The immune system has a remarkable ability to respond to infection, invasion, blood loss, injury and other environmental demands in a rapid and appropriate manner. To do this, hematopoietic stem and progenitor cells must be poised to execute precise lineage differentiation decisions; yet how they achieve this is far from completely understood. Dr. Derek Janssens and colleagues in the Henikoff lab at Fred Hutch have made an exciting step in their transformative single cell chromatin profiling technology to simultaneously detect transcriptional machinery and regulatory histone marks at single cell resolution, revealing how blood stem cells dynamically respond to the body’s needs. The results, recently published in Cell Reports, reveal an orchestrated, two-speed system of gene activation controlled by RNA Polymerase II (Pol II) in coordination with two groups of chromatin regulatory proteins, polycomb and trithorax.
To study this, the researchers developed a new single-cell method called sciCUT&Tag2in1, which simultaneously profiles where Pol II is sitting on the genome and histone modification marks present at those loci. Specifically, they measured tri-methyl histone H3 lysine 27 (H3K27me3), the repressive histone mark deposited by Polycomb group proteins and methylation of histone H3 lysine 4 (H3K4me1, 2 and 3), histone marks associated with active or poised regulatory elements, deposited by trithorax group proteins. Since previous methods could only measure these features separately, how they coordinate to modulate gene regulation is not well understood. The team applied their new method to over 200,000 human CD34+ hematopoietic stem and progenitor cells (HSPC) collected from donors who received G-CSF, a cytokine that mobilizes stem cells from bone marrow into blood by inducing mild inflammatory stress. This allowed the authors to more easily collect these cells and to assess transcriptional effects of inflammatory challenge on blood stem cells.
Their first major finding was that G-CSF-mobilized HSPCs show elevated Pol II occupancy over a set of genes classically associated with rapid inflammatory responses known as the Immediate Early Genes (IEGs). These same genes showed reduced levels of H3K27me3. In more differentiated progenitors, H3K27me3 accumulates over IEG loci, dampening their response to stimulation. In contrast, H3K4me1-2-3 levels over IEGs showed no significant difference between hematopoietic stem cells (HSCs) and committed progenitors, indicating that the activated transcriptional state of IEGs in HSCs is not being driven by changes in the activating histone marks. This suggests HSCs are epigenetically equipped to be sensitive to inflammatory signals, a design feature that may also explain why loss of Polycomb function, as seen in some myeloproliferative neoplasms, can lead to pathological hyperactivation of these same genes.
The authors next examined Pol II and histone marks at genes controlling HSC self-renewal, focusing on the genes MECOM and PRDM16. Although H3K4me3 is known to promote Pol II pause release (to allow transcription), H3K4me1-2-3 levels at both gene promoters remained stable throughout differentiation. What changed instead was a progressive accumulation of H3K27me3 over distal regulatory elements and gene bodies—and despite Pol II remaining present at the promoter—productive transcription into the gene body was lost. H3K4 methylation alone, it turns out, is insufficient to drive pause release when Polycomb repression is simultaneously being imposed at the same locus.
The study's biggest conceptual advance is the definition of two distinct modes of Pol II activation during lineage commitment. The authors focused on two genes involved in differentiation from HSC to megakaryocyte erythrocyte progenitor (MEP), ZFPM1 and GATA1. In ZFPM1, they observed stable H3K4me1-2-3 at the promoter and H3K4me1-2-3 accumulation at distal regulatory elements in a stepwise manner as differentiation state proceeded from HSC to MEP. There was an absence of H3K27me3 and Pol II was present at the promoter but not in the gene body. They defined this pattern as “pause-and-release” mode: Pol II is already loaded and stalled at the promoter in HSCs, held in check without Polycomb repression. Upon lineage commitment, a signal such as phosphorylation of the Pol II C-terminal domain releases the paused polymerase into the gene body, enabling rapid, near-immediate transcription. These genes activate in a sharp early burst.
In contrast, Pol II was absent from the promoter in GATA1 in HSCs but appeared in early MEPs. H3K4me1-2-3 accumulated more rapidly at multiple distal regulatory elements while promoter and distal H3K27me3 was apparent in HSCs but disappeared at the MEP stage. This mode, “initiate-and-release”, governs genes silenced by H3K27me3 in HSCs. Pol II must be newly recruited after dismantling Polycomb repression. These genes activate later and more gradually during differentiation, representing a second wave of gene expression that consolidates lineage identity.
Lead author Derek Janssens shares the serendipitous nature of the findings: “We developed a method for joint single-cell mapping of the gene-expression machinery RNA Polymerase II with activating and repressive modifications to the proteins that package the DNA in human blood stem cells. Initially, we were using RNA Pol II as an “anchor" or “bridge" dataset to combine profiles of multiple different protein modifications, but RNA Pol II turned out to be the star of the show. We found that, in blood stem cells RNA Pol II plays a central role in mounting an inflammatory response, and that RNA Pol II is already sitting in a paused state on the promoters of numerous genes that control the production of innate or adaptive immune cells as well as the cells that carry oxygen in the blood.”
Beyond illuminating normal blood development, this framework has direct implications for understanding hematologic malignancies, where these tightly controlled differentiation circuits are blocked. “One big question is how this paused state of RNA Pol II in blood stem cells relates to disease states such as myelodysplastic syndrome (MDS). In MDS the inflammatory response is often hyper activated, but the production of innate immune cells is stalled or blocked,” Janssens expands. “Our results suggest the interaction of repressive chromatin states and paused RNA Pol II may be disrupted in MDS. This messes up a process called “inflammatory memory,” and causes blood stem cells to become “fixated” on an immune-response program even when that response is no longer needed. It’s like a bad memory the blood stem cells can’t forget. We are interested in following up on this to find ways to restore healthy inflammatory memory through ongoing collaborations with physician scientists.”
The discoveries made possible by sciCUT&Tag2in1 are just beginning.
Fred Hutch/University of Washington/Seattle Children’s Cancer Consortium Members Drs. Lev Silberstein and Steven Henikoff contributed to this research.
The spotlighted research was funded by the Howard Hughes Medical Institute, a Postdoctoral Fellowship from the Hartwell Foundation, and the National Institutes of Health.
Janssens DH, Codomo CA, Otto DJ, Silberstein L, Ahmad K, Henikoff S. 2026. Sequential RNA polymerase II activation drives human hematopoiesis. Cell Rep. doi: 10.1016/j.celrep.2025.116802.
Science Spotlight writer Kelly Mitchell is a postdoctoral fellow in the Paddison Lab at Fred Hutch Cancer Center. She utilizes live cell reporters and CRISPR screening to study how glioblastoma cancer cells resist chemotherapy and radiation treatment. She obtained her PhD in cellular biology from Albert Einstein College of Medicine.