Mol. Cells 2020; 43(2): 107-113
Published online January 10, 2020
https://doi.org/10.14348/molcells.2019.0291
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: wooseok.seo@med.nagoya-u.ac.jp
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The Runt-related transcription factors (RUNX) transcription factors have been known for their critical roles in numerous developmental processes and diseases such as autoimmune disorders and cancer. Especially, RUNX proteins are best known for their roles in hematopoiesis, particularly during the development of T cells. As scientists discover more types of new immune cells, the functional diversity of RUNX proteins also has been increased over time. Furthermore, recent research has revealed complicated transcriptional networks involving RUNX proteins by the current technical advances. Databases established by next generation sequencing data analysis has identified ever increasing numbers of potential targets for RUNX proteins and other transcription factors. Here, we summarize diverse functions of RUNX proteins mainly on lymphoid lineage cells by incorporating recent discoveries.
Keywords development, immune cells, Runx family, transcription factors
The Runt-related transcription factors (RUNX) transcription factors are present in all metazoans, and there are three members of the RUNX family proteins in mammals that play pivotal roles in multiple developmental processes. Initial genetic studies in mice have characterized the essential roles of RUNX1, RUNX2 and RUNX3 in hematopoiesis (Okuda et al., 1996; Wang et al., 1996a), osteopoiesis (Komori et al., 1997; Mundlos et al., 1997; Otto et al., 1997) and neurogenesis (Levanon et al., 2002), respectively. However, as we comprehensively discuss in this review, recent studies have revealed that the functions of the RUNX family proteins in regulating development are much more complex than previously thought. Moreover, these transcription factors have additional functions in regulating cellular function beyond development. For instance, RUNX proteins are shown to be a functional regulator of tissue-resident T cells (Milner et al., 2017), and they have been reported to exert either oncogenic or tumor suppressive roles in the development of hematopoietic cancer as well as solid tumors such as gastric and colon cancers (Ito et al., 2015). One of the mechanisms for their diverse roles is the generation of multiple isoforms from each
RUNX proteins contain a well-conserved DNA-binding domain (Runt domain). Heterodimerization with an essential binding partner protein, core binding factor beta (CBFβ), increases the DNA binding affinity of RUNX proteins (Wang et al., 1996b). Transcripts generated from the
T cell development occurs in the thymus and it begins with early T cell progenitors that are negative for CD4 and CD8 coreceptor expression, thereby referred to as double-negative (DN) thymocytes. A RUNX1-dependent differentiation process leads DN cells to become CD4 and CD8 double-positive (DP) thymocytes that express T cell receptor (TCR) (Fig. 2) (Egawa et al., 2007; Kim et al., 1999). It has been shown that RUNX1 mediates DN3 to DN4 transition by promoting the proliferation of thymocytes that have passed the selection process, known as b-selection (Fig. 2). During b-selection, DN3 cells that failed to express a functional TCRb chain are eliminated. RUNX1 protein also facilitates activation of Tcrb locus by specifically binding to a transcriptional enhancer, known as Eb, located on the 3’ side in the
For many years, we have been trying to understand how TCR signaling in DP progenitors is translated into the exclusive expression of ThPOK versus RUNX3 in MHC class II-restricted and MHC class I-restricted cells, respectively (Fig. 2). The most widely-spread notion on this subject is that the strength and/or duration of TCR signaling during thymocyte differentiation must play a key component. The kinetic signaling model proposed by Alfred Singer group suggest the following. The duration of TCR signaling is translated into the lineage choice. DP thymic progenitors terminate
RUNX proteins continue to function after the T cell lineage decision since continued repression of
As previously mentioned, mature CD4+ T and CD8+ T cells stably express ThPOK and RUNX3, respectively, to maintain their lineage identity. This all-or-none reciprocal expression pattern, however, is variable, as it has been reported that a proportion of intestinal CD4+ intraepithelial lymphocytes (IELs) show much lower expression of ThPOK (Mucida et al., 2013). Instead, these cells acquire RUNX3 expression which is associated with the expression of CD8αα coreceptor (CD8α+CD8β–) (Reis et al., 2013). This indicates that signals from the gut microenvironment control transcriptional program of intraepithelial CD4+ T cells, which make them distinct from conventional CD4+ helper T cells. In addition, the acquisition of RUNX3 expression by CD4+ IELs correlates with the unique expression patterns of CD8αα IEL resembling features of cytotoxic CD8+ T cells. Therefore, suppression of ThPOK and induction of RUNX3 by CD4+ IELs are necessary steps for the acquisition of CTL-like features of CD8αα IELs.
Early works on RUNX proteins focused on primary development of conventional T cells. However, recent studies have given new insight on the importance of RUNX in the differentiation of effector and memory T cells. For example, RUNX3 functions beyond the development of naïve CD8+ T cells and when naïve CD8+ T cells encounter foreign antigens RUNX3 plays a critical role in driving transcriptional programs for their effector functions. In addition, the role of RUNX3 in promoting CD8+ effector function is synergized with the help of T-box proteins such as T-bet (Cruz-Guilloty et al., 2009). Moreover, during the resolution of immune responses, RUNX3 is also required to epigenetically reprogram the surviving cytotoxic CD8+ T cells into effector memory CD8+ T cells. These epigenetic changes mediated by RUNX3 are based on the modification of chromatin accessibility of target gene loci (Wang et al., 2018).
Naïve CD4+ helper T cells differentiate into effector Th1 or Th2 subset depending on the nature of environmental cues. This bifurcation is regulated by two transcription factors, T-box expressed in T cells (T-bet) and GATA-binding protein-3 (GATA-3), respectively. It was reported that RUNX1 negatively regulates GATA-3 and thus inhibits Th2 cell differentiation (Komine et al., 2003). Indeed, RUNX1 overexpression was shown to be enough to enforce Th1 cell differentiation even in Th2-stimulating culture condition
RUNX1 and RUNX3 are also reported to be involved in the differentiation of Th17 cells (Wang et al., 2014). Th17 cells produce IL-17, IL-21 and IL-22 to defend hosts from extracellular pathogens by recruiting macrophages and neutrophils to infected area. The differentiation of Th17 cells requires the transcription factor RAR-related orphan receptor gamma t (RORγt) as well as transforming growth factor beta (TGFβ)/IL-6 signaling pathways. RUNX1 contributes to the differentiation of Th17 cells by cooperating with RORγt to induce
Foxp3 is an indispensable transcription factor for the generation of regulatory T (Treg) cells, a subtype of CD4+ T cells required for the negative regulation of T cell responses.
NKT cells are a subset of non-conventional T cells expressing invariant TCR and exhibit properties of both T and natural killer (NK) cells. Similar to conventional T cells, NKT lymphocytes respond to glycolipid antigens presented by the nonpolymorphic MHC class I-like CD1d molecule. Upon activation, NKT cells produce variety of cytokines such as IFNγ, IL-4, tumor necrosis factor alpha (TNFα), GM-CSF, IL-3 and IL-10. Development of NKT cells from DP thymocytes in the thymus is dependent on RUNX1 (Egawa et al., 2005). Specifically, during the development into mature NKT cells, RUNX1 is required for the positive selection of NKT cells during the development of NKT cells. A recent study further discovered that differentiation of NKT17 cells, a special subtype of NKT cells expressing IL-17, also requires RUNX1 (Thapa et al., 2017).
Epidermis layer of skin contains a variety of special immune cells such as Langerhans cells, dendritic cells (DCs) specialized for skin immunity. RUNX3 has been reported to be essential for differentiation of Langerhans cells, especially with the CBFβ2 isoform (Tenno et al., 2017; Woolf et al., 2007). Another special type of cells in epidermis is dendritic epidermal T cells (DETCs), which are γδ T cells with a shape resembling DCs. Similar to NKT cells, DETCs express invariant TCRs with limited repertoire which recognize antigens in a classical MHC-independent manner. RUNX3 has been shown to regulate expression of CD103, an important molecule for the migration of DETCs, and IL-2Rβ, a receptor for IL-2 or IL-15 necessary for the proliferation of DETCs. Thus RUNX3 plays a crucial role in DETC maturation and maintenance in epidermal layer (Woolf et al., 2007).
It is well established that B lymphocytes develop in a stepwise progression from lymphoid progenitors by the cooperative programing of three core transcription factors, E2A, EBF1 and PAX5 (Mandel and Grosschedl, 2010). More specifically, the earliest step of B lymphoid specification from CLP (common lymphoid progenitors) to pre-pre B cells is regulated by E2A/EBF1. At the molecular levels, it was shown that E2A binds to the promoter of
NK cells are considered as innate lymphocytes because they exhibit features resembling both innate and adaptive immunity (Vivier et al., 2011). Initially, mice with reduced CBFβ expression showed defects in NK cell development, indicating an involvement of RUNX-CBFβ complexes during the NK cell differentiation (Guo et al., 2008). RUNX3 expression is initiated from NK precursors and is maintained throughout the whole developmental processes of NK cells (Ohno et al., 2008). It is elegantly shown that RUNX3 cooperates with ETS and T-box transcription factors to activate the transcription program of NK cells, which is dependent on IL-15 signaling (Levanon et al., 2014). RUNX3 ChIP-seq analysis in resting or IL-15 activated NK cells showed that around 1,000 genes are bound by RUNX3 specifically after IL-15 signaling and many of them were related to NK proliferation and function, indicating that RUNX3 plays pivotal roles in NK cell development. Interestingly, recent findings suggest that NK cells exhibit their lymphocyte-like function by undergoing clonal expansion and memory responses reminiscent of CD8+ T cells (Cooper et al., 2009; Kamimura and Lanier, 2015; O’Sullivan et al., 2015; Sun et al., 2012). This clonal expansion and memory phenotype by NK cells require IL-12 signaling followed by STAT4 signaling. Since RUNX1 and RUNX3 are targets of STAT4, increased expression RUNX proteins after the clonal expansion might be the result of the binding of STAT4 to the promoters of
RUNX1 and RUNX3 have originally been discovered to promote hematopoietic stem cell and cytotoxic T cell development. During the last decade, many studies have reported that RUNX1 and RUNX3 appear to play diverse roles in many other lymphoid lineage cells as we summarized in this review. Although the role of RUNX proteins in non-lymphoid lineage cells was not discussed in this short review, it should be appreciated that RUNX proteins do have important roles in DCs and macrophages function. ChIP-seq powered by next generation sequencing has identified numerous RUNX target genes in several types of immune cells. However, we still lack detailed mechanistic view on how RUNX transcription factors function after they associate with genomic regions in their target loci. Future studies by using biochemical and genetic studies will be required to answer the molecular mechanism of transcriptional regulation conducted by RUNX proteins in this post-genomic era.
This work was supported by grants from Grants-in-Aid for Scientific Research (B) (17H04090) from JSPS (I.T.) and Grants-in-Aid for Scientific Research (C) (18K07186) from JSPS (W.S.).
Authors thank for Aneela Nomura for critical reading of the manuscript.
The authors have no potential conflicts of interest to disclose.
Mol. Cells 2020; 43(2): 107-113
Published online February 29, 2020 https://doi.org/10.14348/molcells.2019.0291
Copyright © The Korean Society for Molecular and Cellular Biology.
Wooseok Seo1,2,* and Ichiro Taniuchi2
1Department of Immunology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan, 2Laboratory for Transcriptional Regulation, RIKEN Center for Integrative Medical Sciences, Yokohama 230-0045, Japan
Correspondence to:*Correspondence: wooseok.seo@med.nagoya-u.ac.jp
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/.
The Runt-related transcription factors (RUNX) transcription factors have been known for their critical roles in numerous developmental processes and diseases such as autoimmune disorders and cancer. Especially, RUNX proteins are best known for their roles in hematopoiesis, particularly during the development of T cells. As scientists discover more types of new immune cells, the functional diversity of RUNX proteins also has been increased over time. Furthermore, recent research has revealed complicated transcriptional networks involving RUNX proteins by the current technical advances. Databases established by next generation sequencing data analysis has identified ever increasing numbers of potential targets for RUNX proteins and other transcription factors. Here, we summarize diverse functions of RUNX proteins mainly on lymphoid lineage cells by incorporating recent discoveries.
Keywords: development, immune cells, Runx family, transcription factors
The Runt-related transcription factors (RUNX) transcription factors are present in all metazoans, and there are three members of the RUNX family proteins in mammals that play pivotal roles in multiple developmental processes. Initial genetic studies in mice have characterized the essential roles of RUNX1, RUNX2 and RUNX3 in hematopoiesis (Okuda et al., 1996; Wang et al., 1996a), osteopoiesis (Komori et al., 1997; Mundlos et al., 1997; Otto et al., 1997) and neurogenesis (Levanon et al., 2002), respectively. However, as we comprehensively discuss in this review, recent studies have revealed that the functions of the RUNX family proteins in regulating development are much more complex than previously thought. Moreover, these transcription factors have additional functions in regulating cellular function beyond development. For instance, RUNX proteins are shown to be a functional regulator of tissue-resident T cells (Milner et al., 2017), and they have been reported to exert either oncogenic or tumor suppressive roles in the development of hematopoietic cancer as well as solid tumors such as gastric and colon cancers (Ito et al., 2015). One of the mechanisms for their diverse roles is the generation of multiple isoforms from each
RUNX proteins contain a well-conserved DNA-binding domain (Runt domain). Heterodimerization with an essential binding partner protein, core binding factor beta (CBFβ), increases the DNA binding affinity of RUNX proteins (Wang et al., 1996b). Transcripts generated from the
T cell development occurs in the thymus and it begins with early T cell progenitors that are negative for CD4 and CD8 coreceptor expression, thereby referred to as double-negative (DN) thymocytes. A RUNX1-dependent differentiation process leads DN cells to become CD4 and CD8 double-positive (DP) thymocytes that express T cell receptor (TCR) (Fig. 2) (Egawa et al., 2007; Kim et al., 1999). It has been shown that RUNX1 mediates DN3 to DN4 transition by promoting the proliferation of thymocytes that have passed the selection process, known as b-selection (Fig. 2). During b-selection, DN3 cells that failed to express a functional TCRb chain are eliminated. RUNX1 protein also facilitates activation of Tcrb locus by specifically binding to a transcriptional enhancer, known as Eb, located on the 3’ side in the
For many years, we have been trying to understand how TCR signaling in DP progenitors is translated into the exclusive expression of ThPOK versus RUNX3 in MHC class II-restricted and MHC class I-restricted cells, respectively (Fig. 2). The most widely-spread notion on this subject is that the strength and/or duration of TCR signaling during thymocyte differentiation must play a key component. The kinetic signaling model proposed by Alfred Singer group suggest the following. The duration of TCR signaling is translated into the lineage choice. DP thymic progenitors terminate
RUNX proteins continue to function after the T cell lineage decision since continued repression of
As previously mentioned, mature CD4+ T and CD8+ T cells stably express ThPOK and RUNX3, respectively, to maintain their lineage identity. This all-or-none reciprocal expression pattern, however, is variable, as it has been reported that a proportion of intestinal CD4+ intraepithelial lymphocytes (IELs) show much lower expression of ThPOK (Mucida et al., 2013). Instead, these cells acquire RUNX3 expression which is associated with the expression of CD8αα coreceptor (CD8α+CD8β–) (Reis et al., 2013). This indicates that signals from the gut microenvironment control transcriptional program of intraepithelial CD4+ T cells, which make them distinct from conventional CD4+ helper T cells. In addition, the acquisition of RUNX3 expression by CD4+ IELs correlates with the unique expression patterns of CD8αα IEL resembling features of cytotoxic CD8+ T cells. Therefore, suppression of ThPOK and induction of RUNX3 by CD4+ IELs are necessary steps for the acquisition of CTL-like features of CD8αα IELs.
Early works on RUNX proteins focused on primary development of conventional T cells. However, recent studies have given new insight on the importance of RUNX in the differentiation of effector and memory T cells. For example, RUNX3 functions beyond the development of naïve CD8+ T cells and when naïve CD8+ T cells encounter foreign antigens RUNX3 plays a critical role in driving transcriptional programs for their effector functions. In addition, the role of RUNX3 in promoting CD8+ effector function is synergized with the help of T-box proteins such as T-bet (Cruz-Guilloty et al., 2009). Moreover, during the resolution of immune responses, RUNX3 is also required to epigenetically reprogram the surviving cytotoxic CD8+ T cells into effector memory CD8+ T cells. These epigenetic changes mediated by RUNX3 are based on the modification of chromatin accessibility of target gene loci (Wang et al., 2018).
Naïve CD4+ helper T cells differentiate into effector Th1 or Th2 subset depending on the nature of environmental cues. This bifurcation is regulated by two transcription factors, T-box expressed in T cells (T-bet) and GATA-binding protein-3 (GATA-3), respectively. It was reported that RUNX1 negatively regulates GATA-3 and thus inhibits Th2 cell differentiation (Komine et al., 2003). Indeed, RUNX1 overexpression was shown to be enough to enforce Th1 cell differentiation even in Th2-stimulating culture condition
RUNX1 and RUNX3 are also reported to be involved in the differentiation of Th17 cells (Wang et al., 2014). Th17 cells produce IL-17, IL-21 and IL-22 to defend hosts from extracellular pathogens by recruiting macrophages and neutrophils to infected area. The differentiation of Th17 cells requires the transcription factor RAR-related orphan receptor gamma t (RORγt) as well as transforming growth factor beta (TGFβ)/IL-6 signaling pathways. RUNX1 contributes to the differentiation of Th17 cells by cooperating with RORγt to induce
Foxp3 is an indispensable transcription factor for the generation of regulatory T (Treg) cells, a subtype of CD4+ T cells required for the negative regulation of T cell responses.
NKT cells are a subset of non-conventional T cells expressing invariant TCR and exhibit properties of both T and natural killer (NK) cells. Similar to conventional T cells, NKT lymphocytes respond to glycolipid antigens presented by the nonpolymorphic MHC class I-like CD1d molecule. Upon activation, NKT cells produce variety of cytokines such as IFNγ, IL-4, tumor necrosis factor alpha (TNFα), GM-CSF, IL-3 and IL-10. Development of NKT cells from DP thymocytes in the thymus is dependent on RUNX1 (Egawa et al., 2005). Specifically, during the development into mature NKT cells, RUNX1 is required for the positive selection of NKT cells during the development of NKT cells. A recent study further discovered that differentiation of NKT17 cells, a special subtype of NKT cells expressing IL-17, also requires RUNX1 (Thapa et al., 2017).
Epidermis layer of skin contains a variety of special immune cells such as Langerhans cells, dendritic cells (DCs) specialized for skin immunity. RUNX3 has been reported to be essential for differentiation of Langerhans cells, especially with the CBFβ2 isoform (Tenno et al., 2017; Woolf et al., 2007). Another special type of cells in epidermis is dendritic epidermal T cells (DETCs), which are γδ T cells with a shape resembling DCs. Similar to NKT cells, DETCs express invariant TCRs with limited repertoire which recognize antigens in a classical MHC-independent manner. RUNX3 has been shown to regulate expression of CD103, an important molecule for the migration of DETCs, and IL-2Rβ, a receptor for IL-2 or IL-15 necessary for the proliferation of DETCs. Thus RUNX3 plays a crucial role in DETC maturation and maintenance in epidermal layer (Woolf et al., 2007).
It is well established that B lymphocytes develop in a stepwise progression from lymphoid progenitors by the cooperative programing of three core transcription factors, E2A, EBF1 and PAX5 (Mandel and Grosschedl, 2010). More specifically, the earliest step of B lymphoid specification from CLP (common lymphoid progenitors) to pre-pre B cells is regulated by E2A/EBF1. At the molecular levels, it was shown that E2A binds to the promoter of
NK cells are considered as innate lymphocytes because they exhibit features resembling both innate and adaptive immunity (Vivier et al., 2011). Initially, mice with reduced CBFβ expression showed defects in NK cell development, indicating an involvement of RUNX-CBFβ complexes during the NK cell differentiation (Guo et al., 2008). RUNX3 expression is initiated from NK precursors and is maintained throughout the whole developmental processes of NK cells (Ohno et al., 2008). It is elegantly shown that RUNX3 cooperates with ETS and T-box transcription factors to activate the transcription program of NK cells, which is dependent on IL-15 signaling (Levanon et al., 2014). RUNX3 ChIP-seq analysis in resting or IL-15 activated NK cells showed that around 1,000 genes are bound by RUNX3 specifically after IL-15 signaling and many of them were related to NK proliferation and function, indicating that RUNX3 plays pivotal roles in NK cell development. Interestingly, recent findings suggest that NK cells exhibit their lymphocyte-like function by undergoing clonal expansion and memory responses reminiscent of CD8+ T cells (Cooper et al., 2009; Kamimura and Lanier, 2015; O’Sullivan et al., 2015; Sun et al., 2012). This clonal expansion and memory phenotype by NK cells require IL-12 signaling followed by STAT4 signaling. Since RUNX1 and RUNX3 are targets of STAT4, increased expression RUNX proteins after the clonal expansion might be the result of the binding of STAT4 to the promoters of
RUNX1 and RUNX3 have originally been discovered to promote hematopoietic stem cell and cytotoxic T cell development. During the last decade, many studies have reported that RUNX1 and RUNX3 appear to play diverse roles in many other lymphoid lineage cells as we summarized in this review. Although the role of RUNX proteins in non-lymphoid lineage cells was not discussed in this short review, it should be appreciated that RUNX proteins do have important roles in DCs and macrophages function. ChIP-seq powered by next generation sequencing has identified numerous RUNX target genes in several types of immune cells. However, we still lack detailed mechanistic view on how RUNX transcription factors function after they associate with genomic regions in their target loci. Future studies by using biochemical and genetic studies will be required to answer the molecular mechanism of transcriptional regulation conducted by RUNX proteins in this post-genomic era.
This work was supported by grants from Grants-in-Aid for Scientific Research (B) (17H04090) from JSPS (I.T.) and Grants-in-Aid for Scientific Research (C) (18K07186) from JSPS (W.S.).
Authors thank for Aneela Nomura for critical reading of the manuscript.
The authors have no potential conflicts of interest to disclose.
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