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.

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.