P. L. Arnold, R. W. F. Kerr, C. Weetman, S. R. Docherty, J. Rieb, F. L. Cruickshank, K. Wang, C. Jandl, M. W. McMullon, A. Pöthig, F. E. Kühn and A. D. Smith, Chem. Sci., 2018, 9, 8035 DOI: 10.1039/C8SC03312A
Even though transcription factors (TFs) are central players of gene regulation and have been extensively studied, their regulatory trans-activation domains (tADs) often remain unknown and a systematic functional characterization of tADs is lacking. Here, we present a novel high-throughput approach tAD-seq to functionally test thousands of candidate tADs from different TFs in parallel. The tADs we identify by pooled screening validate in individual luciferase assays, whereas neutral regions do not. Interestingly, the tADs are found at arbitrary positions within the TF sequences and can contain amino acid (e.g., glutamine) repeat regions or overlap structured domains, including helix-loop-helix domains that are typically annotated as DNA-binding. We also identified tADs in the non-native reading frames, confirming that random sequences can function as tADs, albeit weakly. The identification of tADs as short protein sequences sufficient for transcription activation will enable the systematic study of TF function, which-particularly for TFs of different transcription activating functionalities-is still poorly understood.
Half of the 2018 Nobel Prize in Chemistry was awarded to Professor Frances H. Arnold for her work in the directed evolution of enzymes, while the other half of the prize was split by Professor George P. Smith and Sir Gregory P. Winter for their research into the phage display of peptides and antibodies.
Col. Jeffrey Geraghty, commander, Arnold Engineering Development Complex, makes remarks during the activation Ceremony for the 804th Test Group, the 716th Test Squadron, 717th Test Squadron, 718th Test Squadron and 804th Test Support Squadron at Arnold Air Force Base, Tenn., May 20, 2022. (U.S. Air Force photo by Jill Pickett)
Col. Jeffrey Geraghty, left, commander, Arnold Engineering Development Complex, passes the guidon of the newly-activated 804th Test Group (804 TG) to Col. Lincoln Bonner as Bonner assumes command of the 804 TG during an activation ceremony May 20, 2022, at Arnold Air Force Base, Tenn. The 804 TG was previously designated the Test Division and was led by Bonner. Four other squadrons were activated under the 804 TG during the ceremony. Those squadrons were previously branches under the Test Division. (U.S. Air Force photo by Jill Pickett)
Col. Lincoln Bonner, left, commander, 804th Test Group, passes the guidon of the newly-activated 716th Test Squadron (716 TS) to Lt. Col. John McShane as McShane assumes command of the 716 TS during an activation ceremony May 20, 2022, at Arnold Air Force Base, Tenn. The 716 TS was previously designated the Aerodynamics Test Branch and was led by McShane. (U.S. Air Force photo by Jill Pickett)
Col. Lincoln Bonner, left, commander, 804th Test Group, passes the guidon of the newly-activated 717th Test Squadron (717 TS) to Lt. Col. Lane Haubelt as Haubelt assumes command of the 717 TS during an activation ceremony May 20, 2022, at Arnold Air Force Base, Tenn. The 717 TS was previously designated the Propulsion Test Branch and was led by Haubelt. (U.S. Air Force photo by Jill Pickett)
Col. Lincoln Bonner, left, commander, 804th Test Group, passes the guidon of the newly-activated 718th Test Squadron (718 TS) to Lt. Col. Dayvid Prahl as Prahl assumes command of the 718 TS during an activation ceremony May 20, 2022, at Arnold Air Force Base, Tenn. The 718 TS was previously designated the Space Test Branch and was led by Prahl. (U.S. Air Force photo by Jill Pickett)
Col. Lincoln Bonner, left, commander, 804th Test Group, passes the guidon of the newly-activated 804th Test Support Squadron (804 TSS) to Josh Meeks as Meeks assumes leadership of the 804 TSS during an activation ceremony May 20, 2022, at Arnold Air Force Base, Tenn. The 804 TSS was previously designated the Test Systems Branch and was led by Meeks. (U.S. Air Force photo by Jill Pickett)
Eosinophils are granulocytes arising in the bone marrow from granulocyte-monocyte progenitors; they are released into the peripheral blood as terminally differentiated cells and rapidly migrate to their target tissues. Eosinophils are classically associated with type 2 inflammation that is characteristic of parasite infections and further are known to contribute critically to the pathogenesis of allergic asthma (Lee et al., 2004). In asthma, eosinophils drive multiple hallmarks of the disease, including mucus production, smooth muscle cell hyperplasia, angiogenesis, and fibrosis (Bergeron et al., 2009), and thereby contribute to asthma exacerbations. Targeting eosinophils is now a well-established strategy for the treatment of patients with severe eosinophilic asthma that are refractory to standard of care (i.e., steroid-based treatments; Castro et al., 2011). In the steady state, only small numbers of eosinophils are released from the bone marrow; these numbers increase dramatically during type 2 inflammation (Travers and Rothenberg, 2015). Eosinopoiesis during inflammation and in the steady state is dependent on the cytokine IL-5 (Kopf et al., 1996). IL-5 signaling requires the common β-chain, which is shared with the cytokines IL-3 and GM-CSF. IL-5 acts on eosinophils at multiple time points during their lifespan. In addition to stimulating the differentiation and maturation of eosinophil-committed progenitors in the bone marrow, contributing to eosinophil egress from the bone marrow, IL-5 synergizes with chemotactic factors such as eotaxin-1 (CCL11) to attract eosinophils to tissues, primes eosinophils for activation in response to various mediators, and extends the eosinophil lifespan by blocking apoptosis (Jung and Rothenberg, 2014; Travers and Rothenberg, 2015). IL-5 overexpression is sufficient to induce massive eosinophilia but alone does not induce tissue damage (Dent et al., 1990). IL-5 neutralization or overexpression thus represents a convenient tool to study the contribution of eosinophils to health and disease.
Eosinophils constitute an abundant cellular infiltrate of solid tumors (Lotfi et al., 2007). Interestingly, in historical surveys, tumor-associated tissue eosinophilia tends to be associated with improved prognosis in solid cancers, in particular in malignancies of the gastrointestinal tract such as gastric cancer (Cuschieri et al., 2002; Iwasaki et al., 1986) and colorectal cancer (CRC; Fernández-Aceñero et al., 2000; Nielsen et al., 1999). However, only very few experimental studies have mechanistically addressed eosinophil functions in models of carcinogenesis. In a recent study, eosinophils have been reported to enhance antitumor immune responses by normalizing the tumor vasculature, promoting macrophage polarization toward an inflammatory phenotype and enhancing the infiltration of CD8+ T cells through the release of CCL5, CXCL9, and CXCL10 (Carretero et al., 2015). Eosinophils were also shown to restrict melanoma growth upon IL-33 treatment through the recruitment and activation of cytotoxic T cells and natural killer (NK) cells (Lucarini et al., 2017). In addition, eosinophils have been suggested to exert direct tumoricidal properties by releasing their granular content. To examine possible beneficial or detrimental functions of eosinophils in syngeneic and genetic CRC models, we took advantage of various constitutive and inducible models of eosinophil deficiency or overproduction. We found that CRC cells grow more rapidly and form larger tumors in mice that lack eosinophils; this critical role of eosinophils could be linked to their ability to drive CD4+ and CD8+ T cell responses within the tumor microenvironment (TME). The antitumor activities of eosinophils were found to be activated by GM-CSF signaling through the transcription factor IRF-5 and to be counterregulated by IL-10. The administration of recombinant GM-CSF effectively stimulates antitumor immunity in an eosinophil-dependent manner. The prognostic value of eosinophil infiltration and link between the presence of eosinophils and intratumoral T cell responses could further be confirmed in a large cohort of CRC patients, implicating this cell type in tumor immunity and making it an attractive target in immunotherapy against cancer.
GM-CSF is a potent cytokine promoting the differentiation of myeloid cells, and both pro- and antitumor effects have been reported (Hong, 2016). While GM-CSF can exert stimulatory effects on tumor progression depending on the tumor type or cancer model, it can also be used as an immunostimulatory adjuvant to elicit antitumor immunity. GM-CSF is already administered in clinical trials, with clear benefit in certain cancer settings, particularly when used in combination with checkpoint inhibitors (Hodi et al., 2014). Both immune-dependent and independent mechanisms have been advanced to explain the benefit of GM-CSF therapy (Yamashita et al., 1989; Mach et al., 2000). Interestingly, GM-CSF is found to be overexpressed in one third of CRCs, and patients whose tumors express both GM-CSF and the GM-CSF receptor have excellent 5-yr survival rates (Urdinguio et al., 2013). In the TME, we have identified GM-CSF and IL-10 as critical regulators of eosinophil activity with opposing effects. Our data add to a growing body of evidence implicating GM-CSF in many aspects of eosinophil biology, including their development and survival at steady state (Willebrand and Voehringer, 2016) and their activation (Griseri et al., 2015) and migration (Liu et al., 2015) during inflammation. Studying GM-CSF is challenging due to the shared use of the common β-chain of the GM-CSF receptor by IL-5 and IL-3; we have been able to at least partly overcome this challenge through the use of a mouse strain lacking the (specific) α-chain and of a Cre recombinase that is expressed only after eosinophils have exited the bone marrow. Indeed, eosinophil counts in the bone marrow, blood and spleen are not significantly different in Eo-Cre × Csf2rbfl/fl mice and their WT littermates but rather are specifically reduced in the TME, along with the expression of activation markers by the residual intratumoral eosinophil population. Furthermore, we show that the beneficial effects of GM-CSF administration on tumor control require the expression of the GM-CSF receptor β-chain on eosinophils. The two observations combined indicate that eosinophil activation at and migration to the tumor site both require GM-CSF signaling, with GM-CSF-activated eosinophils driving CD4+ and CD8+ T cell responses. 2b1af7f3a8