Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press PERSPECTIVE Tyrosyl phosphorylation toggles a Runx1 switch Benjamin G. Neel1,2 and Nancy A. Speck3,4 1Campbell Family Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto, Ontario M5G 1L7, Canada; 2Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada; 3Abramson Family Cancer Research Institute, Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA The Runx1 transcription factor is post-translationally core-binding factors (Runx1), Src family kinases (SFKs), modified by seryl/threonyl phosphorylation, acetylation, and the protein tyrosine phosphatase Shp2. and methylation that control its interactions with tran- Runx1 is a sequence-specific DNA-binding transcrip- scription factor partners and epigenetic coregulators. In tion factor involved in multiple developmental processes this issue of Genes & Development, Huang and col- in mammals, including hematopoiesis, neurogenesis, hair leagues (pp. 1587–1601) describe how the regulation of follicle development, and chondrogenesis. In hematopoie- Runx1 tyrosyl phosphorylation by Src family kinases and sis, Runx1 is essential for the formation of hematopoietic the Shp2 phosphatase toggle Runx1’s interactions be- stem cells (HSCs) from endothelium during embryonic tween different coregulatory molecules. development (North et al. 1999), although it is no longer critically required in HSCs once they form, and its deletion only minimally impacts functional HSC num- bers (Ichikawa et al. 2004; Growney et al. 2005; Cai et al. The connection between signaling and the gene expression 2011). Runx1 is essential, though, for the terminal circuitry often contains a transcription factor ‘‘switch’’ differentiation of several blood cell lineages, including that is regulated by phosphorylation. This can be an on–off T cells, B cells, invariant natural killer cells (Va14i NKT), switch in which a transcription factor subunit is inactive basophils, and megakaryocytes (Ichikawa et al. 2004; or sequestered in one state, and phosphorylation or de- Growney et al. 2005; Djuretic et al. 2009; Mukai et al. phosphorylation activates it. Mechanisms by which a 2012). Runx1 is required in multiple T-cell developmen- change in phosphorylation status activates a transcription tal stages. The initial steps of T-cell differentiation in the factor complex can include translocation of one of its thymus involve progression through a series of four CD4À components to the nucleus, protection from proteolytic CD8À (‘‘double-negative’’) T-cell precursor stages (DN1– degradation, increased DNA-binding affinity, or associa- DN4), followed by a CD4+ CD8+ ‘‘double-positive’’ stage. tion with/disassociation from transcription factor part- T cells then choose to become either CD4+ (helper T cells) ners or coregulatory molecules. Classic examples of on– or CD8+ (cytotoxic T cells). Runx1 is required for two off switches are the signal transducers and activators of steps within the double-negative progression, from DN2 transcription (STAT) proteins, which are phosphorylated to DN3 and from DN3 to DN4 (Ichikawa et al. 2004; by the Janus-activated kinases (JAKs). Unphosphorylated, Growney et al. 2005; Egawa et al. 2007). As discussed inactive STAT usually resides in the cytoplasm and is below, Runx1 is also involved in differentiation of the unable to bind DNA. Upon phosphorylation by JAKs, the CD8+ T-cell lineage and has major roles in the elaboration STATs homodimerize or heterodimerize, translocate into of helper T-cell subsets in the periphery, particularly the nucleus, bind DNA, and activate gene expression. Th17 and regulatory T cells. The transcriptional net- The JAK/STAT pathway is unusual in that it is activated works in which Runx1 participates in T-cell development by tyrosyl phosphorylation, whereas the great majority of are complex, and we refer interested readers to more transcription factor switches toggled by phosphorylation comprehensive reviews of this topic by other investigators use seryl/threonyl phosphorylation. In this issue of Genes (Collins et al. 2009; Djuretic et al. 2009; Wong et al. 2011). & Development, Huang et al. (2012) describe a new tran- Deletion of Runx1 in the fetal liver or adult bone marrow scription factor switch driven by tyrosyl phosphorylation, also severely impairs megakaryocyte differentiation consisting of a DNA-binding subunit of the heterodimeric (Ichikawa et al. 2004; Growney et al. 2005). In the absence of Runx1, circulating platelet numbers are reduced, [Keywords: RUNX1; tyrosine phosphorylation; c-Src; Shp2; megakaryo- megakaryocyte progenitor numbers increase, and large, cyte; T cell] polyploid megakaryocytes are replaced by lower-ploidy 4Corresponding author E-mail [email protected] micromegakaryocytes in the bone marrow. Autosomal Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.198051.112. dominant mutations in RUNX1 cause familial platelet 1520 GENES & DEVELOPMENT 26:1520–1526 Ó 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Tyrosyl phosphorylation regulates Runx1 disorder (FPD) with propensity to acute myeloid leuke- et al. 1998; Yoshida and Kitabayashi 2008; Wang et al. mia (AML), in which patients present with thrombocy- 2009; Bakshi et al. 2010; Guo and Friedman 2011; Huang topenia and/or functional platelet abnormalities and have et al. 2011; Yu et al. 2012). Previous studies have shown a 35% lifetime incidence of AML, a striking demonstra- that interactions with these epigenetic regulators are tion of the importance of RUNX1 dosage in maintaining modulated by post-translational modifications of Runx1, normal hematopoiesis and suppressing leukemia (Song which include seryl/threonyl phosphorylation, methyla- et al. 1999). tion, and acetylation (Wang et al. 2009). Now, Huang et al. Many somatically acquired mutations in RUNX1 are (2012) add tyrosyl phosphorylation to this list. found in various hematological malignancies, including de Previously, these investigators identified Runx1-inter- novo and therapy-related AML, myelodysplastic syndrome acting proteins by biotin-tagging Runx1 and purifying it (MDS), chronic myelomonocytic leukemia (CMML), and with its associated proteins from a megakaryocytic pre- acute lymphocytic leukemia (both pre-B-ALL and T-ALL) cursor cell line by streptavidin affinity chromatography (Zelent et al. 2004; Grossmann et al. 2011a; Mangan and (Huang et al. 2009). They discovered some of the usual Speck 2011; Zhang et al. 2012). Mutations include chro- suspects on the list of associated proteins—transcription mosomal rearrangements and loss-of-function (amorphic), factors, epigenetic coregulators, and cyclin-dependent neomorphic, and anti-morphic mutations. RUNX1 muta- kinases (CDK)—but in addition, they identified the non- tions in chronic myelogenous leukemia (CML) are seen in receptor tyrosine kinase c-Src and the tyrosine phospha- the context of conversion of the chronic phase to blast tase Shp2 (Ptpn11). Although there have been numerous crisis (Grossmann et al. 2011b). RUNX1 mutations are reports of seryl/threonyl phosphorylation of Runx1 by categorized as ‘‘class II’’ mutations that primarily serve to several different kinases, including cdk1/cyclin B and impair hematopoietic differentiation. cdk6/cyclin D3, Erk, and the homeodomain-interacting Runx1 is both an activator and repressor of transcrip- protein kinases (Hipk1/Hipk2), (Tanaka et al. 1996; tion and can toggle between these modes of action in Aikawa et al. 2006; Biggs et al. 2006; L Zhang et al. various developmental contexts and on different target 2008; Guo and Friedman 2011), the inclusion of c-Src and genes. Runx1 also has distinct effects on gene regulation Shp2 on the list of Runx1-binding partners suggested that as cells differentiate down a particular pathway. For exam- it might also be tyrosyl phosphorylated. The investigators ple, Runx1 regulates transcription of the Cd4 gene differ- confirmed this supposition by performing phosphotyro- ently at successive stages of T-cell development. Runx1 sine immunoblotting and then mapped the tyrosyl phos- protein is present in CD4À CD8À (double-negative) phorylation sites on Runx1 by a combination of mass T cells, CD4+ CD8+ (double-positive) T cells, and CD4+ spectrometry and mutational analysis. They also ob- and CD8+ T cells (Lorsbach et al. 2004). Runx1 actively served Runx1 tyrosyl phosphorylation in another mega- silences Cd4 in CD4À CD8À cells (Taniuchi et al. 2002) karyocyte cell line and in murine thymocytes. but no longer represses Cd4 expression in double-positive The phoshorylated tyrosine residues lie in two of the cells. Moreover, when double-positive cells choose to less well-characterized regions of Runx1, both of which differentiate down the CD8 pathway, Runx1, in collabo- function to dampen Runx1 activity (Fig. 1). One region ration with its sibling, Runx3, again silences Cd4 expres- was shown in biochemical studies to inhibit DNA sion in CD8+ cells (with Runx3 playing the major role) binding. Purified full-length Runx1 assumes an auto- (Taniuchi et al. 2002; Woolf et al. 2003). inhibited conformation that causes it to bind naked Whether Runx1 activates or represses