to form a Y-shaped 3'-branch structure. Therefore, this base pairing-independent ligation is termed 3'-branch ligation (3'BL). In an extensive study of optimal ligation conditions, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency to more than 80%. Ligation efficiency was slightly varied between different donor and acceptor sequences. More interestingly, we discovered that T4 DNA ligase efficiently ligated DNA to the 3'-recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, suggesting a ternary complex formation preference of T4 DNA ligase. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important genomic applications, such as 3'-end labelling by adding a universal sequence; directional tagmentation for NGS library construction that achieve theoretical 100% template usage; and targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.

Development of coupling controlled polymerizations by adapter-ligation in mate-pair sequencing for detection of various genomic variants in one single assay.

The diversity of disease presentations warrants one single assay for detection and delineation of various genomic disorders. Herein, we describe a gel-free and biotin-capture-free mate-pair method through coupling Controlled Polymerizations by Adapter-Ligation (CP-AL). We first demonstrated the feasibility and ease-of-use in monitoring DNA nick translation and primer extension by limiting the nucleotide input. By coupling these two controlled polymerizations by a reported non-conventional adapter-ligation reaction 3' branch ligation, we evidenced that CP-AL significantly increased DNA circularization efficiency (by 4-fold) and was applicable for different sequencing methods but at a faction of current cost. Its advantages were further demonstrated by fully elimination of small-insert-contaminated (by 39.3-fold) with a ∼50% increment of physical coverage, and producing uniform genome/exome coverage and the lowest chimeric rate. It achieved single-nucleotide variants detection with sensitivity and specificity up to 97.3 and 99.7%, respectively, compared with data from small-insert libraries. In addition, this method can provide a comprehensive delineation of structural rearrangements, evidenced by a potential diagnosis in a patient with oligo-atheno-terato-spermia. Moreover, it enables accurate mutation identification by integration of genomic variants from different aberration types. Overall, it provides a potential single-integrated solution for detecting various genomic variants, facilitating a genetic diagnosis in human diseases.

Gene length as a biological timer to establish temporal transcriptional regulation.

Transcriptional timing is inherently influenced by gene length, thus providing a mechanism for temporal regulation of gene expression. While gene size has been shown to be important for the expression timing of specific genes during early development, whether it plays a role in the timing of other global gene expression programs has not been extensively explored. Here, we investigate the role of gene length during the early transcriptional response of human fibroblasts to serum stimulation. Using the nascent sequencing techniques Bru-seq and BruUV-seq, we identified immediate genome-wide transcriptional changes following serum stimulation that were linked to rapid activation of enhancer elements. We identified 873 significantly induced and 209 significantly repressed genes. Variations in gene size allowed for a large group of genes to be simultaneously activated but produce full-length RNAs at different times. The median length of the group of serum-induced genes was significantly larger than the median length of all expressed genes, housekeeping genes, and serum-repressed genes. These gene length relationships were also observed in corresponding mouse orthologs, suggesting that relative gene size is evolutionarily conserved. The sizes of transcription factor and microRNA genes immediately induced after serum stimulation varied dramatically, setting up a cascade mechanism for temporal expression arising from a single activation event. The retention and expansion of large intronic sequences during evolution have likely played important roles in fine-tuning the temporal expression of target genes in various cellular response programs.

Capturing the dynamic nascent transcriptome during acute cellular responses: The serum response.

Dynamic regulation of gene expression via signal transduction pathways is of fundamental importance during many biological processes such as cell state transitioning, cell cycle progression and stress responses. In this study we used serum stimulation as a cell response paradigm to apply the nascent RNA Bru-seq technique in order to capture early dynamic changes in the nascent transcriptome. Our data provides an unprecedented view of the dynamics of genome-wide transcription during the first two hours of serum stimulation in human fibroblasts. While some genes showed sustained induction or repression, other genes showed transient or delayed responses. Surprisingly, the dynamic patterns of induction and suppression of response genes showed a high degree of similarity, suggesting that these opposite outcomes are triggered by a common set of signals. As expected, early response genes such as those encoding components of the AP-1 transcription factor and those involved in the circadian clock were immediately but transiently induced. Surprisingly, transcription of important DNA damage response genes and histone genes were rapidly repressed. We also show that RNA polymerase II accelerates as it transcribes large genes and this was independent of whether the gene was induced or not. These results provide a unique genome-wide depiction of dynamic patterns of transcription of serum response genes and demonstrate the utility of Bru-seq to comprehensively capture rapid and dynamic changes of the nascent transcriptome.

Identifying transcription start sites and active enhancer elements using BruUV-seq.

BruUV-seq utilizes UV light to introduce transcription-blocking DNA lesions randomly in the genome prior to bromouridine-labeling and deep sequencing of nascent RNA. By inhibiting transcription elongation, but not initiation, pre-treatment with UV light leads to a redistribution of transcription reads resulting in the enhancement of nascent RNA signal towards the 5'-end of genes promoting the identification of transcription start sites (TSSs). Furthermore, transcripts associated with arrested RNA polymerases are protected from 3'-5' degradation and thus, unstable transcripts such as putative enhancer RNA (eRNA) are dramatically increased. Validation of BruUV-seq against GRO-cap that identifies capped run-on transcripts showed that most BruUV-seq peaks overlapped with GRO-cap signal over both TSSs and enhancer elements. Finally, BruUV-seq identified putative enhancer elements induced by tumor necrosis factor (TNF) treatment concomitant with expression of nearby TNF-induced genes. Taken together, BruUV-seq is a powerful new approach for identifying TSSs and active enhancer elements genome-wide in intact cells.