Inherited blood cancer predisposition through altered transcription elongation.

Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a ∼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.

Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

Massively Systematic Transcript End Readout, "MASTER": Transcription Start Site Selection, Transcriptional Slippage, and Transcript Yields.

We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 4(7) (∼ 16,000) barcoded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields (massively systematic transcript end readout, "MASTER"). Using MASTER, we define full inventories of transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo; we define the TSS-region DNA sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield; and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves transcription-bubble expansion ("scrunching") and transcription-bubble contraction ("anti-scrunching").

Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs.

Prokaryotic and eukaryotic RNA polymerases can use 2- to ∼4-nt RNAs, "nanoRNAs," to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.

NanoRNAs prime transcription initiation in vivo.

It is often presumed that, in vivo, the initiation of RNA synthesis by DNA-dependent RNA polymerases occurs using NTPs alone. Here, using the model Gram-negative bacterium Pseudomonas aeruginosa, we demonstrate that depletion of the small-RNA-specific exonuclease, Oligoribonuclease, causes the accumulation of oligoribonucleotides 2 to ∼4 nt in length, "nanoRNAs," which serve as primers for transcription initiation at a significant fraction of promoters. Widespread use of nanoRNAs to prime transcription initiation is coupled with global alterations in gene expression. Our results, obtained under conditions in which the concentration of nanoRNAs is artificially elevated, establish that small RNAs can be used to initiate transcription in vivo, challenging the idea that all cellular transcription occurs using only NTPs. Our findings further suggest that nanoRNAs could represent a distinct class of functional small RNAs that can affect gene expression through direct incorporation into a target RNA transcript rather than through a traditional antisense-based mechanism.

A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq.

Transcription initiation that involves the use of a 2- to ~4-nt oligoribonucleotide primer, "primer-dependent initiation," (PDI) has been shown to be widely prevalent at promoters of genes expressed during the stationary phase of growth in Escherichia coli. However, the extent to which PDI impacts E. coli physiology, and the extent to which PDI occurs in other bacteria is not known. Here we establish a physiological role for PDI in E. coli as a regulatory mechanism that modulates biofilm formation. We further demonstrate using high-throughput sequencing of RNA 5' ends (5' RNA-seq) that PDI occurs in the pathogenic bacterium Vibrio cholerae. A comparative global analysis of PDI in V. cholerae and E. coli reveals that the pattern of PDI is strikingly similar in the two organisms. In particular, PDI is detected in stationary phase, is not detected in exponential phase, and is preferentially apparent at promoters carrying the sequence T-1A+1 or G-1G+1 (where position +1 corresponds to the position of de novo initiation). Our findings demonstrate a physiological role for PDI and suggest PDI may be widespread among Gammaproteobacteria. We propose that PDI in both E. coli and V. cholerae occurs though a growth phase-dependent process that leads to the preferential generation of the linear dinucleotides 5´-UA-3´ and 5´-GG-3´.

PRO-seq: Precise Mapping of Engaged RNA Pol II at Single-Nucleotide Resolution.

Gene regulation is dependent on the production of mRNAs and a repertoire of non-coding RNAs by RNA polymerase II (RNAPII). Precision run-on sequencing (PRO-seq) maps the position of engaged RNAPII complexes at single-nucleotide resolution and can reveal direct targets of regulation, locations of enhancers, and transcription mechanisms that are difficult or impossible to measure by analysis of total cellular RNA. Briefly, this method first involves permeabilizing cells with mild detergents to remove intracellular NTPs and halt transcription. Transcription is then resumed in the presence of biotin-NTPs and sarkosyl to allow transcriptional incorporation of a single biotinylated NTP by RNAPII. The biotin moiety is then bound to streptavidin beads to stringently enrich for nascent RNAs. Sequencing libraries are then generated such that the first base read corresponds to the 3' end of the nascent transcript. Here, we describe our current protocol for generating PRO-seq libraries from metazoan cells, including adaptations of previously published protocols to incorporate unique molecular identifiers, reduce ligation bias, and improve library yields. Additional commentary describes quality control and processing of PRO-seq data and references for more advanced downstream analysis such as gene and enhancer identification. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Cell permeabilization for PRO-seq Basic Protocol 2: Construction of PRO-seq libraries Support Protocol: Adenylation of 3' adapter.

A ubiquitous disordered protein interaction module orchestrates transcription elongation.

During eukaryotic transcription elongation, RNA polymerase II (RNAP2) is regulated by a chorus of factors. Here, we identified a common binary interaction module consisting of TFIIS N-terminal domains (TNDs) and natively unstructured TND-interacting motifs (TIMs). This module was conserved among the elongation machinery and linked complexes including transcription factor TFIIS, Mediator, super elongation complex, elongin, IWS1, SPT6, PP1-PNUTS phosphatase, H3K36me3 readers, and other factors. Using nuclear magnetic resonance, live-cell microscopy, and mass spectrometry, we revealed the structural basis for these interactions and found that TND-TIM sequences were necessary and sufficient to induce strong and specific colocalization in the crowded nuclear environment. Disruption of a single TIM in IWS1 induced robust changes in gene expression and RNAP2 elongation dynamics, which underscores the functional importance of TND-TIM surfaces for transcription elongation.

Negative elongation factor regulates muscle progenitor expansion for efficient myofiber repair and stem cell pool repopulation.

Negative elongation factor (NELF) is a critical transcriptional regulator that stabilizes paused RNA polymerase to permit rapid gene expression changes in response to environmental cues. Although NELF is essential for embryonic development, its role in adult stem cells remains unclear. In this study, through a muscle-stem-cell-specific deletion, we showed that NELF is required for efficient muscle regeneration and stem cell pool replenishment. In mechanistic studies using PRO-seq, single-cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of regeneration whereby it modulates p53 signaling to permit massive expansion of muscle progenitors. Strikingly, transplantation experiments indicated that these progenitors are also necessary for stem cell pool repopulation, implying that they are able to return to quiescence. Thus, we identified a critical role for NELF in the expansion of muscle progenitors in response to injury and revealed that progenitors returning to quiescence are major contributors to the stem cell pool repopulation.

Differential regulation by magnesium of the two MsbB paralogs of Shigella flexneri.

Shigella flexneri, a gram-negative enteric pathogen, is unusual in that it contains two nonredundant paralogous genes that encode the myristoyl transferase MsbB (LpxM) that catalyzes the final step in the synthesis of the lipid A moiety of lipopolysaccharide. MsbB1 is encoded on the chromosome, and MsbB2 is encoded on the large virulence plasmid present in all pathogenic shigellae. We demonstrate that myristoyl transferase activity due to MsbB2 is detected in limited magnesium medium, but not in replete magnesium medium, whereas that due to MsbB1 is detected under both conditions. MsbB2 increases overall hexa-acylation of lipid A under limited magnesium conditions. Regulation of MsbB2 by magnesium occurs at the level of transcription and is dependent on the conserved magnesium-inducible PhoPQ two-component regulatory pathway. Direct hexanucleotide repeats within the promoter upstream of msbB2 were identified as a putative PhoP binding site, and mutations within the repeats led to diminished PhoP-dependent expression of a transcriptional fusion of lacZ to this promoter. Thus, the virulence plasmid-encoded paralog of msbB is induced under limited magnesium in a PhoPQ-dependent manner. PhoPQ regulates the response of many Enterobacteriaceae to environmental signals, which include modifications of lipid A that confer increased resistance of the organism to stressful environments and antimicrobial peptides. The findings reported here are the first example of gene duplication in which one paralog has selectively acquired the mechanism for differential regulation by PhoPQ. Our findings provide molecular insight into the mechanisms by which each of the two MsbB proteins of S. flexneri likely contributes to pathogenesis.

Analysis of Bacterial Transcription by "Massively Systematic Transcript End Readout," MASTER.

A systems-level view of cellular gene expression requires understanding the mechanistic principles governing each step of transcription. In this chapter, we describe a massively multiplexed method for the analysis of the relationship between nucleic acid sequence and transcription termed "MASTER," for massively systematic transcript end readout. MASTER enables parallel measurements of transcription output from at least 410 (~1,000,000) individual template sequences in vitro and in vivo. MASTER involves constructing a DNA template library of barcoded sequences, generating RNA transcripts from the library during transcription in vitro or in vivo, and analyzing the relative abundance and 5'-end sequences of the RNA transcripts by high-throughput sequencing. MASTER provides a powerful, rapid, and versatile method to identify sequence determinants of each step of transcription and to define the mechanistic basis by which these sequence determinants dictate transcription output.

Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5' ends.

We provide a detailed protocol for preparing cDNA libraries suitable for high-throughput sequencing that are derived specifically from the 5' ends of RNA (5' specific RNA-seq). The protocol describes how cDNA libraries for 5' specific RNA-seq can be tailored to analyze specific classes of RNAs based upon the phosphorylation status of the 5' end. Thus, the analysis of cDNA libraries generated by these methods provides information regarding both the sequence and phosphorylation status of the 5' ends of RNAs. 5' specific RNA-seq can be used to analyze transcription initiation and posttranscriptional processing of RNAs with single base pair resolution on a genome-wide level.

The primary σ factor in Escherichia coli can access the transcription elongation complex from solution in vivo.

The σ subunit of bacterial RNA polymerase (RNAP) confers on the enzyme the ability to initiate promoter-specific transcription. Although σ factors are generally classified as initiation factors, σ can also remain associated with, and modulate the behavior of, RNAP during elongation. Here we establish that the primary σ factor in Escherichia coli, σ(70), can function as an elongation factor in vivo by loading directly onto the transcription elongation complex (TEC) in trans. We demonstrate that σ(70) can bind in trans to TECs that emanate from either a σ(70)-dependent promoter or a promoter that is controlled by an alternative σ factor. We further demonstrate that binding of σ(70) to the TEC in trans can have a particularly large impact on the dynamics of transcription elongation during stationary phase. Our findings establish a mechanism whereby the primary σ factor can exert direct effects on the composition of the entire transcriptome, not just that portion that is produced under the control of σ(70)-dependent promoters.

Interactions between RNA polymerase and the "core recognition element" counteract pausing.

Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native elongating transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G₋10Y₋1G(+1) (where -1 corresponds to the position of the RNA 3' end). We demonstrate that sequence-specific interactions between RNAP core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a posttranslocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.

Direct detection of abortive RNA transcripts in vivo.

During transcription initiation in vitro, prokaryotic and eukaryotic RNA polymerase (RNAP) can engage in abortive initiation-the synthesis and release of short (2 to 15 nucleotides) RNA transcripts-before productive initiation. It has not been known whether abortive initiation occurs in vivo. Using hybridization with locked nucleic acid probes, we directly detected abortive transcripts in bacteria. In addition, we show that in vivo abortive initiation shows characteristics of in vitro abortive initiation: Abortive initiation increases upon stabilizing interactions between RNAP and either promoter DNA or sigma factor, and also upon deleting elongation factor GreA. Abortive transcripts may have functional roles in regulating gene expression in vivo.

Modulation of an outer membrane protease contributes to the virulence defect of Shigella flexneri strains carrying a mutation in the virK locus.

The Shigella actin assembly protein IcsA is removed from the bacterial surface by the protease IcsP. We show that decreased intracellular spreading of virK::Tn10 mutants is due in part to significant increases in IcsP and IcsP-mediated cleavage of IcsA and that IcsP expression is a critical determinant of Shigella virulence.

Regulation of IcsP, the outer membrane protease of the Shigella actin tail assembly protein IcsA, by virulence plasmid regulators VirF and VirB.

The Shigella outer membrane protease IcsP removes the actin assembly protein IcsA from the bacterial surface, and consequently modulates Shigella actin-based motility and cell-to-cell spread. Here, we demonstrate that IcsP expression is undetectable in mutants lacking either of two transcriptional activators, VirF and VirB. In wild-type Shigella spp., virB expression is entirely dependent on VirF; therefore, to circumvent this regulatory cascade, we independently expressed VirF or VirB in Shigella strains lacking both activators and measured both IcsP levels and transcription from the icsP promoter. Our results show that VirB significantly enhanced icsP transcription, even in the absence of VirF. In contrast, when VirF was induced in the absence of VirB, VirF had variable effects. The regulation of icsP is distinctly different from the regulation of the gene encoding its major substrate, icsA, which is activated by VirF and not VirB. We propose that the different pathways regulating icsA and icsP may be critical to the modulation of IcsA-mediated actin-based motility by IcsP.

Transport capabilities encoded within the Bacillus subtilis genome.

We here describe all recognized established and putative transport proteins encoded within the genome of Bacillus subtilis. These fall into four classes of established transporter types: (1) channel proteins, (2) secondary active transporters, (3) primary active transporters, and (4) group translocators of the sugar-transporting phosphotransferase system (PTS). Additionally, some transporters are recognized that utilize an unknown mode of action or energy coupling mechanism. The secondary carriers (which represent the majority of Bacillus transporters) are subdivided according to substrate specificity and family association. Characteristics of the families as well as the individual transport systems are presented when sufficient information is available. The recognized transporters fall into 58 families including 4 channel types, 42 secondary carrier types, 3 primary carrier types, 4 PTS-types and 5 unknown types.