Transcription start site mapping using super-low input carrier-CAGE

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). Accurate detection of TSSs enhances identification and discovery of core promoters. In addition, active enhancers can be detected through signatures of bidirectional transcription initiation. Described here is a protocol for performing super-low input carrier-CAGE (SLIC-CAGE). This SLIC adaptation of the CAGE protocol minimizes RNA losses by artificially increasing the RNA amount through use of an in vitro transcribed RNA carrier mix that is added to the sample of interest, thus enabling library preparation from nanogram-amounts of total RNA (i.e., thousands of cells). The carrier mimics the expected DNA library fragment length distribution, thereby eliminating biases that could be caused by the abundance of a homogenous carrier. In the last stages of the protocol, the carrier is removed through degradation with homing endonucleases and the target library is amplified. The target sample library is protected from degradation, as the homing endonuclease recognition sites are long (between 18 and 27 bp), making the probability of their existence in the eukaryotic genomes very low. The end result is a DNA library ready for next-generation sequencing. All steps in the protocol, up to sequencing, can be completed within 6 days. The carrier preparation requires a full working day; however, it can be prepared in large quantities and kept frozen at -80 °C. Once sequenced, the reads can be processed to obtain genome-wide single-nucleotide resolution TSSs. TSSs can be used for core promoter or enhancer discovery, providing insight into gene regulation. Once aggregated to promoters, the data can also be used for 5’-centric expression profiling.publishedVersio

The physiological target for LeuRS translational quality control is norvaline

The fidelity of protein synthesis depends on the capacity of aminoacyl-tRNA synthetases (AARSs) to couple only cognate amino acid-tRNA pairs. If amino acid selectivity is compromised, fidelity can be ensured by an inherent AARS editing activity that hydrolyses mischarged tRNAs. Here we show that the editing activity of Escherichia coli leucyl-tRNA synthetase (EcLeuRS) is not required to prevent incorrect isoleucine incorporation. Rather, as shown by kinetic, structural and in vivo approaches, the prime biological function of LeuRS editing is to prevent mis-incorporation of the non-standard amino acid norvaline. This conclusion follows from a reassessment of the discriminatory power of LeuRS against isoleucine and the demonstration that a LeuRS editing- deficient E. coli strain grows normally in high concentrations of isoleucine but not under oxygen deprivation conditions when norvaline accumulates to substantial levels. Thus, AARS- based translational quality control is a key feature for bacterial adaptive response to oxygen deprivation. The non-essential role for editing under normal bacterial growth has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site

Transcription start site mapping using super-low input carrier-CAGE

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). Accurate detection of TSSs enhances identification and discovery of core promoters. In addition, active enhancers can be detected through signatures of bidirectional transcription initiation. Described here is a protocol for performing super-low input carrier-CAGE (SLIC-CAGE). This SLIC adaptation of the CAGE protocol minimizes RNA losses by artificially increasing the RNA amount through use of an in vitro transcribed RNA carrier mix that is added to the sample of interest, thus enabling library preparation from nanogram-amounts of total RNA (i.e., thousands of cells). The carrier mimics the expected DNA library fragment length distribution, thereby eliminating biases that could be caused by the abundance of a homogenous carrier. In the last stages of the protocol, the carrier is removed through degradation with homing endonucleases and the target library is amplified. The target sample library is protected from degradation, as the homing endonuclease recognition sites are long (between 18 and 27 bp), making the probability of their existence in the eukaryotic genomes very low. The end result is a DNA library ready for next-generation sequencing. All steps in the protocol, up to sequencing, can be completed within 6 days. The carrier preparation requires a full working day; however, it can be prepared in large quantities and kept frozen at -80 °C. Once sequenced, the reads can be processed to obtain genome-wide single-nucleotide resolution TSSs. TSSs can be used for core promoter or enhancer discovery, providing insight into gene regulation. Once aggregated to promoters, the data can also be used for 5’-centric expression profiling

Saccharomyces cerevisiae displays a stable transcription start site landscape in multiple conditions

One of the fundamental processes that determine cellular fate is regulation of gene transcription. Understanding these regulatory processes is therefore essential for understanding cellular responses to changes in environmental conditions. At the core promoter, the regulatory region containing the transcription start site (TSS), all inputs regulating transcription are integrated. Here, we used Cap Analysis of Gene Expression (CAGE) to analyze the pattern of TSSs at four different environmental conditions (limited in ethanol, limited in nitrogen, limited in glucose and limited in glucose under anaerobic conditions) using the Saccharomyces cerevisiae strain CEN.PK113-7D. With this experimental setup, we were able to show that the TSS landscape in yeast is stable at different metabolic states of the cell. We also show that the spatial distribution of transcription initiation events, described by the shape index, has a surprisingly strong negative correlation with measured gene expression levels, meaning that genes with higher expression levels tend to have a broader distribution of TSSs. Our analysis supplies a set of high-quality TSS annotations useful for metabolic engineering and synthetic biology approaches in the industrially relevant laboratory strain CEN.PK113-7D, and provides novel insights into yeast TSS dynamics and gene regulation

Global regulatory transitions at core promoters demarcate the mammalian germline cycle

Core promoters integrate regulatory inputs of genes 1–3 . Global dynamics of promoter usage can reveal systemic changes in how genomic sequence is interpreted by the cell. We previously discovered that early development in fish is associated with a genome-wide shift in how promoters are read: the switch from the oocyte-specific to the somatic-specific promoter usage observed during Zygotic Genome Activation (ZGA) represents one of the most dramatic instances of transcriptional regulatory rewiring known in the animal life cycle 4 However, the key question of how and when the oocyte-specific pattern is established in the germ line has been hitherto impossible to answer, as well as whether this switch is universal across vertebrate development. Here we report the first analysis of promoter dynamics and code switching in the mammalian germ line. Using the Super Low Input Carrier-CAGE 5, 6 (SLIC-CAGE) we show that mouse germline development starts with the somatic promoter code with a prominent switch to the maternal code occurring during follicular oogenesis. The sequence features underlying the shift from somatic to maternal code are conserved across vertebrates, despite large differences in promoter nucleotide compositions. Surprisingly, we also find that the promoters of gonadal germ cells diverge from the canonical somatic transcription initiation. This divergence is distinct from the promoter code used later by developing oocytes and reveals genome-wide promoter remodelling driven by alternative nucleosome positioning during early female and male germline development. Collectively, our findings establish promoter-level regulatory transitions as a central, conserved feature of the vertebrate life cycle.Core promoters integrate regulatory inputs of genes 1–3 . Global dynamics of promoter usage can reveal systemic changes in how genomic sequence is interpreted by the cell. We previously discovered that early development in fish is associated with a genome-wide shift in how promoters are read: the switch from the oocyte-specific to the somatic-specific promoter usage observed during Zygotic Genome Activation (ZGA) represents one of the most dramatic instances of transcriptional regulatory rewiring known in the animal life cycle 4 However, the key question of how and when the oocyte-specific pattern is established in the germ line has been hitherto impossible to answer, as well as whether this switch is universal across vertebrate development. Here we report the first analysis of promoter dynamics and code switching in the mammalian germ line. Using the Super Low Input Carrier-CAGE 5, 6 (SLIC-CAGE) we show that mouse germline development starts with the somatic promoter code with a prominent switch to the maternal code occurring during follicular oogenesis. The sequence features underlying the shift from somatic to maternal code are conserved across vertebrates, despite large differences in promoter nucleotide compositions. Surprisingly, we also find that the promoters of gonadal germ cells diverge from the canonical somatic transcription initiation. This divergence is distinct from the promoter code used later by developing oocytes and reveals genome-wide promoter remodelling driven by alternative nucleosome positioning during early female and male germline development. Collectively, our findings establish promoter-level regulatory transitions as a central, conserved feature of the vertebrate life cycle