Holding the line: low CpG content and DNA inaccessibility safeguard T cell commitment

From the Bernstein Lab, Clinical Research Division

Understanding the basis for cell lineage fate decisions, a key objective in the field of developmental biology, has many potential implications for the study and treatment of human disease. For example, hematopoietic stem cells (HSCs) are the progenitor cells for all leukocyte lineages, and precise control of HSC fate decisions is critical to the maintenance of blood and immune homeostasis. Differentiation of HSCs into T cells, an important immune effector cell type, occurs only within a specialized organ called the thymus. It is known that thymic restriction of T cell development is dependent on high levels of Notch transcription factor signaling in this tissue. Prior work has indicated disparities in Notch dose-responsiveness in determining cell fate outcome, as, in contrast to T cell development, low Notch dosage is capable of inducing HSC self-renewal and the inhibition of myeloid differentiation. Importantly, the mechanisms underpinning the dose-dependent responses to Notch signaling have not been elucidated. Utilizing an ex vivo T cell differentiation system that they had previously developed, members of the Bernstein Laboratory, including staff scientist Dr. Suzanne Furuyama, in the Fred Hutch Clinical Research Division, measured Notch dose-dependent changes in DNA accessibility as HSCs progress through the early stages of T cell development. Their work, recently published in Stem Cell Reports, reveals that T cell lineage commitment genes requiring high doses of Notch signaling for their activation are associated with inaccessible promoter regions containing low CpG content.

Schematic depicting dose-dependent promoter accessibility in response to Notch signaling.
Inaccessible, low CpG content promoters restrict T cell development to the thymus by limiting activation of T cell lineage genes to environments with high Notch signaling. Image provided by Dr. Furuyama and Dr. Bernstein.

Signal transduction is the process through which a cell responds to external stimuli by altering the expression of its genes. Such systems typically function through downstream signaling cascades, involving a relay between molecules spanning from the site of the stimulus to the nucleus, ultimately impacting the activity of transcription factors at target gene promoters. These signaling cascades allow both for signal amplification and complex crosstalk between different signaling pathways. An exception to this paradigm is the Notch signaling pathway. “Notch is a unique signal transduction system in that activation of Notch receptor results in its own proteolytic cleavage, producing an intracellular domain that itself functions as a transcription factor,” explained Dr. Furuyama. “Since each ligand-activated receptor produces only one transcriptional effector, an increase in productive ligand-receptor interactions yields more effector available to alter Notch target gene expression.” Given the quantitative nature of this signaling pathway, the Bernstein group sought to uncover how Notch-responsive genes could exhibit differential dose-dependent activation. “Up to this point, no studies have investigated how target gene expression is modulated in response to Notch dosage, a void we aimed to fill in this study,” said Dr. Furuyama.

T cell differentiation from HSCs is an ideal setting for the study of dose-dependent induction of Notch-responsive genes, since this process specifically requires high levels of Notch signaling. “Our lab had previously developed an ex vivo system to culture hematopoietic stem cells on immobilized Notch ligand and shown that different densities of immobilized ligand impact hematopoietic cell fate outcome,” said Dr. Furuyama. “With this system, we were poised to investigate the basis for target gene selectivity in response to quantitative differences in Notch signal strength and we did so focusing on early T cell development, which is known to be dependent on the high level of Notch signaling induced within the thymic microenvironment.”

The Bernstein lab, in collaboration with Dr. John Stamatoyannopoulos’s labs at UW and the Altius Institute for Biomedical Science, utilized a technique called DNase-seq, which relies on digestion of chromatin with a DNA endonuclease followed by deep sequencing, to map accessible or “open” regions within the genomes of cells at different stages of T cell development. Narrowing in on sites that overlap with gene promoter regions, they, along with Vicky Wu, an Assistant Professor in the Clinical Research and Public Health Divisions here at Fred Hutch, identified Notch-responsive sites by comparing promoter accessibility between developmental stages dependent on different doses of Notch signaling. Finally, they correlated Notch dose-dependent promoter accessibility to gene expression changes, establishing lists of 114 unique low-dose and 38 unique high-dose Notch-responsive genes. Encouragingly, several Notch target genes known to be essential for T cell lineage commitment were included in the high-dose list, and gene ontology (GO) analysis revealed “T cell differentiation” as the most significant GO term enriched in this subset.

Having established a list of high-dose Notch-responsive genes associated with T cell lineage commitment, the group looked for features that could set these genes apart from the low-dose Notch-responsive subset. CpG content is a feature which often distinguishes tightly controlled lineage-associated promoters from those of more broadly expressed genes. Specifically, promoters with low CpG content (LCG) are associated with “closed” or inaccessible DNA under non-differentiation conditions. Operating on a hunch that LCG promoters might play a role in dose-dependent responses to Notch signaling during T cell lineage fate decisions, Dr. Furuyama and colleagues compared the CpG content of low-dose and high-dose Notch-responsive promoters. Indeed, they found that high-dose promoters were significantly lower in CpG content than low-dose promoters.

“We found that Notch target genes differ in their transcriptional competence. In contrast to low Notch dose responsive genes, high Notch dose dependent promoters are inaccessible in hematopoietic stem progenitor cells and only acquire promoter DNA accessibility upon exposure to high dose Notch signaling,” explained Dr. Furuyama. “A distinguishing feature of these high dose dependent genes is low promoter CpG content, a characteristic known to be associated with lineage determining genes.”

These results begin to explain how Notch signaling thresholds function to control T cell lineage fate decisions. “These findings are significant in that they suggest that the DNA inaccessibility of LCG genes may have evolved as a signal safeguard to prevent stochastic lineage gene expression, and consequently lineage commitment, in inappropriate signaling contexts,” said Dr. Furuyama. Moving forward, the Bernstein group plans to investigate the circuitry involved in the Notch dose dependent regulation of T cell fate, a study that will provide tools for the ex vivo engineering of T cell subsets for therapeutic purposes.

This work was funded by the National Institutes of Health.

UW/Fred Hutch Cancer Consortium members John Stamatoyannopoulos and Irwin Bernstein contributed to this work.

Furuyama S, Wu QV, Varnum-Finney B, Sandstrom R, Meuleman W, Stamatoyannopoulos JA, Bernstein ID. Inaccessible LCG Promoters Act as Safeguards to Restrict T Cell Development to Appropriate Notch Signaling Environments. Stem Cell Reports. 2021 Apr 13;16(4):717-726. doi: 10.1016/j.stemcr.2021.02.017. Epub 2021 Mar 25. PMID: 33770495; PMCID: PMC8072033.